Pandemic Outbreaks in the 21st Century

Epidemiology, Pathogenesis, Prevention, and Treatment

This page intentionally left blank
Pandemic Outbreaks in the
21st Century
Epidemiology, Pathogenesis, Prevention, and
Treatment

Edited by
Buddolla Viswanath
Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr. Buddolla’s Educational Society,
Tirupati, India
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, United Kingdom
525 B Street, Suite 1650, San Diego, CA 92101, United States
50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States
The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom
Copyright © 2021 Elsevier Inc. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including
photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher.
Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with
organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.
elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be
noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding,
changes in research methods, professional practices, or medical treatment may become necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information,
methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their
own safety and the safety of others, including parties for whom they have a professional responsibility.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury
and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of
any methods, products, instructions, or ideas contained in the material herein.
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the British Library
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress
ISBN: 978-0-323-85662-1

For Information on all Academic Press publications

visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff

Acquisitions Editor: Linda Versteeg-Buschman
Editorial Project Manager: Timothy Bennett
Production Project Manager: Maria Bernard
Cover Designer: Miles Hitchen
Typeset by MPS Limited, Chennai, India
 Dedication
Dedicated to
THE BEST TEACHER WHO TAUGHT FROM THE
HEART BUT NOT FROM THE BOOK

Prof. Pothur Sreenivasulu (1950–2020)

Former Professor of VIROLOGY, Sri Venkateswara University, India
vi Dedication

Obituary by friends, colleagues, students, and family

I have known Prof. Sreenivasulu for over 40 years. He was a rare combination
of outstanding scientific abilities with impeccable integrity and completely
trustworthy. In my 45 years of career, I did not come across another person
who excelled him in the qualities I mentioned. His passing away is an
irreparable loss for me.

Dodla Venkata Raghava Reddy
Former Leader of Virology Program, ICRISAT,
Hyderabad, India
Email: dvrreddy369@gmail.com

I was associated with Prof. P. Sreenivasulu, Department of Virology,

S.V. University for almost 40 years. He was the anchor and pillar of the
department and was ever willing to share his vast knowledge in virology. He
was extremely committed to teaching and training young students. One of the
most significant contributions by him is the development of various methods
for diagnosis of viral diseases. The Covid-19 pandemic has underscored the
importance of his research. I fondly cherish the memory of my association with
him and other colleagues in the Department of Virology.

H.S. Savithri
NASI Senior Scientist, Department of Biochemistry,
Indian Institute of Science, Bangalore, India
Email: hss@alumni.iisc.ac.in

It is a great privilege and honor to write few words about my beloved teacher and
research supervisor, Pothur Sreenivasulu, who is a distinguished academician,
renowned virologist, and cofounder of first MSc Virology postgraduate degree
course in India. I regard him as a “God Father”; he shaped my career in virology
and encouraged me all along. I joined MSc Virology at S.V. University in 1990
with a strong intension to learn a new subject and I am fortunate enough to have
Prof. P. Sreenivasulu as one of the teachers there. He taught me general virology,
plant virology, molecular biology, and recombinant DNA technology, and his
subject knowledge was vast and incredible. He is a man with principles and
believed in only actions but not words. He is extremely patient and kind by nature
and has always helped several students in many ways. He was my guide for MSc,
 Dedication vii

PhD, and CSIR-RA from 1992 to 2002, and under his able guidance, I learnt
several aspects that are important for a successful professional as well as personal
life and those experiences turned my life altogether and made me what I am
today. He instilled passion and infused courage in me to accept challenges in
research and stood by me to find the solutions for the problems that I encountered
throughout my career. Through his guidance and support, I maintained my dignity
and integrity intact, and learnt to handle both success and failure equally in the
professional life. I admired him the most for his simplicity, dignity, honesty,
punctuality, patience, perseverance, and work-minded nature. He is my source of
inspiration and support and I am deeply thankful to him for everything that he did
for my growth for all these 30 long years. Among all his students, I am so blessed
to have longest association with Prof. P. Sreenivasulu and I am privileged to take
over his lab in the Department of Virology after his superannuation in 2010.
I have several great memories with him to cherish for the rest of my life.
We published several articles together and his last article appeared recently in
Encyclopedia of Virology during March 2021. In conclusion, Prof. P. Sreenivasulu
is not only an eminent teacher who ignited passion toward research in his
students, helping them to shape their career, but also a committed researcher who
made a mark in the field of plant virology with his great contributions. He is a
truly inspiring individual who has taught so much more than simply curriculum.
All the hard work, efforts, and care invested by him to bring best in all of us can
never be repaid in mere words. We are grateful for having a teacher like
Prof. P. Sreenivasulu. Apart from all these, he is a great human being and touched
many hearts with his kind gesture; Prof. P. Sreenivasulu lives on forever in the
hearts of virology student fraternity.

Hema Masarapu
Professor and Head, Department of Virology,
Sri Venkateswara University, Tirupati, India
Email: hemamasarapu70@gmail.com

Prof. Sreenivasulu is a dedicated scientist and teacher. I had a chance of closely

interacting with him while studying viruses associated with sugarcane,
especially mosaic viruses. I gained basic understanding on plant virus
purification, characterization, and diagnostics from him in the pre
genomics
era. He had contributed immensely on plant viruses and developed Virology
viii Dedication

Department at S.V. University and made it reach to a greater height.

Furthermore, he has trained many scholars who are in top positions in various
institutions in different countries. The country has lost an eminent teacher and
able researcher.

Rasappa Viswanathan
Head and Principal Scientist (Plant Pathology),
Division of Crop Protection, ICAR-Sugarcane Breeding Institute,
Coimbatore, India
Email: rasaviswanathan@yahoo.co.in

In academics, I am a year junior to Prof. P. Sreenivasulu, and my relationship

with him spans more than four decades. With his benevolent attitude, I became
more intimate during our PhD program. From a basic botanist, I saw him grow
as a reputed Virologist all with his scholastic abilities. He was the backbone for
the establishment of the Department of Virology together with his mentor
Prof. M. V. Nayudu. He was also instrumental in the starting of the Department
of Biotechnology with his expertise in molecular biology. He was a role model
for all the researchers, and he trained a band of virologists of international
reputation. I trust they carry forward his legacies to noble heights.
Prof. P. Sreenivasulu was a compassionate, trustworthy, sincere person and a
man of great perseverance. His sudden demise is a great loss to me.

Ghanta Ram Gopal
Former Professor of Botany, Sri Venkateswara University,
Tirupati, India
Email: ghantargopal@gmail.com

It is very fitting to honor Dr. Sreenivasulu with publishing this book.

Hanu Pappu
Professor of Plant Pathology, Washington State University,
Pullman, WA, United States
Email: hrp@wsu.edu
 Dedication ix

I just wish that everyone have had the chance to be a student of Dr. Pothur
Sreenivasulu. He was a great mentor and an extraordinary man whose work
and legacy will live on for many years. He will be greatly missed!

Vijaya Krishna Singu
Director, Laboratory Services, Central States Research Center Inc.,
Oakland, NE, United States
Email: vijay@mvsinc.net

Dr. Sreenivasulu was great motivator and inspiring personality. Learnt a lot
from him as a PhD student on how to conduct research, work aptitude, and
aptitude. He remains to be my Guru forever and greatly indebted for the work
ethics instilled. My last 30 years research career, I am still following my
teacher’s valuable lessons. He was simply great and admirable.

Sivaprasad Vankadara
Central Sericulture Research & Training Institute,
Central Silk Board, Berhampur, India
Email: siva.nsso@gmail.com

A great teacher, guide, and a friend, who believed in me, inspired me, and
encouraged me to become who I am today. Eternally grateful to my humble
teacher!

Lava Kumar
International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria
Email: L.kumar@cgiar.org

Prof. P. Sreenivasulu was the best mentor any student could ask for and I was
fortunate to be his last PhD student. Prof. P. Sreenivasulu was a personification
of knowledge and dignity. Many are knowledgeable but to be able to impart
that knowledge into others makes them a great teacher and he was a great one
in every sense. Let his legacy live on through his and his students’ work.

Srinivas KP
Department of Microbiology, New York University School of Medicine,
NY, United States
Email: dilip.srinivaskp@gmail.com
x Dedication

It has been quite a daunting task for me to gather myself and put down a few
words about my father. Also, it was all the more difficult to recollect the
association as a memory, but I have tried. He was an extremely poised and self-
reliant person, intending to do everything by himself. As the saying goes “when
you really want something to happen the whole universe will conspire so that
your wish comes true. . .”. His grit to be self-reliant seems to have got granted till
the last breath. I have grown up seeing the grit, determination, and dedication of
my father toward his work. He used to spend a minimum of
4 hours (outside of university working hours) on a daily basis in preparing
content for teaching, writing articles, books, correcting documents, etc., till his
superannuation. I believe satisfaction of the job well done used to be the end in
itself for him as I have never seen him referring to the laurels out of his work. As
a father, he used to have oodles of patience and love to handle all the crankiness
and mischievousness from me and my brother. He had a strong memory and
very critical observation skills. Once he did his legwork and took a position on
something, he never used to vacillate from it. He always motivated us to learn,
develop skills, and be independent. He had been the unwavering support behind
every aspect and step of mine and continues to be. . ..

Hima Bindu Pothur (Daughter)
#209, Sindhu Amazon, Bellandur, Bangalore
Email: himapothur@gmail.com

I like my grandfather a lot. He always helps me in studies and he is like a

“Google” for me. He can answer anything that I ask him. He plays with me as
well and he is my best grandfather.

Srisreshta Gorrepati (Granddaughter)
#209, Sindhu Amazon, Bellandur, Bangalore, India
Email: himapothur@gmail.com

My grandfather used to help me study and guide me in all subjects. He also

used to answer any number of questions without getting frustrated or annoyed.
He explained concepts in a very clear and easy way and I can recall whatever
he said at any time. He also encouraged me to do small science experiments to
increase my interest in science. I miss him a lot!

Srihitha Gorrepati (Granddaughter)
#209, Sindhu Amazon, Bellandur, Bangalore
Email: himapothur@gmail.com
 Dedication xi

My association with Prof. P. Sreenivasulu is dated back to 1971 when we joined

to pursue postgraduation in Botany at S.V. University. Later it continued till we
were awarded PhD degrees in 1978. He was elevated to the position of
Assistant Professor in Botany and attained the age of superannuation as
Professor of Virology in 2010. He was an outstanding academician and a
researcher par excellence. Dynamism and administrative capacity in managing,
organizing, and monitoring educational activities are his virtues. Patience and
perseverance, dignity and duty mindedness, honesty, and hospitality are the
noteworthy features of this great gentleman teacher. Blessed with two children,
brought up in the shade of his unassuming character brought him a memorable
parenthood. I have lost a benevolent and an immortal friend, whom I loved the
most in my life.

N. Raja Kumar
Chittoor, India

My father was passionate about virology and he was an eminent scientist in the
field of Plant Virology. He dedicated his life to teaching and research and
helped several students to come up in their lives. My father’s dedication and
perseverance will be an inspiration to many. I am proud to be his son and we
all dearly miss him.

Kodanda Pani Pothur (Son)
Miramar, FL 33029, United States
Email: panipothur@gmail.com

I am intimately associated with Professor Sreenivasulu in initiating innovative

Virology Programme in S.V. University. He is meticulous in planning and
implementing new programs, which is praise worthy. Several of his students
occupy important positions internationally. He laid solid foundation for
virology in India by creating wonderful human resource to take it forward.
Prof. Sreenivasulu richly deserves to dedicate a book on pandemic virus
outbreaks in his name.

Venkata Subba Rao Mandava
Ex-Dean of Veterinary Science, Acharya N. G. Ranga
Agricultural University, Hyderabad, India
Email: mandava_vs@yahoo.com
xii Dedication

It was 2003, I joined masters in virology with a lot of excitement and full of
energy with renewed hopes on my future life. In the department, I found many
people are easy going, friendly seniors, accommodative scholars, and helpful
permanent staff. However, I found one place peculiar, since people were
walking across this room with pin-drop silence unlike other places in the
department. With curiosity, I looked into the room and saw an old man with
big specs pretty busy in reading and writing. I smiled and moved on. As the
days passed, I accumulated more respect for him; me and my colleagues loved
his classes so much that his mannerism became ours, as part of it, one of his
manneristic words became permanent with us “Ok then”.
By then, my age was equivalent to his experience, even then he prepared a few
hours and came to the class with updated knowledge, filling the board with
notable points with the shaky hands. He showed us what excellence is. He was
a great teacher who teaches with detailing, crystal, crisp to name a few in that
master class. He maintained an enthusiastic classroom time. Once he stepped
out of the classroom, I used to be frustrated for the fact that he does not go
easy with us, which would have facilitated learning a lot from him. There used
to be some unknown arena gathered around him with passion and excellence.
But it took years for me to understand that, if you want to have a conversation
or communication with a genius, you must have a bit of it. I wish I had some.
Dear passionate teacher, you are always in our thoughts and you will remain!

Purushotham Guroji
Researcher V, Department of Dermatology,
University of Alabama Birmingham (UAB), Birmingham,
AL, United States
Email: pguroji@uabmc.edu

I’ve never met a professor like him before; he’s very punctual, interactive,
positive, and responsive. He never believed in memorizing facts; instead, he
focused on learning ideas, which made him one of my favorite professors.
I remember him fondly.

Naveen Thanjavur
Department of Virology, Sri Venkateswara University, Tirupati, India
Email: naveenthanjaoor@gmail.com
 Dedication xiii

I was a student with Prof. P. Sreenivasulu Sir from 2009 to 2011. During the
coursework, the seminars and the presentations, I was fortunate to interact
with him. Though I did not realize then, looking back, Sreenivasulu Sir was
indeed a great teacher. He was highly intelligent, yet looked for simple
solutions. He always encouraged the innovation.

Thathireddy Krishna Reddy
Department of Virology, Sri Venkateswara University,
Tirupati, India
Email: krishnasvu09@gmail.com

Prof. P. Sreenivasulu Sir was a great inspiring teacher, mentor, and researcher.
He was always there to help out his students in all ways possible. It is very hard
to believe he is no more with us. May God give strength to his family to bear
this loss. Your spirit and teachings and contagious passion will always inspire
us, Sir. Rest in Peace!

Praveen Bellam
The Hebrew University of Jerusalem, Jerusalem, Israel
Email: praveen.virology@gmail.com

Respect is the only word that sums up all of my emotions for

Prof. P. Sreenivasulu Sir. This is a huge loss for scientist-starved scientific
community of India. But he is alive within us through his teaching and will be
alive for eternal times! May his Soul rest in peace!

Yagani Jayavardhana Rao
Department of Virology, Sri Venkateswara University, Tirupati, India
Email: jayavardhanvirology@gmail.com
This page intentionally left blank
Contents

List of contributors xxi Acknowledgments 20

Foreword xxiii Conflict of interest 20
Preface xxv References 20
Acknowledgments xxvii
3. Biology, prevention, and treatment of
1. Lessons learned from the first pandemic SARS-CoV-2 (COVID-19) 25
of the 21st century, global experience, Kalanghad P. Srinivas
recommendations, and future
directions 1 3.1 Introduction 25
3.2 Origin of SARS-CoV-2 25
Kandati Kusuma, Praveen Belagal, Buddolla 3.3 SARS-CoV-2 evolution and genomic
Viswanath and Divi Venkata Ramana Sai Gopal analyses 26
1.1 Introduction 1 3.4 Molecular biology of SARS-CoV-2 27
1.2 First pandemic of the 21st century, severe 3.5 Tissue tropism and molecular
acute respiratory syndrome 3 pathogenesis of SARS-CoV-2 30
1.2.1 Structure of SARS-CoV 3 3.6 Pulmonary system 30
1.2.2 SARS-COV: mechanism of action 3 3.7 Gastrointestinal system 31
1.2.3 Global experiences 5 3.8 Nervous system 31
1.3 Future directions 6 3.9 Cardiovascular system 32
1.4 Conclusion 7 3.10 Renal system 33
References 7 3.11 Transmission 34
3.11.1 Contact and droplet transmission 34
2. Epidemiology of COVID-19 in Latin 3.11.2 Airborne transmission 35
3.11.3 Fomite transmission 35
America 11
3.11.4 Fecal
oral transmission 35
Alfonso J. Rodriguez-Morales and D. Katterine 3.11.5 Other modes of transmission 35
Bonilla-Aldana 3.12 Prevention and treatment 36
3.12.1 Vaccines 36
2.1 Introduction 11
3.12.2 Antiviral therapy 36
2.2 Previous epidemiological situation of major
3.12.3 Immune-based therapy 38
infectious diseases in Latin America 12
3.12.4 Adjunctive therapy 39
2.3 COVID-19 arrival at Latin America 13
References 39
2.4 Genomic and molecular epidemiology of
COVID-19 in Latin America 15
2.5 Emerging situations during the COVID-19 4. Avian influenza A virus infections in
pandemic in Latin America: coinfections humans: current knowledge to
and reinfections 17 enhance host innate immunity to
2.6 Health care workers infections due to control Avian influenza 43
SARS-CoV-2 17
Bosetty Anjana, Buddolla Viswanath and
2.7 Pharmacoepidemiology of the therapeutic
Soumya Dakshinamurthy
approaches in the region 18
2.8 Epidemiology of the vaccinations against 4.1 Introduction 43
COVID-19 in Latin America 18 4.2 Major IAV lineages 43
2.9 Conclusion 19 4.3 Epidemiology 44

xv
xvi Contents

4.4 Exposure risk factors to humans 45 5.5.5 Nucleotide sequencing and

4.5 Pathophysiology 46 phylogenetic analysis 68
4.5.1 Viral replication in host 46 5.6 Biosensors 69
4.6 Innate immunity and adaptive immunity 48 5.7 Prevention and control 71
4.7 Diagnosis 50 5.7.1 Vaccination 71
4.8 Clinical findings in H5N1 infection 50 5.8 Clinical management 72
4.9 Detection of Avian influenza 50 5.8.1 M2 inhibitors 73
4.9.1 Biosensors 51 5.8.2 Rimantadine 73
4.9.2 Electrical biosensors 51 5.8.3 Finding an ultimate cure for the
4.9.3 Immunosensors 51 disease 74
4.9.4 Enzymatic biosensors 51 5.8.4 Shikimic acid 74
4.9.5 Geno sensors 52 5.8.5 Importance and uses of shikimic
4.9.6 Whole-cell biosensors 52 acid 74
4.10 Conclusion 52 5.8.6 Limitations of shikimic acid
Acknowledgment 52 production 74
Conflict of interest 52 5.8.7 Alternative approach 74
References 52 5.9 Threats and challenges 75
5.10 Conclusion 79
5. Swine-origin influenza A (H1N1) virus: References 79
current status, threats, and
challenges 57 6. Molecular mechanisms of Zika fever
Praveen Belagal, Hemanth Naick Banavath and in inducing birth defects: an update 87
Buddolla Viswanath Hema Masarapu and Naga Charan Konakalla
5.1 Introduction 57 6.1 Introduction 87
5.2 Genome, structure, and functions 58 6.2 Molecular biology of ZIKV 88
5.2.1 Gene functions 59 6.2.1 Genome organization of ZIKV 88
5.2.2 Virion structure 59 6.2.2 Replication of ZIKV 89
5.2.3 Viral attachment 59 6.3 ZIKV transmission 89
5.2.4 Fusion and entry 60 6.3.1 Vector-borne transmission 89
5.2.5 Trafficking to the host cell nucleus 60 6.3.2 Nonvector transmission 90
5.2.6 Replication and transcription 60 6.4 Clinical manifestations associated with
5.2.7 Host-cell translation of vmRNAs 61 ZIKV infection 91
5.2.8 Packaging of RNA and assembly of 6.5 Molecular mechanisms underlying
virus 61 ZIKV-induced birth defects 92
5.2.9 Virus budding and release 61 6.5.1 Cellular targets and entry of ZIKV 92
5.3 Epidemiology 61 6.5.2 Induction and suppression of innate
5.3.1 Origin of “2009 swine flu” or “A immune mechanisms mediated by
(H1N1) p09” 61 ZIKV 93
5.3.2 Incidence and mortality 62 6.5.3 ZIKV-mediated mechanisms to
5.4 Clinical features 64 induce congenital Zika syndrome 96
5.4.1 Transmission 64 6.6 Summary 102
5.4.2 Infectious versus incubation period 64 Acknowledgments 103
5.4.3 Symptoms 65 References 103
5.4.4 Complications 66
5.5 Diagnostics 66
5.5.1 Diagnostics of H1N1 swine flu 7. Middle East respiratory syndrome:
(pdm09) 66 outbreak response priorities, treatment
5.5.2 Surveillance methods: advance and strategies, and clinical management
quick methods for influenza approaches 111
detection 68
Kandati Kusuma, Pandeeti Emmanuel Vijay Paul
5.5.3 Rapid influenza detection tests 68
and Buddolla Viswanath
5.5.4 Non-polymerase chain reaction-based
RNA detection methods 68 7.1 Introduction 111
 Contents xvii

7.2 Epidemiology 112 9.2.2 The worst diseases outbreaks in

7.3 Ecology and spreading of MERS-CoV history 140
virus 113 9.2.3 Prehistoric pandemic 141
7.4 Virus structure and life cycle 113 9.2.4 Historic pandemics 141
7.5 Molecular mechanisms of pathogenesis 115 9.2.5 Plague 141
7.6 Immune responses to MERS infection 116 9.2.6 Leprosy 142
7.7 MERS—initial and postinfection 9.2.7 Influenza 143
manifestations 116 9.2.8 Cholera 145
7.8 Outbreak response priorities 117 9.2.9 Ebola 146
7.9 Diagnostics 117 9.2.10 Lessons learned from Ebola
7.10 Vaccines 117 outbreaks 147
7.11 Treatment strategies 118 9.2.11 HIV/AIDS 147
7.12 Clinical management approaches 118 9.2.12 Swine flu 148
7.12.1 Prevention and control of MERS 118 9.2.13 Zika 149
7.13 Summary and future prospective 119 9.2.14 Coronaviruses 149
References 119 9.2.15 COVID-19 150
9.2.16 Lessons 150
8. Advances in vaccination to combat 9.2.17 Future perspectives 155
pandemic outbreaks 123 References 155

Subramanyam Dasari
10. Immunological mechanisms associated
8.1 Introduction 123 with clinical features of Ebola virus
8.2 Human immunodeficiency virus/acquired disease and its control and
immunodeficiency syndrome 124 prevention 159
8.3 Dengue virus 124
8.4 Chikungunya virus 124 Nayaka Boramuthi Thippeswamy
8.5 Zika virus 125 10.1 Introduction 159
8.6 Severe acute respiratory syndrome 125 10.2 Epidemiology 159
8.7 Ebola viral disease 125 10.2.1 Ecology and spreading of Ebola
8.8 Middle East respiratory syndrome virus 160
coronavirus 126 10.3 Virus structure 161
8.9 Human coronavirus 127 10.4 Life cycle 162
8.10 Evolution of vaccine technologies 127 10.5 Molecular mechanisms of Ebola
8.11 Box 1: ideal characteristics of a vaccine 128 pathogenesis 162
8.12 Box 2: strategies for the development of 10.5.1 Dysregulation of the innate
vaccines 128 immune response during Ebola
8.13 Viral vector-based vaccines 129 infection 162
8.14 Adenovirus vectors 129 10.5.2 Subversion of IFN-induced
8.15 Poxviruses as vaccine vector 129 signaling by EBOV 163
8.16 Frontrunners in COVID-19 vaccine race 129 10.5.3 Degradation of IRF3 and IRF7
8.17 Vector-based vaccines come to the fore by VP35-mediated
in the COVID-19 pandemic 130 SUMOylation 165
8.18 Conclusion 134 10.5.4 VP24 inhibits KPNA-mediated
References 134 IFN response signaling 167
10.6 Adaptive immune response during
9. Pandemics of the 21st century: EBOV infection 167
lessons and future perspectives 139 10.6.1 Dysregulation of the adaptive
immune response 169
Hunasanahally Puttaswamygowda Gurushankara
10.7 Vascular permeability and coagulation
9.1 The legacy of an epidemic and pandemic 139 defects 170
9.2 Origin of communicable diseases 139 10.7.1 EBOLA-postinfection
9.2.1 Clio-epidemiology to neo- manifestation 171
epidemiology 140 10.8 Diagnosis 172
xviii Contents

10.9 Vaccines 172 12. Importance of in silico studies on the

10.10 Therapeutics 173 design of novel drugs from medicinal
10.11 Prevention and control of EVD 175 plants against 21st-century pandemics:
10.12 Summary 175 past, present, and future 211
Acknowledgments 176
References 176 Mallikarjuna Nimgampalle, Vasudharani
Devanathan and Ambrish Saxena
12.1 Introduction 211
11. SARS-CoV-2—host cell interactions and 12.2 Pandemic outbreaks of 21st century 212
pathways: understanding its physiology, 12.2.1 Severe acute respiratory
pathology, and targeted drug syndrome 213
therapy 185 12.2.2 Avian influenza 213
12.2.3 The Middle East respiratory
Rhea Conchita Gonsalves, Himavani Pacharla, syndrome 213
Sai Manohar, Siva Kumar Belliraj, Ekta Tripathi, 12.2.4 Swine flu 213
Prashanthi Karyala and Suresh B. Pakala 12.2.5 Ebola virus disease 214
11.1 Introduction 185 12.2.6 Zika fever 214
11.2 COVID-19 disease 186 12.2.7 COVID-19 214
11.2.1 History and epidemiology 186 12.3 Plant-derived compounds as source of
11.2.2 Transmission and course of drugs to treat pandemics 214
infection 187 12.3.1 Plant-derived antiviral compounds
11.2.3 Pathogenesis 187 as therapeutics for coronaviruses
11.2.4 SARS-CoV-2 association with (SARS, MERS, SARS-CoV-2) 214
other comorbidities 188 12.3.2 Plant-derived compounds as
11.3 The molecular biology of SARS-CoV-2 188 antiviral drugs for influenza viruses
11.3.1 Overview of SARS-CoV-2 (swine flu and Avian flu) 216
replication cycle 188 12.3.3 Antiviral activity of plant-derived
11.4 Identifying SARS-CoV-2 host cell interface, compounds against Ebola and Zika
host dependency factors and cytokine viruses 216
storm: high-throughput and 12.4 Computational approaches in identifying
low-throughput approaches 194 novel drugs using plant-derived
11.4.1 Requirement of host factors for compounds 216
SARS-CoV-2 life cycle 194 12.4.1 In silico screening of plant-derived
11.4.2 Analysis of the viral transcriptome antiviral compounds against
of SARS-CoV-2 194 pandemics of the 21st century 218
11.4.3 Identification of virus
host 12.4.2 SARS and MERS 218
proteome and interactome using 12.4.3 Swine flu and Avian flu 219
high-throughput technologies 196 12.4.4 Ebola virus disease 219
11.4.4 Identification of virus
host 12.4.5 Zika fever 219
dependency factors using CRISPR/ 12.4.6 COVID-19 220
Cas9 technology 196 12.5 Future prospective and limitations of
11.4.5 Identification of host factors in in silico studies 220
COVID-19 patient samples 197 12.6 Conclusion 221
11.4.6 Cytokine storm and COVID-19 198 Acknowledgment 221
11.4.7 Chemical compounds virtual References 221
screening 198
11.5 Host cell factors and viral proteins as 13. Recent developments in the diagnosis
target for antiviral agents 200 of COVID-19 with micro- and
11.5.1 Viral life cycle as drug targets 200 nanosystems 225
11.5.2 Virus-based targets 201
Manpreet Singh, Kamal Kishore and
11.5.3 Host-based druggable targets 202
Seshadri Reddy Ankireddy
11.6 Concluding remarks 204
References 204 13.1 Introduction 225
 Contents xix

13.2 SARS CoV-19 structure 226 15.5.6 Others probable potential

13.3 Micro- and nanosystems for the retasking agents for the treatment
diagnosis of COVID-19 226 of COVID-19 249
13.4 Limitations and future prospectus 230 15.6 Limitations to drug repurposing
13.5 Conclusion 231 approach 250
Acknowledgments 231 15.7 COVID-19 vaccination programs and
Conflict of interest 231 repurposing of existing vaccines 250
References 232 15.8 Evolving strains of coronavirus genome
and ineffectiveness of the vaccines 251
14. Recent trends in the development of 15.9 Conclusion 252
vaccine technologies to combat Acknowledgments 252
pandemic outbreaks and challenges 235 Conflict of interest 252
References 253
Gayathri Chellasamy, Rose Mary Kiriyanthan,
Saravanan Govindaraju, A. Radha and Kyusik Yun
16. Genetics of coronaviruses 257
14.1 Introduction 235
14.2 Pandemic outbreaks and challenges in Shanthala Mallikarjunaiah, Basavaraja Metikurki
vaccine development 235 and Hunasanahally Puttaswamygowda
14.3 Vaccine technologies and its types 237 Gurushankara
14.3.1 Viral vector vaccines 238
16.1 History of coronaviruses 257
14.3.2 Viral-like particles 239
16.2 Taxonomy of coronaviruses 258
14.3.3 Nucleic acid vaccine 240
16.3 Naming of coronaviruses 258
14.4 Challenges in the success of vaccination
16.4 Genome of coronaviruses 260
toward pandemic outbreaks 241
16.5 Coronavirus diversity 261
14.5 Conclusion and future perspectives 241
16.6 Genetics of coronavirus infection 262
Acknowledgment 242
16.7 Potential genes for pathogenesis of
References 242
COVID-19 262
16.7.1 Chromosome 3P21.31
15. Could repurposing existing vaccines gene locus 263
and antibiotics help to control the 16.8 ABO blood group genes 263
COVID-19 pandemic? 245 16.8.1 Human leukocyte antigen
Kajal Rathod, Niyati Dhingra, Soumya genes 263
Dakshinamurthy and Buddolla Viswanath 16.8.2 X-chromosomal Toll-like
receptor 7 gene 264
15.1 Introduction 245 16.8.3 Apolipoprotein E 264
15.1.1 An upsurge of SARS CoV-2 246 16.8.4 Interferon-induced transmembrane
15.2 Genome of coronavirus 246 protein 3-encoding gene 264
15.3 Drug repurposing 247 16.8.5 Transmembrane protein 189-
15.4 Therapeutic targets 247 ubiquitin-conjugating enzyme E2
15.5 Therapeutic options for COVID-19 variant 1 264
management 247 16.8.6 ACE2 and TMPRSS2 receptor
15.5.1 Repurposed drugs that act through genes 265
virus-related targets such as RNA 16.8.7 Interferon-α and β receptor
genome 248 subunit 2 265
15.5.2 Repurposed drugs acting through 16.8.8 20 -50 oligoadenylate synthetase
polypeptide packing 248 family 265
15.5.3 Repurposed drugs acting through 16.8.9 DPP9 and FOXP4 genes 266
host target such as antiviral 16.9 New SARS-CoV-2 variant 266
immunity 248 16.10 Impact of genetics on COVID-19
15.5.4 Repurposed drugs targeting the treatment 266
virus uptake pathways 249 16.11 Future perspectives 267
15.5.5 Drugs acting on host Acknowledgment 267
pro-inflammatory cytokines 249 References 267
xx Contents

17. Spike in electronic sports during the 18.3 Preparedness and leveraging digital
coronavirus disease pandemic 273 health in COVID-19 management:
a case of Singapore 284
Neha Singh 18.4 What was different in Singapore’s
17.1 COVID-19 pandemic, lockdown, and response and what lessons can other
e-sports 273 countries learn? 284
17.2 E-sports: an introduction and research in 18.5 Conclusion 285
different academic disciplines 273 References 286
17.3 Effect of COVID-19 on economic
growth of e-sports or gaming industry 274 19. COVID-19 and its effects on
17.4 Advances in e-sports during the neurological expressions 287
COVID-19 pandemic 276
Roopkumar Sangubotla and Jongsung Kim
17.5 E-sports: tool for social connectedness and
psychological healing in the COVID-19 19.1 Introduction 287
pandemic 276 19.1.1 Significance of “S” proteins in
17.6 E-sports: role in health and well-being COVID-19 288
during the pandemic 277 19.2 Routes of entry for COVID-19 into the
References 278 brain 288
19.3 Neuroinflammation and immune
18. How digital health and pandemic responses in COVID-19 289
preparedness proved a game changer? 19.4 Limitations for clinical performances
A case of Singapore in COVID-19 during COVID-19 289
management 281 19.5 Conclusion and future prospects 290
Acknowledgments 290
Sibasis Hense, Pratik Mukherjee and Conflict of interest 290
Hunasanahally Puttaswamygowda Gurushankara References 290
18.1 The context 281
18.2 Digital health and COVID-19 pandemic 283 Index 293
List of contributors

Bosetty Anjana Department of Biotechnology, Sri Tirupati, India; Vice-chancellor of Cluster University,
Padmavathi Mahila Visvavidyalayam (Women’s Kurnool, Andhra Pradesh, India
University), Tirupati, India; Dr. Buddolla’s Institute of Saravanan Govindaraju Department of Bionanotechnology,
Life Sciences, A Unit of Dr. Buddolla’s Educational Gachon University, Seongnam, South Korea
Society, Tirupati, India
Hunasanahally Puttaswamygowda Gurushankara
Seshadri Reddy Ankireddy Department of Chemical Department of Zoology, School of Biological
and Biological Sciences, Dr. Buddolla’s Institute of Sciences, Central University of Kerala, Tejaswini
Life Sciences, A Unit of Dr. Buddolla’s Educational Hills, Kasaragod, India
Society, Tirupati, India
Sibasis Hense Department of Public Health and
Hemanth Naick Banavath Department of Sports Bio- Community Medicine, Central University of Kerala,
Sciences, Central University of Rajasthan, Ajmer, India Kasaragod, India
Praveen Belagal DST PURSE Centre, Sri Venkateswara Prashanthi Karyala Department of Biotechnology,
University, Tirupati, India Faculty of Life and Health Science, Ramaiah
Siva Kumar Belliraj Grey Scientific Labs, University of Applied Sciences, Bengaluru, India
Visakhapatnam, India Jongsung Kim Department of Chemical and Biological
D. Katterine Bonilla-Aldana Semillero de Investigación Engineering, Gachon University, Seongnam-Si, South
en Zoonosis (SIZOO), Grupo de Investigación Korea
GISCA, Fundacion Universitaria Autonoma de las Rose Mary Kiriyanthan PG and Research Department
Americas, Pereira, Risaralda, Colombia of Botany, Bharathi Women’s College, Tamil Nadu,
Gayathri Chellasamy Department of Bionanotechnology, India
Gachon University, Seongnam, South Korea Kamal Kishore Department of Chemistry, Akal
Soumya Dakshinamurthy Department of Microbiology, College of Basic Sciences, Eternal University,
Sri Venkateswara Institute of Medical Sciences Sirmour, India
(SVIMS), Tirupati, India Naga Charan Konakalla Department of Plant Protection
Subramanyam Dasari Indiana University School of Biology, Swedish University of Agricultural Sciences,
Medicine, Bloomington, IN, United States Alnarp, Sweden

Vasudharani Devanathan Department of Biology, Kandati Kusuma Department of Biotechnology, Sri

Indian Institute of Science Education and Research Padmavati Mahila Visvavidyalayam, Tirupati, India;
Tirupati (IISER T), Tirupati, India Dr. Buddolla’s Institute of Life Sciences, A Unit of
Dr. Buddolla’s Educational Society, Tirupati, India
Niyati Dhingra Department of Biotechnology, Sri
Padmavati Mahila Visvavidyalayam, Tirupati, India; Shanthala Mallikarjunaiah Center for Applied
Dr. Buddolla’s Institute of Life Sciences, A Unit of Genetics, Department of Zoology, Bangalore
Dr. Buddolla’s Educational Society, Tirupati, India University, Bengaluru, India
Rhea Conchita Gonsalves Department of Biochemistry, Sai Manohar Department of Chemistry, Sri Sathya Sai
Indian Academy Degree College—Autonomous, Institute of Higher Learning, Anantapur, India
Bengaluru, India Hema Masarapu Department of Virology, Sri
Divi Venkata Ramana Sai Gopal DST PURSE Centre, Venkateswara University, Tirupati, India
Sri Venkateswara University, Tirupati, India; Basavaraja Metikurki Nitte College of Pharmaceutical
Department of Virology, Sri Venkateswara University, Sciences, Bengaluru, India

xxi
xxii List of contributors

Pratik Mukherjee Woodlands Health Campus, Singapore Roopkumar Sangubotla Department of Chemical and
Mallikarjuna Nimgampalle Department of Biology, Biological Engineering, Gachon University,
Indian Institute of Science Education and Research Seongnam-Si, South Korea
Tirupati (IISER T), Tirupati, India Ambrish Saxena Center for Sponsored Research and
Consultancy, Indian Institute of Technology (IIT)
Himavani Pacharla Department of General Medicine,
Tirupati, Tirupati, India
Apollo Hospital, Hyderabad, India
Manpreet Singh Department of Chemistry, Akal
Suresh B. Pakala Biology Division, Indian Institute of
College of Basic Sciences, Eternal University,
Science Education and Research (IISER) Tirupati,
Sirmour, India
Tirupati, India
Neha Singh Department of Sports Biosciences, Central
Pandeeti Emmanuel Vijay Paul Albus Eco Projects,
University of Rajasthan, Ajmer, India
LLP, BioNEST, University of Hyderabad, Hyderabad,
India Kalanghad P. Srinivas Department of Microbiology,
NYU School of Medicine, Alexandria Center for Life
A. Radha PG and Research Department of Botany, Sciences, New York, NY, United States
Bharathi Women’s College, Tamil Nadu, India
Nayaka Boramuthi Thippeswamy Department of
Kajal Rathod Department of Biotechnology, Sri Postgraduate Studies and Research in Microbiology,
Padmavati Mahila Visvavidyalayam, Tirupati, India; Kuvempu University, Shivamogga, India
Dr. Buddolla’s Institute of Life Sciences, A Unit
of Dr. Buddolla’s Educational Society, Tirupati, Ekta Tripathi Department of Biotechnology, Faculty of
India Life and Health Science, Ramaiah University of
Applied Sciences, Bengaluru, India
Alfonso J. Rodriguez-Morales Grupo de Investigación
Biomedicina, Faculty of Medicine, Fundacion Buddolla Viswanath Dr. Buddolla’s Institute of Life
Universitaria Autonoma de las Americas, Pereira, Sciences, A Unit of Dr. Buddolla’s Educational
Risaralda, Colombia; Universidad Cientı́fica del Sur, Society, Tirupati, India
Lima, Peru; Universidad Privada Franz Tamayo, Kyusik Yun Department of Bionanotechnology, Gachon
Cochabamba, Bolivia University, Seongnam, South Korea
Foreword

Throughout history, epidemics and pandemics have been part of human life. Although mostly with negative impacts,
pathogens emergence and reemergence are also related to multiple factors, including anthropogenic ones. Today, even
more than ever, in a globalized world, highly connected by air traffic but at the same time by telecommunications, the
consequences of such emerging conditions, as has been especially the case of the coronavirus disease 2019 (COVID-19),
that began with a small outbreak in a localized place of the earth, Wuhan, China in late 2019, may potentially affect in
few weeks and months, the whole planet, as with COVID-19 occurred. The book Pandemic Outbreaks in the 21st
Century: Epidemiology, Pathogenesis, Prevention, and Treatment, edited by Buddolla Viswanath, is a timely piece that
amid the COVID-19 pandemic greatly summarize in 19 chapters highly relevant topics covering multiple aspects of
COVID-19, MERS-CoV, and other coronaviruses, Avian and Swine Influenza, Ebola, Zika, among others.
As usual, the efforts behind a book like this, with a wide number of international contributors, reflect a global view
on a problem that represents a serious health issue and a matter of global interest for society. Focused on the recent pan-
demics, those of the “21 century,” this book is a relevant work, with a clear invitation to be prepared for coming epi-
demics and pandemics, to invest more in pandemic preparedness and research, and to in general increase the knowledge
on these emerging global situations.

Alfonso J. Rodriguez-Morales1,2,3
1
Grupo de Investigación Biomedicina, Faculty of Medicine, Fundacion Universitaria Autonoma
de las Americas, Pereira, Colombia, 2Universidad Cientı´fica del Sur, Lima, Peru,
3
Universidad Privada Franz Tamayo, Cochabamba, Bolivia

xxiii
This page intentionally left blank
Preface

Pandemic Outbreaks in the 21st Century: Epidemiology, Pathogenesis, Prevention, and Treatment is a book published
by Academic Press, Elsevier to honor one of the renowned professors of virology, Prof. Pothur Sreenivasulu, for his
dedication toward teaching and research. The 21st century has seen a number of viral pandemics that have impacted the
health of millions of people around the world. Reviewing the evidence on epidemiology, pathogenesis, prevention, and
treatment is critical for understanding factors that may lead to virus transmission and addressing long-term health conse-
quences for survivors. This book aims to summarize the many pandemics that have plagued the world during the last
two decades.
Divided into 19 chapters, the book emphasizes on understanding viral infectious disease epidemiology, characteriza-
tion of the outbreak of viruses, importance of early detection, prevention measures, and modern vaccines.
Chapter 1, Lessons Learned From the First Pandemic of the 21st Century, Global Experience, Recommendations,
and Future Directions, by Prof. Divi Venkata Ramana Sai Gopal and group, concentrated primarily on SARS-CoV in
this chapter, with references to other respiratory infections such as MERS-CoV and SARS-CoV-2. The authors also
summarized the SARS-CoV knowledge gained so far, with a focus on future directions.
Chapter 2, Epidemiology of COVID-19 in Latin America, by Prof. Alfonso J. Rodriguez-Morales and
Dr. D. Katterine Bonilla-Aldana, focuses on the main epidemiological features of the COVID-19 during the first year of
the pandemic in the region of Latin America.
Chapter 3, Biology, Prevention, and Treatment of SARS-CoV-2 (COVID-19), by Dr. Kalanghad P. Srinivas, reveals
the basic biological features of the COVID-19 including how it replicates and causes the disease. In addition, the chap-
ter looks at how the virus spreads and what interventions can be used to prevent it. The most promising drugs from clin-
ical trials and preclinical research to date were also discussed by the author.
Chapter 4, Avian Influenza A Virus Infections in Humans: Current Knowledge to Enhance Host Innate Immunity to
Control Avian Influenza, by Bosetty Anjana, Buddolla Viswanath, and Soumya Dakshinamurthy, gives an overview of
Avian influenza A virus infections in humans with an emphasis on host innate immunity to control Avian influenza.
Chapter 5, Swine-Origin Influenza A (H1N1) Virus: Current Status, Threats, and Challenges, by Dr. Praveen
Belagal et al., presents a detailed overview of the role of the swine
human interface in causing new pandemics, as well
as threats and challenges in fostering public preparedness.
Chapter 6, Molecular Mechanisms of Zika Fever in Inducing Birth Defects: An Update, by Prof. Hema Masarapu
and Naga Charan Konakalla, summarizes the recent progress that has been made to define the ZIKV-mediated cellular
apoptotic and immune response evasion pathways and the plausible molecular mechanisms underlying ZIKV-associated
neurological disorders and birth defects in the developing fetuses and new-born babies, respectively, using several inde-
pendent in vitro and in vivo animal studies.
Chapter 7, Middle East Respiratory Syndrome: Outbreak Response Priorities, Treatment Strategies, and Clinical
Management Approaches, by Ms. Kusuma Kandati and team, covers the major MERS-CoV outbreaks, mode of trans-
mission, human infection route, virus structure, and life cycle with a focus on molecular pathogenesis, diagnostics, vac-
cines, treatment, and control strategies.
Chapter 8, Advances in Vaccination to Combat Pandemic Outbreaks, by Dr. Subramanyam Dasari, discusses some
of the world’s most recent pandemics as well as emerging vaccine production techniques as potential solutions to these
public health challenges.
Chapter 9, Pandemics of the 21st Century: Lessons and Future Perspectives, by Dr. Hunasanahally
Puttaswamygowda Gurushankara, gives an overview of the challenges posed by 21-century pandemics with an empha-
sis on prevention and response.
Chapter 10, Immunological Mechanisms Associated With Clinical Features of Ebola Virus Disease and Its Control
and Prevention, by Prof. Nayaka Boramuthi Thippeswamy, attempts to disseminate information about the molecular

xxv
xxvi Preface

basis of EVD pathogenesis, with an emphasis on dysregulation of innate and adaptive immune response signaling path-
ways, clinical manifestations, vaccine production, and long-term health consequences for EVD survivors.
Chapter 11, SARS-CoV2—Host Cell Interactions and Pathways: Understanding Its Physiology, Pathology, and
Targeted Drug Therapy, by Dr. Suresh B. Pakala et al., provides a detailed overview of the current status of SARS-
CoV-2 research, encompassing every stage of the viral replication cycle with a particular emphasis on virus
host cell
interaction. In addition, the author discusses chemical inhibitors that target cellular factors and processes relevant to the
SARS-CoV-2 replication cycle, as well as their potential for use in the production of next-generation antivirals.
Chapter 12, Importance of In Silico Studies on the Design of Novel Drugs From Medicinal Plants Against 21st-
Century Pandemics: Past, Present, and Future, by Vasudharani Devanathan et al., addressed various pandemic outbreaks
and trials to use plant-derived compounds as a source of drugs to treat pandemics in detail.
Chapter 13, Recent Developments in the Diagnosis of COVID-19 With Micro- and Nanosystems, by Dr. Seshadri
Reddy Ankireddy, highlights ongoing research efforts on developing integrated micro and nanosystems for nucleic
acid
based virus detection, as well as promising technologies that could improve COVID-19 and other viral infectious
disease diagnoses.
Chapter 14, Recent Trends in the Development of Vaccine Technologies to Combat Pandemic Outbreaks and
Challenges, by Gayathri Chellasamy et al., deals with a thorough overview of pandemic outbreaks, various vaccine
methodologies, and their progress platforms.
Chapter 15, Could Repurposing Existing Vaccines and Antibiotics Help to Control the COVID-19 Pandemic?, by
Buddolla Viswanath et al., compiles a useful collection of data on COVID-19 drugs and vaccines, including their repur-
posing properties and the various stages of clinical trials they are undergoing around the world.
Chapter 16, Genetics of Coronaviruses, by Prof. Shanthala Mallikarjunaiah et al., emphasizes the history, taxonomy,
naming, genetic diversity of CoVs, the genome of CoVs, and potential genes for diseases’ pathogenesis.
Chapter 17, Spike in E-sports During the Coronavirus Disease Pandemic, by Dr. Neha Singh, evaluates the effects
of the COVID-19 pandemic on digital games and e-sports using a variety of sources and evidence.
Chapter 18, How Digital Health and Pandemic Preparedness Proved a Game Changer? A Case of Singapore in
COVID-19 Management, by Sibasis Hense et al., answers the question of how Singapore, a small country in Southeast
Asia, displayed outstanding pandemic preparedness skills and adopted digital health technologies to achieve remarkable
progress in combating the virus.
Chapter 19, COVID-19 and Its Effects on Neurological Expressions, by Roopkumar Sangubotla and Jongsung Kim,
reviews the neurological effects that are caused by COVID-19. Moreover, this chapter deals with the possible limita-
tions and future perspectives for therapeutic strategies and treatment options against COVID-19.

Buddolla Viswanath
Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr. Buddolla’s Educational Society, Tirupati, India
Acknowledgments

All chapters in this volume are clearly illustrated and contain accessible information for a wide audience. The editor
wishes to extend heartfelt thanks to all the contributed authors for their time, support, and their excellent contributions.
Also grateful to Mrs. Linda Versteeg, Mr. Timothy Bennett, Mrs. Maria Bernadette Vidhya Bernard, and the entire
team of Elsevier/Academic Press for all their incentive, patience, and support in all cases that needed specific consider-
ation and in producing this book. The editor’s experiences with Academic Press/Elsevier with this project have been
excellent. The editor hopes that this volume will help scientists, faculties, and students from around the world.
Suggestions for improving future volumes are welcome.

xxvii
This page intentionally left blank
Chapter 1

Lessons learned from the first pandemic

of the 21st century, global experience,
recommendations, and future directions
Kandati Kusuma1,2, Praveen Belagal3, Buddolla Viswanath1 and Divi Venkata Ramana Sai Gopal3,4,5
1
Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr. Buddolla’s Educational Society, Tirupati, India, 2Department of Biotechnology, Sri
Padmavati Mahila Visvavidyalayam, Tirupati, India, 3DST PURSE Centre, Sri Venkateswara University, Tirupati, India, 4Department of Virology, Sri
Venkateswara University, Tirupati, India, 5Vice-chancellor of Cluster University, Kurnool, Andhra Pradesh, India

1.1 Introduction
Many infectious diseases have swept the world, taking the lives of millions of people. Viral outbreaks of varying fre-
quencies and severities have caused panic and havoc across the globe throughout history [1]. The 21st century wit-
nessed a few pathogenic and contagious virus outbreaks of zoonotic origin including severe acute respiratory syndrome
coronavirus (SARS-CoV-1&2), Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and Nipah
virus [1]. SARS is an airborne virus and can spread through small droplets of saliva in a similar way to the cold and
influenza. It was the first severe and readily transmissible new disease to emerge in the 21st century and showed a sus-
tained human
human transmission along the routes of international air travel [2]. The 2003 outbreak of SARS shocked
the world as it spread swiftly from continent to continent, resulting in .8000 infections, a total of 916 deaths globally
with B10% mortality and affecting local and regional economies [3]. In November 2002 the first case of SARS
occurred in Foshan, China, and in June 2012, the first case of MERS died at a hospital in Jeddah, Saudi Arabia [4]. In
November 2002 unusual cases of atypical pneumonia of unknown cause occurred in Foshan City, Guangdong province,
in China, where many health care workers were infected [5]. Three laboratories—one each in Hong Kong, Germany,
and the Centres for Disease Control and Prevention (CDCs) in Atlanta, Georgia, United States—nearly simultaneously
isolated an apparently new coronavirus as the causative pathogen of SARS [3]. The infection was brought to Hong
Kong on February 21, 2003, by a physician who had looked after similar cases of atypical pneumonia in the mainland
China, leading to outbreaks in Hong Kong. On March 15, 2003, WHO officially declared an epidemic and labeled it as
a SARS (later referred as SARS-CoV) [6
8]. The SARS-CoV epidemic quickly spread to 29 countries, but the global
public health, medical, and scientific communities were not adequately prepared for the emergency. Chains of human-
to-human transmission occurred in Toronto, Canada, Hong Kong Special Administrative Region of China, Chinese
Taipei, Singapore, and Hanoi, Vietnam. The duration of the SARS epidemic was short and WHO declared the end of
the SARS epidemic in July 2003 with a total of 8096 SARS cases and 774 deaths reported across 29 countries and
regions [4]. The scientific effort demonstrated unusual international cooperation and was in turn facilitated by electronic
communication. Media coverage provided accurate worldwide pictures to augment scientific data. As of March 1, 2004,
there were 1695 citations related to SARS seen in the Medline [9]. One reason why SARS-CoV-2 spread is evidently
much wider compared to SARS is the rapid urbanization and world trade network resulting in increased international
travel during the last two decades. Hence, the control measures applied at the time of SARS-CoV-2 are no longer ade-
quate in the current days, and more vigorous actions are required to control SARS-CoV-2 [10]. Besides, the duration in
the infectious period between patients infected with SARS and those infected with SARS-CoV-2 is not the same. While
in the former case, viral shedding peaks only when the patient’s illness is advanced and respiratory symptoms appear
[10], for SARS-CoV-2, the transmission can occur in the early phase of the illness, when the patients are completely
asymptomatic [11]. Hence, isolation after the onset of symptoms might be ineffective in preventing virus transmission
Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00010-0
© 2021 Elsevier Inc. All rights reserved. 1
2 Pandemic Outbreaks in the 21st Century

and this also makes temperature screening less effective [12]. In comparison to other betacoronaviridae members
SARS-CoV-2 has been shown to have higher transmissibility and a wider population distribution [13]. Despite being
highly infectious and having higher transmissibility (designated as Reproduction number; R0), the severity of SARS-
CoV-2 is much lesser compared to SARS. Though SARS caused major disruptions to international air travel, and
impact on the health services and merchandise in the affected countries by 2004, SARS cases were hardly reported any-
where in the world [4]. Hence, any attempts of developing vaccines were stopped. In September 2012 Saudi Arabia
reported the first case of the MERS, which was caused by MERS-CoV, another type of betacoronavirus. MERS-CoV
spread to 27 countries and caused 2519 infections and 866 deaths by January 2020, with a Coronary Flow Rate (CFR)
of 34.4% [14]. Both SARS-CoV and SARS-CoV-2 are closely related and originated in bats, which most likely are
serving as a reservoir host for these two viruses [15
18]. SARS-CoV causes atypical pneumonia that spreads rapidly
throughout or parts of Asia, North America, and Europe (Sino Biological). SARS-CoV was produced by recombination
within bats and then transmitted to palm civets or other mammals via fecal
oral transmission. When virus-infected
civets were transported to the Guangdong market, the infection spread among the civets in the market where it has
probably acquired further new mutations before transmission to humans [19]. The concern is magnified by rapid popu-
lation growth in areas with weak health systems, urbanization, globalization, climate change, civil conflict, and the
changing nature of pathogen transmission between human and animal populations [20]. Influenza, smallpox, measles,
and yellow fever reverberated for centuries, causing a huge burden on economies. As successive epidemics have swept
the world, the scientific community has quickly learned about the emergence and transmission of communicable dis-
eases. Epidemics usually occur when health systems are unprepared. During an unexpected epidemic, health authorities
engage in damage control, fear drives action, and the desire to understand the threat is greatest [21]. Hong Kong was
among the first cities affected by SARS, and its health care community suffered greatly from the epidemic. Some les-
sons from their experiences included recognition of the value of real-time information in a rapidly progressing epidemic
with a large number of cases and the need for frequent patient updates, challenges of national efforts to maintain entry
and exit health screening among international travelers, and implementation of home quarantine as an effective tool to
control SARS transmission [3]. A novel beta CoV (SARS-CoV) of lineage B was confirmed as the cause of the atypical
pneumonia cases on March 22, 2003 [4,6]. Outbreaks of various zoonotic viruses occur as a result of the influence of
several factors including human-to-human contact and animal interaction with severe environmental changes [1]. At the
end of 2019, an outbreak of severe respiratory illness occurred in Wuhan City, China. SARS-CoV-2 is responsible for
the outbreak of severe respiratory illness (COVID-19) and is still spreading rapidly throughout the world [22]. On
January 9, 2020, the Chinese CDC declared the identification of a novel Coronavirus [23]. As of January 29, 2021,
around 100,455,529 confirmed cases of COVID-19 worldwide including 2,166,440 deaths, reported to WHO (Fig. 1.1).
As of March 31, 2020, the SARS-CoV-2 has infected over a million and has caused more than 50,000 deaths (https://
www.who.int/emergencies/diseases/novel-coronavirus-2019). Infectious disease threats, the fear, and panic in the public
that may accompany various economic and social risks. With respect to outbreaks and epidemics (whether naturally
occurring or human-initiated), there are obvious costs to the health care system in terms of medical treatment and out-
break control [20]. The virus that causes COVID-19, known as SARS-CoV-2, was first identified in Wuhan, China, on
31 December 2019 with the presentation of symptoms of atypical pneumonia. This case was further confirmed to be
caused by the novel coronavirus, SARS-CoV-2 [24]. There are also other types of human coronaviruses. Coronaviruses
have been found in many different animal species including birds and mammals. SARS-CoV-2 is thought to have
reached from animals to humans through close contact, butchering, or eating undercooked meat in parts of Southern

14,0000 FIGURE 1.1 Covid-19 outbreaks across the world.

Global
120,000

100,000
United States of
80,000 America
60,000
India
40,000

20,000
Brazil
0
Cases - cumulave total per 1
million populaon
 Lessons learned from the first pandemic of the 21st century Chapter | 1 3

China. The most potential risk for the spread of COVID-19 worldwide is related to travel that is causing the regional
and global spread of the disease (Bai et al., 2020). According to current observed epidemiologic characteristics, every-
one is susceptible to Covid-19 and the median age is about 50 years [25
27].

1.2 First pandemic of the 21st century, severe acute respiratory syndrome
SARS is a SARS-associated coronavirus-caused viral respiratory disease. It was first observed at the end of February
2003 during an epidemic that started in China and spread to other nations. It is a member of the Coronaviridae family,
which also encompasses many of the viruses that cause the common cold [9].

1.2.1 Structure of SARS-CoV

Coronavirus genomes are the largest among RNA viruses [28]. This family has been classified into at least three pri-
mary genera (alpha, beta, and gamma). Within this family, seven viruses are currently known to infect humans, namely,
NL63 and 229E from the alpha genus and OC43, HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2 from the beta
genus. SARS-CoV has similar structural proteins as three previously known groups of coronaviruses: spike glycoprotein
(S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N) (Fig. 1.2). Coronavirus N protein is
required for coronavirus RNA synthesis and has RNA chaperone activity that may be involved in template switch.
SARS-CoV spike glycoprotein is 1255 amino acids long, with low (20
27%) amino acid similarity among other coro-
naviruses. Its carboxyl terminus (C-terminus) comprises the transmembrane region and the cytoplasmic tail (https://
www.sinobiological.com/research/virus/sars-coronavirus-overview). In addition to the original genes, the SARS-CoV
genome encodes another eight putative accessory proteins, known as ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b, which vary
in length from 39 to 274 amino acids [30].

1.2.2 SARS-COV: mechanism of action

Coronaviruses appear as crown-like structures under electron microscope hence named coronavirus. They have positive-
stranded RNA as their genomic material and have an outer envelope [24]. Coronaviruses belong to a family that comes
under the order Nidovirales. Nidovirales order includes the viruses that use replication [23]. The life cycle of the virus
with the host consists of the following five steps (Fig. 1.3): attachment, penetration, biosynthesis, maturation, and release.
Once viruses bind to host receptors (attachment), they enter host cells through endocytosis or membrane fusion (penetra-
tion). Once viral contents are released inside the host cells, viral RNA enters the nucleus for replication. Viral mRNA is
used to make viral proteins (biosynthesis). Then, new viral particles are made (maturation) and released. Coronaviruses
consist of four structural proteins; spike (S), membrane (M), envelop (E), and nucleocapsid (N) [32]. Spike is composed
of a transmembrane trimetric glycoprotein protruding from the viral surface, which determines the diversity of corona-
viruses and host tropism. Spike comprises two functional subunits; S1 subunit is responsible for binding to the host cell
receptor and S2 subunit is for the fusion of the viral and cellular membranes. Angiotensin-converting enzyme 2 (ACE2)

FIGURE 1.2 Representation of SARS-CoV structure [29]. SARS-

CoV, Severe acute respiratory syndrome coronavirus.
4 Pandemic Outbreaks in the 21st Century

FIGURE 1.3 Mechanism of viral entry and replication and RNA packing in the human cell or invasion of SARS-CoV into the host cell [31]. SARS-
CoV, Severe acute respiratory syndrome coronavirus.

was identified as a functional receptor for SARS-CoV [32]. The extracellular domain of the SARS-CoV spike glycopro-
tein consists of two heptad repeat regions that are known as heptad repeat region 1 and heptad repeat region 2. SARS-
CoV spike glycoprotein has two functional domains: S1 and S2. S1 is responsible for the binding with its receptor ACE2
on host cells and defines the host range of the virus. S2 is the transmembrane subunit that facilitates viral and cellular
membrane fusion. Membrane fusion occurs when there is a conformational change in the HRs to form a fusion core. The
HRs of the protein fold into a coiled-coil structure—called the fusogenic state—causing the HR domains of the S protein
to fold into a hairpin-like formation. This hairpin structure results in the cellular and viral membranes being pulled
together and ultimately fusing. SARS-CoV infection can cause bronchial epithelial cell peeling, cilia damage, the forma-
tion of multinucleated giant cells, squamous cell aplasia, alveolar interstitial fiber cell hyperplasia, and fibrotic lung dis-
ease [33]. The SARS-CoV genome encodes 28 proteins in three distinct classes, many of them with unknown functions
and sharing low similarity to other proteins. The structures of 16 SARS-CoV proteins or functional domains have been
determined to date [34]. This virus was rapidly identified and characterized by a combination of classical virological meth-
ods and cutting-edge molecular biology. Electron microscopic examination of swabs and sputum specimens from affected
patients revealed the presence of viral particles. Fortuitously, this newly identified agent replicated in Vero cells, in con-
trast to other human coronaviruses [9]. Moreover, cataloging the genome from human cases assisted in the search for the
origin of this disease, when viruses related to the SARS-CoV were identified in animals [Himalayan palm civets (Paguma
larvata) and raccoon dogs (Nyctereutes procyonoides)] in a live animal market in Shenzhen, China [35]. Viral genomes
from nasal swabs from palm civets were 99.8% homologous to the human SARS-CoV and represented a distinct phyloge-
netic group from the human isolates [4].
The virus can also spread through indirect contact transmission. Virus-containing droplets contaminate hands, people
then contact the mucous membranes of the mouth, nose, and eyes, causing infection [36]. The genome of CoVs
 Lessons learned from the first pandemic of the 21st century Chapter | 1 5

(27
32 kb) is a single-stranded positive-sense RNA that is larger than any other RNA viruses [23]. The SARS-CoV-2
genome sequence shares B80% sequence identity with SARS-CoV [18,37]. The transmission of SARS-CoV-2 is not
limited to the respiratory tract [36]. Scientists aligned the full-length genome sequence of SARS-CoV-2 and other avail-
able genomes of betacoronaviruses. Results indicate the closest relationship of SARS-CoV-2 with the bat SARS-like
coronavirus strain BatCov RaTG13, with an identity of 96%. These studies suggest that SARS-CoV-2 could be of bat
origin, and SARS-CoV-2 might be naturally evolved from bat coronavirus RaTG13 [38]. Compared to SARS-CoV,
many SARS-CoV-2 patients develop low levels of neutralizing antibodies and suffer prolonged illness [25]. Once the
genome is released into the host cytosol, ORF1a and ORF1b are translated into viral replicase proteins, which are
cleaved into individual Nsps (via host and viral proteases: PLpro); these form the RNA-dependent RNA polymerase
(Nsp12 derived from ORF1b) [39]. Here, the replicase components rearrange the endoplasmic reticulum into double-
membrane vesicles that facilitate viral replication of genomic and subgenomic RNAs; the latter are translated into
accessory and viral structural proteins that facilitate virus particle formation [40]. Epidemiological studies have shown
that mortalities are higher in the elder population [18] and the incidence is much lower in children [41].

1.2.3 Global experiences

Members of a GOARN mission to China in late March warned that country’s health authorities that if SARS was not
brought under control in China, there would be no chance of controlling the global threat of SARS. Within days, the
GOARN team announced that Chinese authorities had agreed to join the GOARN collaborative effort to contain the
outbreak and prevent further international spread [42]. Although Chinese officials acknowledged that SARS had
emerged in their country, they continued to downplay the extent and severity of the outbreak. This led the WHO team
in Beijing to take the unusual measure of publicly expressing strong concern over inadequate reporting of SARS cases
on April 16 [43]. The emergence of SARS in 2003 had a particularly devastating impact, both on human health and on
the global economy, and demonstrated how rapidly viruses can spread around the world [34]. The outbreak also pro-
vided a stark warning of how ill-prepared we were at the time against a newly emerging infectious disease such as
SARS [44]. The paucity of available scientific data for coronaviruses was a considerable disadvantage, but scientists
mounted a rapid international response to the threat of SARS [45]. For instance, the SARS coronavirus was quickly
identified and its genome was sequenced within weeks [6,46]. Now COVID-19 pandemic is a major health crisis and
the world is experiencing an unprecedented challenge due to the coronavirus disease (COVID-19) pandemic [47].
However, the emerging global health system has made significant contributions to the protection and promotion of
human health. According to UNESCO’s monitoring, more than 160 countries implemented nationwide closures, which
impacted over 87% of the world’s student population [48].
However, successful public health prevention strategies eventually brought the SARS disease under control. Since
then, researchers from all over the world have worked hard to learn more about the virus origins, inner workings, and
interactions with host cells [34]. At the end of the SARS outbreak, the cases of over 1700 health care workers who had
been affected were reported to the WHO, from China (19% of total cases), Canada (43%), France (29%), and Hong
Kong (22%). During this epidemic, insufficient or inappropriate infection control measures, such as inconsistent use of
personal protective equipment, reuse of N95 masks, and lack of adequate infection control, were related to the high risk
of infection among health care workers [49]. The relative effectiveness of various strategies applied to SARS contain-
ment—the use of standardized case definitions and laboratory testing to identify the infected, the isolation of ill persons,
and the quarantine of contacts—remains to be determined. Based on current knowledge, asymptomatic infected people
only transmit SARS at a low rate. Thus in 2004 after the epidemic was contained, the WHO released a framework that
was prepared according to the six phases of an epidemic, moving from preparedness, planning, and routine surveillance
for cases, through to the prevention of the consequent international spread, to the disruption of global transmission [50].
The WHO, in collaboration with the Food and Agriculture Organization of the United Nations (FAO), the World
Organization for Animal Health (OIE), and national governments, have been working with health care workers and
scientists in affected countries to gather and share scientific evidence based on the previous coronavirus epidemic. This
information gathering process has been beneficial for a better understanding of the virus and the disease it causes and
for the regulation of outbreak response priorities, treatment approaches, and clinical management tactics [49]. When
WHO declared on July 5 that all chains of SARS transmission had been broken, the disease was thought to have spread
to more than 30 countries, only eight of which—Canada, China, Hong Kong, the Philippines, Singapore, Taiwan, the
United States, and Vietnam—reported more than 10 probable cases [51].
In the case of Covid-19, global lockdowns helped respective countries to flatten the curve so that health care sys-
tems get ready with planning and infrastructure. In a highly populated country like India early lockdown largely slowed
6 Pandemic Outbreaks in the 21st Century

down the infections. One best example is New Zealand which implemented a scientific approach [52] to successfully
contain the Covid-19. On the contrary, many developed countries equipped with the preparedness and infrastructure,
like United Kingdom, Italy, Spain, France, and the United States could not contain the spread effectively in the first
wave of the pandemic. This could be largely due to an unscientific approach, lack of leadership, wrong decisions on
prophylaxis, and so on. Since the 1918 pandemic, we have not improved much except in having ventilators, improved
ICU facilities, blood thinners, few repurposed drugs, antigen/antibody testing, and so on. Yet, the three major nonmedi-
cal interventions such as masks, physical distancing, and hand washing were largely helpful during Covid-19. As a con-
sequence, during the lockdowns for 4
6 months, the Covid-19 spread was relatively slowed down but the seasonal
influenzas disappeared across continents as R0 of seasonal influenzas is less than that of Covid-19. No single interven-
tion may be efficient to control pandemics like Covid-19.
Although accumulated knowledge and risk preparedness from the SARS/MERS epidemics allowed researchers to
examine the effectiveness of strategic plans in dealing with the ongoing pandemic of COVID-19, several challenges
have been raised in preventing the spread of COVID-19, such as the lack of medical supplies and laboratory facilities
for the assessment of the disease and the presentation of a high number of asymptomatic cases [30]. The WHO Health
Cluster framework is a gateway to useful resources to support COVID-19 preparedness and response [53]. Generally,
each pandemic/epidemic has presented a public health emergency of uncertain scope and effect; thus essential elements
of current approaches to pandemic preparedness and extenuation, such as the development of vaccines and stockpiling
of antiviral drugs, necessitate detailed virological and immunological data on viruses with apparent pandemic potential.
However, the development of vaccines against new strains is challenging. Therefore physicians and health workers
have found themselves facing the massive challenge of preventing infections or stabilizing patients’ conditions [30].
Several other countries implemented localized school closures; should these closures become nationwide, millions of
additional learners will experience education disruption [48]. The real danger, however, may be in the long-term effects
of the epidemic. Years of budget cuts and failure to meet students’ basic needs make higher education especially vul-
nerable and potentially unequipped to handle a crisis like this [54].

1.3 Future directions

Advancements in science and technology related to diagnostic capacities, vaccines, and antiviral have provided more
effective tools for preventing and responding to infectious disease threats. Effective preventive measures must be imple-
mented to control it from global spreading. In addition, great effort should be made on the development of vaccines and
antiviral drugs [55]. The use of a systems biology approach may enable us to finally understand the intricate dynamics
between the cell and virus proteome that eventually constitute a particular disease phenotype [56]. The integration of
transcriptome data, proteomic profiles, and detailed interaction networks between viral and cellular proteins should pro-
vide a system view of the intricate communication networks regulating virus infection at the cellular level. Platform
approaches for each of these key system components are available, providing high-throughput identification of the inter-
action networks and the impact on host expression [57]. Comparing the responses to infection in lungs from SARS-
CoV, H5N1 influenza virus, Ebola virus, and respiratory syncytial virus-infected hosts can further our understanding of
how each virus modulates the innate immune response. The use of a systems genetic approach may be our best chance
at identifying the host genes responsible for coronavirus disease and age-related susceptibility phenotypes [56]. The
onset of the SARS epidemic in different continents has led to the formation of a successful laboratory network to iden-
tify the molecular mechanisms underlying the SARS infection [58]. Next to the development of early diagnostic tests
and effective treatment strategies, it is most important to orchestrate research activities that lead to the development of
vaccines and antiviral agents, as there is no established therapy to date. Even now in a situation of only a handful of
new cases, SARS remains a major global health hazard that may reappear [58]. In the future the development of multi-
plex tests that differentiate among the members of coronaviruses and also influenzas with one patient sample would
reduce testing burdens.
The Ministry of Family Welfare of the Government of India has set forth guidance for fighting COVID-19. The
strategy involves establishing three types of facilities: fever clinic/COVID care center, dedicated COVID health center,
and dedicated COVID hospital [60]. Plasma technology is now being considered as an alternative. Blood is collected
from a person who has recovered from COVID-19. Separation of serum and screening is done for virus-neutralizing
antibodies 59,60. The COVID-19 pandemic can be viewed as a crisis opportunity for the development of India [60].
There is a great experience in every country as far as the fight against COVID-19 is concerned. A combined effort to
collect all these experiences and lessons learned from almost 213 countries will be a resource for future preparedness in
terms of medical infrastructure, handling of quarantine centers, COVID-19 care centers, medicines, personal care
 Lessons learned from the first pandemic of the 21st century Chapter | 1 7

equipment, movable infrastructure, masks for frontline workers and working people, guidelines for social distancing,
doctors, nurses, Asha village health workers maintenance personnel and legislative support. The most important is peo-
ple’s participation in all health care programs and strict adherence to guidelines [60]. Furthermore, the timely reporting
of cases, updates on clinical status and disposition of patients, the real-time analysis of data, and the appropriate dissem-
ination of information are essential for outbreak-managing decisions [30].

1.4 Conclusion
Now the pandemic by COVID-19 is a live issue affecting people worldwide. Strategies for preventing and controlling
pandemic/epidemic viruses can be improved by being well-prepared. Preparedness strategies, which primarily include
the quarantine of infected persons, self-protection (wearing facemasks, using disinfectants, washing hands, and disin-
fecting surfaces with bleach or alcohols), and social distancing are all considered to be important for a comprehensive
plan that can be tested and promoted by conducting exercises to engage the whole of society. Without fundamental ther-
apeutic interventions, current management is to reduce the virus spread and provide supportive care for diseased
patients. There is an urgent need to develop targeted therapies. Understanding the difference in pediatric and adult
responses to this virus may help to direct immune-based therapeutics. The ultimate goal is to develop a resilient global
health infrastructure. Besides acquiring treatments, vaccines, and other preventive medicine, bio-surveillance is critical
to preventing disease emergence and to counteracting its spread. During disasters, there is the utilitarian goal of doing
the most good for as many people as possible with minimal harm. Vaccination is one of the most effective public health
interventions and innovative strategies for the research and development of vaccines. The lessons learned from COVID-
19 are maintaining self-hygiene, using masks, maintaining physical distance, and keeping the surroundings clean.
Preparedness plans are crucial to build frameworks for emergency response, thereby providing countries with the oppor-
tunity to plan, strategize and mobilize human and capital resources before a pandemic occurs. Adequate and thorough
plans ensure that countries can respond immediately when a pandemic is declared.

References
[1] Soman Pillai V, Krishna G, Valiya Veettil M. Nipah virus: past outbreaks and future containment. Viruses 2020;12(4):465.
[2] WHO. Clinical management of severe acute respiratory infection when novel coronavirus (nCoV) infection is suspected: interim guidance, 25
January 2020. World Health Organization; 2020 (No. WHO/nCoV/Clinical/2020.2).
[3] LeDuc JW, Barry MA. SARS, the first pandemic of the 21st century. Emerg Infect Dis 2004;10(11):e26.
[4] Hui DS, Zumla A. Severe acute respiratory syndrome: historical, epidemiologic, and clinical features. Infect Dis Clin 2019;33(4):869
89.
[5] Zhao Z, Zhang F, Xu M, Huang K, Zhong W, Cai W, et al. Description and clinical treatment of an early outbreak of severe acute respiratory
syndrome (SARS) in Guangzhou, PR China. J Med Microbiol 2003;52(8):715
20.
[6] Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, Lim W, et al. Coronavirus as a possible cause of severe acute respiratory syndrome.
Lancet 2003;361(9366):1319
25.
[7] Tsang KW, Ho PL, Ooi GC, Yee WK, Wang T, Chan-Yeung M, et al. A cluster of cases of severe acute respiratory syndrome in Hong Kong.
N Engl J Med 2003;348(20):1977
85.
[8] World Health Organization. Transmission of SARS-CoV-2: implications for infection prevention precautions: scientific brief, 09 July 2020.
World Health Organization; 2020.
[9] Cherry JD, Krogstad P. SARS: the first pandemic of the 21st century. Pediatr Res 2004;56(1):1
5.
[10] Strunk T, Inder T, Wang X, Burgner D, Mallard C, Levy O. Infection-induced inflammation and cerebral injury in preterm infants. Lancet
Infect Dis 2014;14(8):751
62.
[11] Rothe C, Schunk M, Sothmann P, Bretzel G, Froeschl G, Wallrauch C, et al. Transmission of 2019-nCoV infection from an asymptomatic con-
tact in Germany. N Engl J Med 2020;382(10):970
1.
[12] Singanayagam A, Patel M, Charlett A, Bernal JL, Saliba V, Ellis J, et al. Duration of infectiousness and correlation with RT-PCR cycle thresh-
old values in cases of COVID-19, England, January to May 2020. Eurosurveillance 2020;25(32):2001483.
[13] Lai CC, Shih TP, Ko WC, Tang HJ, Hsueh PR. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019
(COVID-19): the epidemic and the challenges. Int J Antimicrob Agents 2020;55(3):105924.
[14] Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet 2015;386(9997):995
1007.
[15] Ge XY, Li JL, Yang XL, Chmura AA, Zhu G, Epstein JH, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the
ACE2 receptor. Nature 2013;503(7477):535
8.
[16] Hu B, Zeng LP, Yang XL, Ge XY, Zhang W, Li B, et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new
insights into the origin of SARS coronavirus. PLoS Pathog 2017;13(11):e1006698.
[17] Yang J. Asymptotic behavior of solutions for competitive models with a free boundary. Discrete Contin Dyn Syst A 2015;35(7):3253.
8 Pandemic Outbreaks in the 21st Century

[18] Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin.
Nature 2020;579(7798):270
3.
[19] Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17(3):181
92.
[20] Bloom DE, Cadarette D. Infectious disease threats in the twenty-first century: strengthening the global response. Front Immunol
2019;10:549.
[21] Gonzalez JP, Souris M, Valdivia-Granda W. Global spread of hemorrhagic fever viruses: predicting pandemics. Hemorrhagic fever viruses.
New York: Humana Press; 2018. p. 3
31.
[22] Chakraborty C, Sharma AR, Sharma G, Bhattacharya M, Lee SS. SARS-CoV-2 causing pneumonia-associated respiratory disorder (COVID-
19): diagnostic and proposed therapeutic options. Eur Rev Med Pharmacol Sci 2020;24(7):4016
26.
[23] Wang Y, Wang Y, Chen Y, Qin Q. Unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (COVID-
19) implicate special control measures. J Med Virol 2020;92(6):568
76.
[24] Rabaan AA, Al-Ahmed SH, Haque S, Sah R, Tiwari R, Malik YS, et al. SARS-CoV-2, SARS-CoV, and MERS-COV: a comparative overview.
Infez Med 2020;28(2):174
84.
[25] Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature
2020;579(7798):265
9.
[26] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Lancet 2020;395(10223):497
506.
[27] Xu X, Chen P, Wang J, Feng J, Zhou H, Li X, et al. Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its
spike protein for risk of human transmission. Sci China Life Sci 2020;63(3):457
60.
[28] Pellett PE, Mitra S, Holland TC. Basics of virology. Handb Clin Neurol 2014;123:45
66.
[29] Florindo HF, Kleiner R, Vaskovich-Koubi D, Acúrcio RC, Carreira B, Yeini E, et al. Immune-mediated approaches against COVID-19. Nat
Nanotechnol 2020;15(8):630
45.
[30] Abdelrahman Z, Li M, Wang X. Comparative review of SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza a respiratory virus. Front
Immunol 2020;11:2309.
[31] Trends in Immunology. Available from: https://www.cell.com/trends/immunology/fulltext/S1471-4906(20)30233-7.
[32] Yuki K, Fujiogi M, Koutsogiannaki S. COVID-19 pathophysiology: a review. Clin Immunol 2020;215:108427.
[33] Luk HK, Li X, Fung J, Lau SK, Woo PC. Molecular epidemiology, evolution and phylogeny of SARS coronavirus. Infect Genet Evol
2019;71:21
30.
[34] Bartlam M, Xu Y, Rao Z. Structural proteomics of the SARS coronavirus: a model response to emerging infectious diseases. J Struct Funct
Genomics 2007;8(2):85
97.
[35] Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, Cheung CL, et al. Isolation and characterization of viruses related to the SARS coronavirus
from animals in southern China. Science 2003;302(5643):276
8.
[36] Du RH, Liang LR, Yang CQ, Wang W, Cao TZ, Li M, et al. Predictors of mortality for patients with COVID-19 pneumonia caused by SARS-
CoV-2: a prospective cohort study. Eur Respir J 2020;55(5):2000524.
[37] Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus
origins and receptor binding. Lancet 2020;395(10224):565
74.
[38] Zhang C, Zheng W, Huang X, Bell EW, Zhou X, Zhang Y. Protein structure and sequence reanalysis of 2019-nCoV genome refutes snakes as
its intermediate host and the unique similarity between its spike protein insertions and HIV-1. J Proteome Res 2020;19(4):1351
60.
[39] Harrison AG, Lin T, Wang P. Mechanisms of SARS-CoV-2 transmission and pathogenesis. Trends Immunol 2020;41(12):1100
15.
[40] Wu HY, Brian DA. Subgenomic messenger RNA amplification in coronaviruses. Proc Natl Acad Sci USA 2010;107(27):12257
62.
[41] Qiu H, Wu J, Hong L, Luo Y, Song Q, Chen D. Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-
19) in Zhejiang, China: an observational cohort study. Lancet Infect Dis 2020;20(6):689
96.
[42] Oberholtzer K, Sivitz L, Mack A, Lemon S, Mahmoud A, Knobler S, editors. Learning from SARS: preparing for the next disease outbreak:
workshop summary. National Academies Press; 2004.
[43] Syndrome SA. Status of the outbreak and lessons for the immediate future. Geneva: WHO; 2003.
[44] Drosten C, Günther S, Preiser W, Van Der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute
respiratory syndrome. N Engl J Med 2003;348(20):1967
76.
[45] Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, et al. A novel coronavirus associated with severe acute respiratory syn-
drome. N Engl J Med 2003;348(20):1953
66.
[46] Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, Van Amerongen G, Van Riel D, et al. Newly discovered coronavirus as the primary
cause of severe acute respiratory syndrome. Lancet 2003;362(9380):263
70.
[47] Hu Z, Lin X, Kaminga AC, Xu H. Impact of the COVID-19 epidemic on lifestyle behaviors and their association with subjective well-being
among the general population in mainland China: cross-sectional study. J Med Internet Res 2020;22(8):e21176.
[48] de Oliveira Araújo FJ, de Lima LSA, Cidade PIM, Nobre CB, Neto MLR. Impact of Sars-Cov-2 and its reverberation in global higher education
and mental health. Psychiatry Res 2020;288:112977.
[49] Suwantarat N, Apisarnthanarak A. Risks to healthcare workers with emerging diseases: lessons from MERS-CoV, Ebola, SARS, and avian flu.
Curr Opin Infect Dis 2015;28(4):349
61.
[50] WHO C. Emergencies preparedness, response. Chikungunya; 2018.
 Lessons learned from the first pandemic of the 21st century Chapter | 1 9

[51] Knobler S, Mahmoud A, Lemon S, Mack A, Sivitz L, Oberholtzer K. Workshop Summary. Learning from SARS: preparing for the next disease
outbreak; 2004. 11.
[52] Baker MG, Wilson N, Anglemyer A. Successful elimination of Covid-19 transmission in New Zealand. N Engl J Med 2020;383(8):e56.
Available from: https://doi.org/10.1056/NEJMc2025203.
[53] Peeri NC, Shrestha N, Rahman MS, Zaki R, Tan Z, Bibi S, et al. The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest
and biggest global health threats: what lessons have we learned? Int J Epidemiol 2020;49(3):717
26.
[54] Inside Higher Ed. 2020. Available from: https://www.insidehighered.com/views/2020/03/12/coronavirus-couldhave-long-term-impact-state-
funding-universities-opinion.
[55] Zheng J. SARS-CoV-2: an emerging coronavirus that causes a global threat. Int J Biol Sci 2020;16(10):1678.
[56] Frieman M, Baric R. Mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation. Microbiol Mol Biol Rev
2008;72(4):672
85.
[57] Tan SL, Ganji G, Paeper B, Proll S, Katze MG. Systems biology and the host response to viral infection. Nat Biotechnol 2007;25(12):1383
9.
[58] Groneberg DA, Hilgenfeld R, Zabel P. Molecular mechanisms of severe acute respiratory syndrome (SARS). Respir Res 2005;6(1):1
16.
[59] Duan K, Liu B, Li C, Zhang H, Yu T, Qu J, et al. Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad
Sci USA 2020;117(17):9490
6.
[60] Munnoli PM, Nabapure S, Yeshavanth G. Post-COVID-19 precautions based on lessons learned from past pandemics: a review. J Public Health
(Bangkok) 2020;1
9.
This page intentionally left blank
Chapter 2

Epidemiology of COVID-19 in Latin

America
Alfonso J. Rodriguez-Morales1,2,3 and D. Katterine Bonilla-Aldana4
1
Grupo de Investigación Biomedicina, Faculty of Medicine, Fundacion Universitaria Autonoma de las Americas, Pereira, Risaralda, Colombia,
2
Universidad Cientı´fica del Sur, Lima, Peru, 3Universidad Privada Franz Tamayo, Cochabamba, Bolivia, 4Semillero de Investigación en Zoonosis
(SIZOO), Grupo de Investigación GISCA, Fundacion Universitaria Autonoma de las Americas, Pereira, Risaralda, Colombia

2.1 Introduction
Coronaviruses have been affecting the world since this family, Coronaviridae, and their first identified members were
discovered more than five decades ago, in the 1960s. According to the International Committee on Taxonomy of
Viruses (ICTV) [1], this family is part of the suborder Cornidovirineae, order Nidovirales, which is part of the class
Pisoniviricetes, belonging to the phylum Pisuviricota, at the kingdom Orthornavirae, in the realm Riboviria (Fig. 2.1).
The family Coronaviridae has only two subfamilies, Letovirinae and Orthocoronavirinae, which included the corona-
viruses of human and animal importance. In particular, this last subfamily has four genera: Alpha, Beta, Gamma, and
Deltacoronavirus. The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the Severe Acute Respiratory
Syndrome Coronavirus 2 (SARS-CoV-2) belong to the genus Betacoronavirus and the subgenus Sarbecovirus (Fig. 2.1).
The Middle East Respiratory Syndrome (MERS-CoV) also belongs to the Betacoronavirus genus but the subgenus
Merbecovirus (Fig. 2.1). Other coronaviruses affecting humans include Human Coronavirus 229E (HCoV-229E), Human
Coronavirus NL63 (HCoV-NL63), Human Coronavirus 229E (HCoV-OC43), and Human Coronavirus HKU1 (HCoV-
HKU1), which cause respiratory tract infection in immunocompromised individuals [2,3]. In addition, recently, porcine
deltacoronavirus (PDCoV) strains in plasma samples of three Haitian children with acute undifferentiated febrile illness
were identified and confirmed by sequencing and phylogenetic analyses [4]. Also, during the validation of a highly sensi-
tive pan-species coronavirus (CoV) semi-nested RT-PCR assay, a study in Sarawak, Malaysia, found canine CoV
(CCoV) RNA in nasopharyngeal swabs from eight (2.5%) of 301 patients hospitalized with pneumonia during 2017-18.
With complete genome sequencing of the virus causing cytopathic effects in A72 cells, it was identified as a novel
canine-feline recombinant alphacoronavirus (genotype II) that authors named CCoV-HuPn-2018 [5].
The SARS-CoV appeared, unexpectedly as other subsequent coronaviruses, and cause epidemics in 2002
03 in China,
also affecting other countries in Asia and beyond, even with cases in North America [2,3]. Later, in 2012
13 the MERS-
CoV emerged in Saudi Arabia, also extending to other countries in the Middle East, East Asia, and Europe, mainly [6
10].
The emergence of the Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 [11,12] occurred appar-
ently in December 2019 in Wuhan, Hubei province, China. In this country it has begun to spread rapidly in different
areas domestically and also in Asia and the rest of the world at the beginning of 2020. Nevertheless, growing evidence
is showing that the SARS-CoV-2 was already circulating in some countries of Europe before December 2019 [13].
Precisely Europe was one of the significantly impacted regions, and among this country, Italy [14], in the northern
provinces, presented a highly concentrated epidemic. For decades, the main routes of outbound travel from Latin
America are North America and Europe, and the main travelers arriving in Latin America are from those other regions
of the world. During February
March 2020, many travelers arrived in different Latin American countries from these
areas in Italy, such as Brazil, Colombia, and Mexico [15
17]. As expected, resource-constrained areas of the world, as
is the case of Latin America, have been more affected given their previous epidemiological context, health care sys-
tems, and the socioeconomic conditions [17]. In this chapter the main epidemiological features of the COVID-19 during
the first year of the pandemic in this region are revised.

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00012-4

© 2021 Elsevier Inc. All rights reserved. 11
12 Pandemic Outbreaks in the 21st Century

FIGURE 2.1 Taxonomical location of the family Coronaviridae and its members, according to the ICTV (https://talk.ictvonline.org/taxonomy/).
ICTV, International Committee on Taxonomy of Viruses.

2.2 Previous epidemiological situation of major infectious diseases in Latin America

Poverty, illiteracy, corruption, weak health care systems, and deep political crises characterize most of the countries in
the region. Before 2020, major simultaneous epidemics of vaccine-preventable diseases, such as measles, whooping
cough, diphtheria, yellow fever, and even some suspected case of polio, affected many territories [18]. This was mainly
linked to the complex humanitarian crisis in Venezuela, once a wealthy nation in the region (included in the global top
of oil producer countries), that forced migration to other nations, especially in South America [19,20], according to
experts in refugees.
Those refugees harbor in a considerable proportion those vaccine-preventable diseases, as immunization programs
in Venezuela have been lacking or are completely shut down, leading to a low immunization coverage among the popu-
lation, especially in those more vulnerable, including native Ameridians, children, pregnant and childbearing age
women, among others [18
24]. In addition to this vector-borne diseases, such as malaria, Chagas disease (previously
only occurring in Latin America but now detected worldwide due to migration), leishmaniasis, and dengue (as well as
other arboviral diseases), have also caused epidemics in Venezuela and generated imported cases in Colombia, Brazil,
Ecuador, Panama, and other distant countries, as a consequence of the forced migration [18,23,25
27]. Over the last
5 years, Venezuela has become a significant problem in terms of malaria for the regional health authorities. The highest
number of cases and the highest incidence rate are now in Venezuela, above Brazil, Mexico, Colombia, and other coun-
tries that are reducing and controlling this disease [27,28].
 Epidemiology of COVID-19 in Latin America Chapter | 2 13

Human immunodeficiency virus (HIV) [29,30], tuberculosis, antimicrobial resistance, and health care-associated
infections caused by Candida auris have been other infectious diseases causing concern [31,32], some of them also in
relation to the forced migration from Venezuela, but the common and regular problems of the countries in the region.
Given this epidemiological scenario, the situation in the region from 2015 to 2019 was incredibly complex. HIV-
positive individuals from Venezuela are also migrating due to the lack of antiretroviral drugs in the country.
In that period (2015
19), two major regional epidemic diseases were affecting the tropical and subtropical coun-
tries, with autochthonous vector transmission, and also in subtropical countries through sexual and vertical ways, as
was the case of Chikungunya and Zika viruses [33
40], that even were locally transmitted, as in the past occurred with
dengue and malaria, in the South Florida, United States. The first, an alphavirus, especially causing acute disease and
long-term rheumatological and nonrheumatological chronic consequences, the second, a flavivirus, generating associ-
ated epidemics of Guillain
Barré syndrome and the microcephaly and congenital Zika syndrome with their multiple
disabilities and long-term consequences in children [41
49]. This was the scenario of Latin America and the Caribbean
before the arrival of COVID-19. Both arboviral diseases arrived in the region as consequence of international travel.
Chikungunya entered via the Caribbean islands, and Zika, from the Pacific islands to Brazil, and then, to the rest of the
region.

2.3 COVID-19 arrival at Latin America

On February 25, 2020, the Brazilian Ministry of Health confirmed the first imported case from Lombardy, northern
Italy, to São Paulo [15
17]. Rapidly, in the next following days, other countries such as Ecuador, Mexico, Chile,
Argentina, Colombia, among others, reported the arrival of their first imported cases [50].
By March 2020, most countries reported imported cases of COVID-19 in the region (Fig. 2.2). Weeks and months
after, all affected, and most with the autochthonous transmission (Fig. 2.3), marching to their first pandemic wave of
cases. In Chile their first wave was in June, while in Brazil and Colombia, it was in August and Argentina in October
(Fig. 2.4). The holidays generated the second wave in most countries in the region for January 2021.

FIGURE 2.2 Daily new confirmed COVID-19 cases per million people in selected countries of Latin America, from February 26 to April 26, 2020.
COVID-19, Coronavirus Disease 2019. Johns Hopkins University CSSE COVID-19 Data—Last updated 18 February, 15:03 (London time).
14 Pandemic Outbreaks in the 21st Century

FIGURE 2.3 Daily new confirmed COVID-19 cases per million people in selected countries of Latin America, from April 26, 2020, to February 17,
2021. COVID-19, Coronavirus Disease 2019. Johns Hopkins University CSSE COVID-19 Data—Last updated 18 February, 15:03 (London time).

FIGURE 2.4 Daily new confirmed COVID-19 cases per million people in Brazil, Argentina, Chile, and Colombia, from February 26, 2020, to February
17, 2021. COVID-19, Coronavirus Disease 2019. Johns Hopkins University CSSE COVID-19 Data—Last updated 18 February, 15:03 (London time).
 Epidemiology of COVID-19 in Latin America Chapter | 2 15

Although during the first weeks and months, most health authorities in the region believed that the countries would
be able to handle the pandemic with the regular capabilities of the health care system, the number of hospitalizations,
and especially the number of patients requiring medical management at the intensive care units (ICU) eventually reach
the collapse in many cities across Latin America [51
53].
Fortunately, the region’s age structure makes most of the cases mild, managed in-home as outpatients, and recovery
quickly, in more than 95% of the cases. However, the rest required hospitalizations and ICU, and between 3% and 7%,
depending on the country, died.
Countries such as Brazil have reported the highest number of cases and deaths in the region (Table 2.1), but Mexico
reported the highest cumulated case fatality rate (%), with 8.75% [54]. Multiple countries reported a cumulated inci-
dence rate above 4000 cases/100,000 pop (4%), including Brazil, Colombia, Argentina, Chile, Costa Rica, and French
Guiana (Table 2.1).

2.4 Genomic and molecular epidemiology of COVID-19 in Latin America

The International Association of Epidemiology defines molecular epidemiology as “the application of epidemiologic
principles to study the molecular, biochemical, cellular and genetic mechanisms that underlie the pathophysiology, eti-
ology and prevention of human diseases and related outcomes, as well as their early detection, treatment, or prognosis”
[55
57]. In this context the molecular diagnosis, sequencing, and phylogeny of the SARS-CoV-2, since the beginning
of the epidemic, has been a critical element in the understanding of the evolution of the pandemic during the time but
also has been applied to the interpretation of transmission, clinical phenotypes, and pathology of COVID-19 [57].

TABLE 2.1 Leading epidemiological indicators of COVID-19 in selected Latin American countries, up to February 18,
2021.

Countries and Cases Deaths Recoveries Population Incidence CFR Mortality

territories rate % rate
Brazil 9,866,710 239,773 8,821,887 211,500,000 4665.1 2.43 113.4
Colombia 2,198,549 57,786 2,090,467 49,400,000 4450.5 2.63 117.0
Argentina 2,029,004 50,327 1,833,404 44,900,000 4518.9 2.48 112.1
Mexico 1,995,892 174,657 1,555,923 128,000,000 1559.3 8.75 136.5
Peru 1,238,501 43,880 1,149,367 32,100,000 3858.3 3.54 136.7
Chile 779,541 19,624 737,126 19,100,000 4081.4 2.52 102.7
Panama 332,679 5642 313,783 4,000,000 8317.0 1.70 141.1
Ecuador 267,701 15,355 230,377 17,500,000 1529.7 5.74 87.7
Bolivia 237,144 11,234 179,057 11,500,000 2062.1 4.74 97.7
Dominican Republic 230,563 2959 178,146 11,000,000 2096.0 1.28 26.9
Costa Rica 200,024 2730 163,334 5,000,000 4000.5 1.36 54.6
Guatemala 167,383 6150 154,446 17,000,000 984.6 3.67 36.2
Honduras 160,983 3893 63,346 10,000,000 1609.8 2.42 38.9
Paraguay 145,095 2953 121,542 7,200,000 2015.2 2.04 41.0
Venezuela 133,577 1285 125,565 32,200,000 414.8 0.96 4.0
Uruguay 49,360 541 43,486 3,500,000 1410.3 1.10 15.5
French Guiana 16,456 80 9995 300,000 5485.3 0.49 26.7
Suriname 8811 167 8242 600,000 1468.5 1.90 27.8
Guyana 8232 186 7399 800,000 1029.0 2.26 23.3

Incidence rate: cases/population 3 100,000; CFR%: case fatality rate, cases/deaths 3 100; mortality rate: deaths/population 3 100,000. Note: Bold values
indicates highest value for the indicator.
Source: https://ourworldindata.org/coronavirus.
16 Pandemic Outbreaks in the 21st Century

More than 3100 SARS-CoV-2 whole-genome sequences are deposited in databases from Brazil, Bolivia, Chile,
Ecuador, Colombia, Uruguay, Suriname, Peru, and Argentina (Fig. 2.5) [58
61]. Genomic epidemiology has been vital in
tracking the emergence of variants of the SARS-CoV-2. In countries such as the United Kingdom and South Africa
emerged new SARS-CoV-2 variants of concern (VOC) 501Y.V1 (B.1.1.7) and 501Y.V2 (B.1.351), respectively. Such var-
iants had mutations (N501Y) at the receptor-binding domain of the spike protein that appeared to be increasing the virus
transmission by nearly 40%
70% and possibly associated with an increased risk of death. In January 2021 other VOC
began to be reported in other countries, such as Brazil [P.1 (501Y.V3)], United States, France, Italy, Denmark, among
others [62
64]. These are other variants, and mutations are currently under the track in Brazil (https://covariants.org/)
(Fig. 2.6) [65].

FIGURE 2.5 Genomic epidemiology of SARS-CoV-2 in South America. SARS-CoV-2, Severe Respiratory Syndrome Coronavirus 2. (https://next-
strain.org/ncov/global).

FIGURE 2.6 Covariants tracking in Brazil (https://covariants.org/).

 Epidemiology of COVID-19 in Latin America Chapter | 2 17

FIGURE 2.7 SARS-CoV-2 genome sequencing phylogenetic analysis and classification up to February 16, 2021, available in the GISAID platform
(https://www.gisaid.org/). SARS-CoV-2, Severe Respiratory Syndrome Coronavirus 2.

Nevertheless, given the uprise detection of the VOC and their implications, there is a concern in Latin America that
there is a lack of SARS-CoV-2 genome sequencing compared to other regions. Currently, there have been sequenced
almost 500,000 genomes of the virus (Fig. 2.7).

2.5 Emerging situations during the COVID-19 pandemic in Latin America:

coinfections and reinfections
As expected from the beginning of the pandemic, the overlapping and syndemics with other endemic pathogens have
generated coinfections, especially with dengue and other pathogens [66
71]. In this context other coinfections, such as
HIV, tuberculosis, and malaria, are also of concern [30].
After August 2020, reinfections began to be confirmed in Hong Kong, United States, India, and other countries.
Later these included Ecuador, Brazil, and Peru [72], among others in Latin America [73
84].

2.6 Health care workers infections due to SARS-CoV-2

As occurred early during the pandemic in China, health care workers in Latin America have been significantly affected
in Brazil, Mexico, Colombia, Peru, and Honduras [85
87]. For example, in Colombia, over the pandemic, more than
42,000 cases have occurred in health care workers (Fig. 2.8), with 208 that have died so far (0.5%) and 8.97% of the
cases asymptomatic. Countries such as Peru or Honduras highlight the high diversity of the impact of COVID-19
18 Pandemic Outbreaks in the 21st Century

FIGURE 2.8 COVID-19 among health care workers in Colombia, up to February 18, 2021, by the source of transmission. COVID-19, Coronavirus
Disease 2019 (https://www.ins.gov.co/Noticias/Paginas/Coronavirus.aspx).

among health care workers. In Peru the fatality rate among health care workers has ranged from 0% up to 8.75% in
some areas (e.g., Arequipa) [85
87].

2.7 Pharmacoepidemiology of the therapeutic approaches in the region

Early during the pandemic, multiple research groups have proposed different therapeutic approaches; in most cases
repurposing old drugs used for different indications [88,89]. Many in vitro suggested different drug candidates be used
as compassionate drugs in COVID-19 and later tested in observational and clinical trials.
Drugs included chloroquine/hydroxychloroquine, azithromycin, arbidol, lopinavir, ribavirin, remdesivir, and iver-
mectin that have not proved significant efficacy in controlled trials [90
93]. Despite this, for a long time during the
pandemic, these drugs have been used without any evidence that may support their recommendation and use in patients
for treatment or prophylaxis. Especially, and even up to February 2021, in countries such as Peru and some Mexico,
Colombia, Brazil, and Venezuela cities, ivermectin is used and recommended broadly.
Fortunately, in other countries, such as the case of Colombia, evidence-based guidelines do not recommend and
even contribute to regulating the use of such not supported therapies. Drugs such as dexamethasone, or recently tocili-
zumab, are used based on the evidence derived from randomized clinical trials [3,94].

2.8 Epidemiology of the vaccinations against COVID-19 in Latin America

With the advance of the pandemic and millions affected in the region, vaccines’ arrival became a critical issue once
these became available [89,95
97]. Now, most of the region’s countries have started their COVID-19 vaccination
national plans (Fig. 2.9).
In countries such as Brazil more than 5.88 million vaccines have been applied. In Chile more than 2.38 million. In
Mexico 1.16 million. Countries such as Colombia have just received their first lots of vaccines. On February 15, 2021,
Colombia received 50,000 doses of the Pfizer/Biontech vaccine. This vaccine arrived also, in South America, to
Ecuador (25,000 doses) and Chile (110,000 doses). Between February 17 and 18, 2021, almost 8000 doses of the vac-
cine in Colombia have been applied. But in the region, Chile highlights rapid growth in the number of doses adminis-
tered in their population (Fig. 2.10), becoming the first in the region and the second in the world in doses administered
per 100 population. Other vaccines in the region that have arrived included the Sputnik V in Venezuela (100,000 doses),
 Epidemiology of COVID-19 in Latin America Chapter | 2 19

FIGURE 2.9 COVID-19 vaccination in North, Central, and South America, February 17, 2021. COVID-19, Coronavirus Disease 2019. Modified
from: Official data collated by Our World in Data—Last updated 18 February, 14:40 (London time).

Argentina (1.2 million doses), and Bolivia (20,000 doses); Sinovac in Brazil (10 million doses), and Chile (4 million
doses); AstraZeneca in Brazil (2 million doses), Argentina (580,000 doses); and Sinopharm in Peru (1 million doses).

2.9 Conclusion
The region’s complex epidemiology for COVID-19, also marked by social and health system inequalities intercountries
and also intranational, still showed that after millions of people affected, there is still high susceptibility, and the so-
called herd immunity, given the cases and the seroprevalences already reported in some cities in the region, is still far.
Vaccination programs will be essential during the year to reduce the gap. However, the challenge is relatively high to
reach a high vaccine coverage in a short time among the population that has not been infected yet by SARS-CoV-2.
Even more, in recent weeks, scandals related to the irregular application of the COVID-19 vaccines to persons not pri-
oritized, in Peru, Argentina, and Colombia, are raising more concerns on the future of the vaccination programs in the
region, to reach appropriately the goals.
Future waves would be perfectly expectable, in some countries, second and third waves. Major mass gatherings,
such as the Holy Week ending March-beginning April 2021, would be an opportune moment to increase transmission,
especially if vaccination does not progress rapidly [98]. This is a period of religious events, massive participation, and
vacations or holy days in touristic areas of the region, beaches, and mountains [8]. Nevertheless, some countries take
measures to avoid such situations. For example, the carnivals of Rio de Janeiro, Brazil, or Barranquilla, Colombia, have
been suspended to avoid an increase in transmission. Some New Year festivals become virtual, as many artists concerts.
Finally, can COVID-19 become endemic in the world, in some countries, in some urban or rural areas? Still is early
to answer this complex question, but more research on the zoonotic origins and human
animal transmission is needed
[99
101]. As this may be a key element in the persistence in nature of the SARS-CoV-2. In Latin America few studies
on these have been performed so far [10,102
105], and more on this is required for a full understanding of the whole
epidemiology of SARS-CoV-2/COVID-19, but already reports from some countries, such as Chile and Mexico, have
20 Pandemic Outbreaks in the 21st Century

FIGURE 2.10 Daily COVID-19 vaccine doses administered per 100 people, up to February 17, 2021. COVID-19, Coronavirus Disease 2019.
Official data collated by Our World in Data—Last updated 18 February, 14:40 (London time).

reported the SARS-CoV-2 infection in domestic animals, such as cats, and this deserves more studies, in order to under-
stand their real role in the transmission cycles of this emerging disease.

Acknowledgments
We acknowledge the support of Fundacion Universitaria Autonoma de las Americas, Pereira, Colombia, and Universidad Cientifica del Sur,
Lima, Peru. We would also like to thank Asociación Colombiana de Infectologı́a for their support.

Conflict of interest
The authors declare that they have no conflict of interest.

References
[1] Coronaviridae Study Group of the International Committee on Taxonomy of V. The species Severe acute respiratory syndrome-related coronavi-
rus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020;5(4):536
44.
[2] Bonilla-Aldana DK, Villamil-Gómez WE, Rabaan AA, Rodriguez-Morales AJ. Una nueva zoonosis viral de preocupación global: COVID-19,
enfermedad por coronavirus 2019. Iatreia 2020;33(2):107
10.
[3] Millan-Oñate J, Rodrı́guez-Morales AJ, Camacho-Moreno G, Mendoza-Ramı́rez H, Rodrı́guez-Sabogal IA, Álvarez-Moreno C. A new emerging
zoonotic virus of concern: the 2019 novel Coronavirus (COVID-19). Infectio 2020;24(3):187
92.
[4] Lednicky JA, Tagliamonte MS, White SK, Elbadry MA, Alam MM, et al. Emergence of porcine delta-coronavirus pathogenic infections among
children in Haiti through independent zoonoses and convergent evolution. medRxiv [Preprint] 2021;2021.03.19.21253391. Available from:
https://doi.org/10.1101/2021.03.19.21253391.
 Epidemiology of COVID-19 in Latin America Chapter | 2 21

[5] Vlasova AN, Diaz A, Damtie D, Xiu L, Toh TH, et al. Novel Canine Coronavirus Isolated from a Hospitalized Pneumonia Patient, East
Malaysia. Clin Infect Dis 2021;ciab456. Available from: https://doi.org/10.1093/cid/ciab456.
[6] Bonilla-Aldana DK, Cardona-Trujillo MC, Garcia-Barco A, Holguin-Rivera Y, Cortes-Bonilla I, Bedoya-Arias HA, et al. MERS-CoV and
SARS-CoV infections in animals: a systematic review and meta-analysis of prevalence studies. Infez Med 2020;28(suppl 1):71
83.
[7] Rabaan AA, Al-Ahmed SH, Haque S, Sah R, Tiwari R, Malik YS, et al. SARS-CoV-2, SARS-CoV, and MERS-COV: a comparative overview.
Infez Med 2020;28(2):174
84.
[8] Al-Tawfiq JA, Rodriguez-Morales AJ. Super-spreading events and contribution to transmission of MERS, SARS, and SARS-CoV-2 (COVID-19).
J Hosp Infect 2020;105(2):111
12.
[9] Bonilla-Aldana DK, Quintero-Rada K, Montoya-Posada JP, Ramirez-Ocampo S, Paniz-Mondolfi A, Rabaan AA, et al. SARS-CoV, MERS-CoV
and now the 2019-novel CoV: have we investigated enough about coronaviruses? - a bibliometric analysis. Travel Med Infect Dis 2020;33:101566.
[10] Dhama K, Patel SK, Sharun K, Pathak M, Tiwari R, Yatoo MI, et al. SARS-CoV-2 jumping the species barrier: zoonotic lessons from SARS,
MERS and recent advances to combat this pandemic virus. Travel Med Infect Dis 2020;37:101830.
[11] Franco-Paredes C, Marcos LA, Henao-Martinez AF, Rodriguez-Morales AJ, Villamil-Gomez WE, Gotuzzo E, et al. Cutaneous mycobacterial
infections. Clin Microbiol Rev 2018;32(1):e00069-18.
[12] Rodriguez-Morales AJ, Cardona-Ospina JA, Gutierrez-Ocampo E, Villamizar-Pena R, Holguin-Rivera Y, Escalera-Antezana JP, et al. Clinical,
laboratory and imaging features of COVID-19: a systematic review and meta-analysis. Travel Med Infect Dis 2020;34:101623.
[13] Carrat F, Figoni J, Henny J, Desenclos J-C, Kab S, de Lamballerie X, et al. Evidence of early circulation of SARS-CoV-2 in France: findings
from the population-based “CONSTANCES” cohort. Eur J Epidemiol 2021;36:219
22.
[14] Martellucci CA, Sah R, Rabaan AA, Dhama K, Casalone C, Arteaga-Livias K, et al. Changes in the spatial distribution of COVID-19 incidence
in Italy using GIS-based maps. Ann Clin Microbiol Antimicrob 2020;19(1):30.
[15] Rodriguez-Morales AJ, Sánchez-Duque JA, Hernández-Botero S, Pérez-Dı́az CE, Villamil-Gómez WE, Méndez CA, et al. Preparación y control
de la enfermedad por coronavirus 2019 (COVID-19) en América Latina. Acta Medica Peruana 2020;37(1):3
7.
[16] Sanchez-Duque JA, Arce-Villalobos LR, Rodriguez-Morales AJ. [Coronavirus disease 2019 (COVID-19) in Latin America: Role of primary
care in preparedness and response]. Aten Primaria 2020;52(6):369
72.
[17] Rodriguez-Morales AJ, Gallego V, Escalera-Antezana JP, Mendez CA, Zambrano LI, Franco-Paredes C, et al. COVID-19 in Latin America:
The implications of the first confirmed case in Brazil. Travel Med Infect Dis 2020;35:101613.
[18] Rodriguez-Morales AJ, Suarez JA, Risquez A, Delgado-Noguera L, Paniz-Mondolfi A. The current syndemic in Venezuela: measles, malaria
and more co-infections coupled with a breakdown of social and healthcare infrastructure. Quo vadis? Travel Med Infect Dis 2019;27:5
8.
[19] Mougenot B, Amaya E, Mezones-Holguin E, Rodriguez-Morales AJ, Cabieses B. Immigration, perceived discrimination and mental health: evi-
dence from Venezuelan population living in Peru. Global Health 2021;17(1):8.
[20] Rodriguez-Morales AJ, Bonilla-Aldana DK, Morales M, Suarez JA, Martinez-Buitrago E. Migration crisis in Venezuela and its impact on HIV
in other countries: the case of Colombia. Ann Clin Microbiol Antimicrob 2019;18(1):9.
[21] Noya-Alarcon O, Bevilacqua M, Rodriguez-Morales AJ. Trachoma in 3 Amerindian communities, Venezuelan Amazon, 2018. Emerg Infect
Dis 2019;25(1):182
3.
[22] Rodriguez-Morales AJ, Suarez JA, Risquez A, Cimerman S, Valero-Cedeno N, Cabrera M, et al. In the eye of the storm: infectious disease
challenges for border countries receiving Venezuelan migrants. Travel Med Infect Dis 2019;30:4
6.
[23] Rodriguez-Morales AJ, Suarez JA, Risquez A, Villamil-Gomez WE, Paniz-Mondolfi A. Consequences of Venezuela’s massive migration crisis
on imported malaria in Colombia, 2016
2018. Travel Med Infect Dis 2019;28:98
9.
[24] Tobon-Giraldo M, Salazar MI, Aguirre-Florez M, Montilla-Trejos CA, Suarez JA, Rodriguez-Morales AJ. The dilemmas and care challenges of
Venezuelan pregnant migrants presenting in Colombia. Travel Med Infect Dis 2019.
[25] Molina-Ortiz K, Puerta-Laverde V, Gutierrez-Ocampo E, Villamizar-Pena R, Rodriguez-Morales AJ. Should we talk more about imported
malaria in Bogota, Colombia? Infect Dis Health 2020;25(2):130
1.
[26] Franco-Herrera D, Gonzalez-Ocampo D, Restrepo-Montoya V, Gomez-Guevara JE, Alvear-Villacorte N, Rodriguez-Morales AJ. Relationship
between malaria epidemiology and the human development index in Colombia and Latin America. Infez Med 2018;26(3):255
62.
[27] Gutierrez-Ocampo E, Villamizar-Pena R, Holguin-Rivera Y, Molina-Ortiz K, Puerta-Laverde V, Rodriguez-Morales AJ. Malaria in Bogota,
Colombia (2007
2017) - an analysis of notified domestic and international cases. Travel Med Infect Dis 2020;33:101560.
[28] Suárez J, Carreño L, Paniz-Mondolfi A. Infectious diseases, social, economic and political crises, anthropogenic disasters and beyond:
Venezuela 2019
implications for public health and travel. Rev Panam Enferm Infecciosas 2018;1(2).
[29] Beerenwinkel N, Daumer M, Sing T, Rahnenfuhrer J, Lengauer T, Selbig J, et al. Estimating HIV evolutionary pathways and the genetic barrier
to drug resistance. J Infect Dis 2005;191(11):1953
60.
[30] Salvo CP, Di Lella N, Solveyra Lopez F, Hugo J, Gigena Zito J, Vilela A. [Dengue and SARS-CoV-2 coinfection in an HIV positive patient].
Medicina (B Aires) 2020;80(Suppl 6):94
6.
[31] Rodriguez-Morales AJ, Sabogal-Roman JA, Alvarez-Moreno CA. What has been researched about MDR-Candida auris? A bibliometric analysis
on the ‘new kid on the block’ in hospital-associated infections. J Infect Public Health 2018;11(2):295
6.
[32] Alvarado-Socarras JL, Vargas-Soler JA, Franco-Paredes C, Villegas-Lamus KC, Rojas-Torres JP, Rodriguez-Morales AJ. A cluster of neonatal
infections caused by Candida auris at a large referral center in Colombia. J Pediatric Infect Dis Soc 2021;10(5):549
55.
[33] Rodriguez-Morales AJ, Bonilla-Aldana DK, Bonilla-Aldana JC, Mondragon-Cardona A. Chikungunya and Zika in Huila: mapping their inci-
dence in a neglected area of Colombia. Arch Med Res 2018;49(7):512
13.
22 Pandemic Outbreaks in the 21st Century

[34] Alvarez MF, Bolivar-Mejia A, Rodriguez-Morales AJ, Ramirez-Vallejo E. Cardiovascular involvement and manifestations of systemic
Chikungunya virus infection: a systematic review. F1000Res 2017;6:390.
[35] Escalera-Antezana JP, Murillo-Garcia DR, Gomez C, Unzueta-Quiroga RC, Rodriguez-Morales AJ. Chikungunya in Bolivia: domestic imported
case series in Cochabamba. J Formos Med Assoc 2018;117(12):1133
4.
[36] Zambrano LI, Sierra M, Lara B, Rodriguez-Nunez I, Medina MT, Lozada-Riascos CO, et al. Estimating and mapping the incidence of dengue
and chikungunya in Honduras during 2015 using Geographic Information Systems (GIS). J Infect Public Health 2017;10(4):446
56.
[37] Rodriguez-Morales AJ, editor. Letter to the editor: chikungunya virus infection-an update on chronic rheumatism in Latin America. Rambam
Maimonides Med J 2017;8:1.
[38] Bonilla-Aldana DK, Bonilla-Aldana JL, Garcia-Bustos JJ, Lozada CO, Rodriguez-Morales AJ. Geographical trends of chikungunya and Zika in
the Colombian Amazonian gateway department, Caqueta, 2015
2018—implications for public health and travel medicine. Travel Med Infect
Dis 2020;35:101481.
[39] Cabrera M, Cordova-Lepe F, Valero-Cedeno N, Reyes-Baque J, Rodriguez-Morales AJ. Chikungunya in Ecuador, 2014
2017: maps and more.
Travel Med Infect Dis 2019;29:63
6.
[40] Cardona-Ospina JA, Jimenez-Canizales CE, Vasquez-Serna H, Garzon-Ramirez JA, Alarcon-Robayo JF, Ceron-Pineda JA, et al. Fatal dengue,
chikungunya and leptospirosis: the importance of assessing co-infections in febrile patients in tropical areas. Trop Med Infect Dis 2018;
3(4):123.
[41] Blohm GM, Lednicky JA, Marquez M, White SK, Loeb JC, Pacheco CA, et al. Evidence for mother-to-child transmission of Zika virus through
breast milk. Clin Infect Dis 2018;66(7):1120
1.
[42] Blohm GM, Lednicky JA, Marquez M, White SK, Loeb JC, Pacheco CA, et al. Complete genome sequences of identical Zika virus isolates in a
nursing mother and her infant. Genome Announc 2017;5(17):e00231-17.
[43] Cardona-Ospina JA, Henao-SanMartin V, Acevedo-Mendoza WF, Nasner-Posso KM, Martinez-Pulgarin DF, Restrepo-Lopez A, et al. Fatal
Zika virus infection in the Americas: a systematic review. Int J Infect Dis 2019;88:49
59.
[44] Villamil-Gomez WE, Sanchez-Herrera AR, Hernandez-Prado H, Hernandez-Iriarte J, Diaz-Ricardo K, Vergara-Serpa O, et al. Zika virus and
HIV co-infection in five patients from two areas of Colombia. J Formos Med Assoc 2018;117(9):856
8.
[45] Alvarado-Socarras JL, Aux-Cadena CP, Murillo-Garcia DR, Rodriguez-Morales AJ. Ophthalmologic evaluation in infants of mothers with
Zika: a report from Colombia. Travel Med Infect Dis 2019.
[46] Alvarado-Socarras JL, Idrovo AJ, Contreras-Garcia GA, Rodriguez-Morales AJ, Audcent TA, Mogollon-Mendoza AC, et al. Congenital micro-
cephaly: a diagnostic challenge during Zika epidemics. Travel Med Infect Dis 2018;23:14
20.
[47] Rodriguez-Morales AJ, Cardona-Ospina JA, Ramirez-Jaramillo V, Gaviria JA, Gonzalez-Moreno GM, Castrillon-Spitia JD, et al. Diagnosis and
outcomes of pregnant women with Zika virus infection in two municipalities of Risaralda, Colombia: Second report of the ZIKERNCOL study.
Travel Med Infect Dis 2018;25:20
5.
[48] Villamil-Gomez WE, Sanchez-Herrera AR, Hernandez H, Hernandez-Iriarte J, Diaz-Ricardo K, Castellanos J, et al. Guillain-Barre syndrome
during the Zika virus outbreak in Sucre, Colombia, 2016. Travel Med Infect Dis 2017;16:62
3.
[49] Zambrano LI, Vasquez-Bonilla WO, Fuentes-Barahona IC, Claudio da Silva J, Valle-Reconco JA, Medina MT, et al. Spatial distribution of
Zika in Honduras during 2016
2017 using geographic information systems (GIS)—implications for public health and travel medicine. Travel
Med Infect Dis 2019;31:101382.
[50] Millan-Onate J, Millan W, Mendoza LA, Sanchez CG, Fernandez-Suarez H, Bonilla-Aldana DK, et al. Successful recovery of COVID-19 pneu-
monia in a patient from Colombia after receiving chloroquine and clarithromycin. Ann Clin Microbiol Antimicrob 2020;19(1):16.
[51] Cimerman S, Chebabo A, Cunha CAD, Rodriguez-Morales AJ. Deep impact of COVID-19 in the healthcare of Latin America: the case of
Brazil. Braz J Infect Dis 2020;24(2):93
5.
[52] Singh RK, Rani M, Bhagavathula AS, Sah R, Rodriguez-Morales AJ, Kalita H, et al. Prediction of the COVID-19 pandemic for the top 15
affected countries: Advanced Autoregressive Integrated Moving Average (ARIMA) model. JMIR Public Health Surveill 2020;6(2):e19115.
[53] Diaz-Guio DA, Villamil-Gomez WE, Dajud L, Perez-Diaz CE, Bonilla-Aldana DK, Mondragon-Cardona A, et al. Will the Colombian intensive
care units collapse due to the COVID-19 pandemic? Travel Med Infect Dis 2020;38:101746.
[54] Callejas D, Echevarria JM, Carrero Y, Rodriguez-Morales AJ, Moreira R. The SARS-CoV-2 pandemic in Latin America: the need for multidis-
ciplinary approaches. Curr Trop Med Rep 2020;1
6.
[55] Rodriguez-Morales AJ, Balbin-Ramon GJ, Rabaan AA, Sah R, Dhama K, Paniz-Mondolfi A, et al. Genomic epidemiology and its importance
in the study of the COVID-19 pandemic. Infez Med 2020;28(2):139
42.
[56] Rabaan AA, Al-Ahmed SH, Sah R, Al-Tawfiq JA, Haque S, Harapan H, et al. Genomic epidemiology and recent update on nucleic acid-based
diagnostics for COVID-19. Curr Trop Med Rep 2020;1
7.
[57] Rodriguez-Morales AJ, Rodriguez-Morales AG, Mendez CA, Hernandez-Botero S. Tracing new clinical manifestations in patients with
COVID-19 in Chile and its potential relationship with the SARS-CoV-2 divergence. Curr Trop Med Rep 2020;1
4.
[58] Ramirez JD, Florez C, Munoz M, Hernandez C, Castillo A, Gomez S, et al. The arrival and spread of SARS-CoV-2 in Colombia. J Med Virol
2021;93(2):1158
63.
[59] Ramirez JD, Sordillo EM, Gotuzzo E, Zavaleta C, Caplivski D, Navarro JC, et al. SARS-CoV-2 in the Amazon region: a harbinger of doom for
Amerindians. PLoS Negl Trop Dis 2020;14(10):e0008686.
[60] Paniz-Mondolfi A, Munoz M, Florez C, Gomez S, Rico A, Pardo L, et al. SARS-CoV-2 spread across the Colombian-Venezuelan border. Infect
Genet Evol 2020;86:104616.
 Epidemiology of COVID-19 in Latin America Chapter | 2 23

[61] Laiton-Donato K, Villabona-Arenas CJ, Usme-Ciro JA, Franco-Munoz C, Alvarez-Diaz DA, Villabona-Arenas LS, et al. Genomic epidemiol-
ogy of severe acute respiratory syndrome Coronavirus 2, Colombia. Emerg Infect Dis 2020;26(12):2854
62.
[62] Fontanet A, Autran B, Lina B, Kieny MP, Karim SSA, Sridhar D. SARS-CoV-2 variants and ending the COVID-19 pandemic. Lancet
2021;397(10278):952
4.
[63] Volz E, Mishra S, Chand M, Barrett JC, Johnson R, Geidelberg L, et al. Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: insights
from linking epidemiological and genetic data. medRxiv 2021; 2020.12.30.20249034.
[64] Tegally H, Wilkinson E, Giovanetti M, Iranzadeh A, Fonseca V, Giandhari J, et al. Emergence and rapid spread of a new severe acute respiratory
syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv 2020; 2020.12.21.20248640.
[65] Schlagenhauf P, Patel D, Rodriguez-Morales A, Gautret P, Grobusch MP, Leder K. Variants, vaccines and vaccination passports: challenges
and chances for travel medicine in 2021. Travel Med Infect Dis 2021;40:101996.
[66] Cardona-Ospina JA, Arteaga-Livias K, Villamil-Gomez WE, Perez-Diaz CE, Katterine Bonilla-Aldana D, Mondragon-Cardona A, et al.
Dengue and COVID-19, overlapping epidemics? An analysis from Colombia. J Med Virol 2020.
[67] Haqqi A, Awan UA, Ali M, Saqib MAN, Ahmed H, Afzal MS. COVID-19 and dengue virus coepidemics in Pakistan: a dangerous combination
for an overburdened healthcare system. J Med Virol 2020;93(1):80
2.
[68] Epelboin L, Blonde R, Nacher M, Combe P, Collet L. COVID-19 and dengue co-infection in a returning traveller. J Travel Med 2020;27:6.
[69] Lorenz C, Azevedo TS, Chiaravalloti-Neto F. COVID-19 and dengue fever: a dangerous combination for the health system in Brazil. Travel
Med Infect Dis 2020;35:101659.
[70] Navarro JC, Arrivillaga-Henriquez J, Salazar-Loor J, Rodriguez-Morales AJ. COVID-19 and dengue, co-epidemics in Ecuador and other coun-
tries in Latin America: pushing strained health care systems over the edge. Travel Med Infect Dis 2020;37:101656.
[71] Vasquez-Chavesta AZ, Moran-Marinos C, Rodrigo-Gallardo PK, Toro-Huamanchumo CJ. COVID-19 and dengue: pushing the peruvian health
care system over the edge. Travel Med Infect Dis 2020;36:101808.
[72] Arteaga-Livias K, Panduro-Correa V, Pinzas-Acosta K, Perez-Abad L, Pecho-Silva S, Espinoza-Sanchez F, et al. COVID-19 reinfection? a sus-
pected case in a Peruvian patient. Travel Med Infect Dis 2020;39:101947.
[73] European Centre for Disease Prevention and Control. Reinfection with SARS-CoV: considerations for public health response. ECDC; 2020.
[74] Tillett RL, Sevinsky JR, Hartley PD, Kerwin H, Crawford N, Gorzalski A, et al. Genomic evidence for reinfection with SARS-CoV-2: a case
study. Lancet Infect Dis 2021;21(1):52
8.
[75] Bao L, Deng W, Gao H, Xiao C, Liu J, Xue J, et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. bioRxiv 2020;
2020.03.13.990226.
[76] Gupta V, Bhoyar RC, Jain A, Srivastava S, Upadhayay R, Imran M, et al. Asymptomatic reinfection in two healthcare workers from India with
genetically distinct SARS-CoV-2. Clin Infect Dis 2020.
[77] Van Elslande J, Vermeersch P, Vandervoort K, Wawina-Bokalanga T, Vanmechelen B, Wollants E, et al. Symptomatic SARS-CoV-2 reinfec-
tion by a phylogenetically distinct strain. Clin Infect Dis 2020.
[78] Rodriguez-Morales AJ, Cardona-Ospina JA, Villamil-Gómez WE. Should we concern about reinfection in COVID-19? Infectio 2020;25
(2):77
8.
[79] Kumar A, Shiwalkar N, Shaikh JD, Kaur R, Vommaro AFL, Persaud P, et al. Reinfection after SARS-CoV2 infection: a looming concern. J
Exp Biol Agric Sci 2020;8:S114
18.
[80] Goldman JD, Wang K, Roltgen K, Nielsen SCA, Roach JC, Naccache SN, et al. Reinfection with SARS-CoV-2 and failure of humoral immu-
nity: a case report. medRxiv 2020; 2020.09.22.20192443.
[81] Osman AA, Al Daajani MM, Alsahafi AJ. Re-positive coronavirus disease 2019 PCR test: could it be a reinfection? New Microbes New Infect
2020;37:100748.
[82] Bonifacio LP, Pereira APS, Araujo D, Balbao V, Fonseca B, Passos ADC, et al. Are SARS-CoV-2 reinfection and Covid-19 recurrence possi-
ble? a case report from Brazil. Rev Soc Bras Med Trop 2020;53:e20200619.
[83] Deng W, Bao L, Liu J, Xiao C, Liu J, Xue J, et al. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science
2020;369(6505):818
23.
[84] Alvarez-Moreno CA, Rodriguez-Morales AJ. Testing dilemmas: post negative, positive SARS-CoV-2 RT-PCR—is it a reinfection? Travel Med
Infect Dis 2020;35:101743.
[85] Henriquez-Marquez KI, Lainez-Murillo DC, Sierra M, Munoz-Lara F, Valenzuela-Rodriguez G, Pecho-Silva S, et al. High impact of SARS-
CoV-2 or COVID-19 in the Honduran health personnel. J Med Virol 2020;93(4):1885
7.
[86] Valenzuela-Rodriguez G, Zambrano LI, Munoz-Lara F, Pecho-Silva S, Arteaga-Livias K, Rodriguez-Morales AJ. Intranational differences in
the case fatality rates for COVID-19 among Peruvian physicians. Int J Infect Dis 2020;101:226
7.
[87] Erdem H, Lucey DR. Healthcare worker infections and deaths due to COVID-19: a survey from 37 nations and a call for WHO to post national
data on their website. Int J Infect Dis 2021;102:239
41.
[88] Sharun K, Dhama K, Patel SK, Pathak M, Tiwari R, Singh BR, et al. Ivermectin, a new candidate therapeutic against SARS-CoV-2/COVID-19.
Ann Clin Microbiol Antimicrob 2020;19(1):23.
[89] Rabaan AA, Al-Ahmed SH, Sah R, Tiwari R, Yatoo MI, Patel SK, et al. SARS-CoV-2/COVID-19 and advances in developing potential thera-
peutics and vaccines to counter this emerging pandemic. Ann Clin Microbiol Antimicrob 2020;19(1):40.
[90] Hernandez AV, Roman YM, Pasupuleti V, Barboza JJ, White CM. Hydroxychloroquine or chloroquine for treatment or prophylaxis of COVID-
19: a living systematic review. Ann Intern Med 2020;173(4):287
96.
24 Pandemic Outbreaks in the 21st Century

[91] Hernandez AV, Roman YM, Pasupuleti V, Barboza JJ, White CM. Update alert 2: hydroxychloroquine or chloroquine for the treatment or pro-
phylaxis of COVID-19. Ann Intern Med 2020;173(7):W128
9.
[92] Hernandez AV, Roman YM, Pasupuleti V, Barboza JJ, White CM. Update alert 3: hydroxychloroquine or chloroquine for the treatment or pro-
phylaxis of COVID-19. Ann Intern Med 2020;173(11):W156
7.
[93] Hernandez AV, Roman YM, Pasupuleti V, Barboza JJ, White CM. Update alert: hydroxychloroquine or chloroquine for the treatment or pro-
phylaxis of COVID-19. Ann Intern Med 2020;173(4):W78
9.
[94] Smith BC, Vigotsky AD, Apkarian AV, Schnitzer TJ. Temporal factors associated with opioid prescriptions for patients with pain conditions
in an urban emergency department. JAMA Netw Open 2020;3(3):e200802.
[95] Patel SK, Pathak M, Tiwari R, Yatoo MI, Malik YS, Sah R, et al. A vaccine is not too far for COVID-19. J Infect Dev Ctries 2020;14(5):
450
3.
[96] Sah R, Shrestha S, Mehta R, Sah SK, Raaban AA, Dharma K, et al. AZD1222 (Covishield) vaccination for COVID-19: experiences, chal-
lenges, and solutions in Nepal. Travel Med Infect Dis 2021;40:101989.
[97] Petersen E, Lucey D, Blumberg L, Kramer LD, Al-Abri S, Lee SS, et al. COVID-19 vaccines under the international health regulations—we
must use the WHO international certificate of vaccination or prophylaxis. Int J Infect Dis 2021;104:175
7.
[98] Rodriguez-Morales AJ, Sah R, Paniz-Mondolfi A. Should the Holy Week 2020 be cancelled in Latin America due to the COVID-19 pan-
demic? Travel Med Infect Dis 2020;36:101633.
[99] Bonilla-Aldana DK, Suarez JA, Franco-Paredes C, Vilcarromero S, Mattar S, Gomez-Marin JE, et al. Brazil burning! What is the potential
impact of the Amazon wildfires on vector-borne and zoonotic emerging diseases?—a statement from an international experts meeting. Travel
Med Infect Dis 2019;31:101474.
[100] Ahmad T, Khan M, Haroon, Musa TH, Nasir S, Hui J, et al. COVID-19: zoonotic aspects. Travel Med Infect Dis 2020;36:101607.
[101] Rodriguez-Morales AJ, Bonilla-Aldana DK, Balbin-Ramon GJ, Rabaan AA, Sah R, Paniz-Mondolfi A, et al. History is repeating itself: proba-
ble zoonotic spillover as the cause of the 2019 novel coronavirus epidemic. Infez Med 2020;28(1):3
5.
[102] Bonilla-Aldana DK, Jimenez-Diaz SD, Patel SK, Dhama K, Rabaan A, Sah R, et al. Importance of bats in wildlife: not just carriers of pan-
demic SARS-CoV-2 and other viruses. J Pure Appl Microbiol 2020;14(suppl 1):709
12.
[103] Bonilla-Aldana DK, Holguin-Rivera Y, Perez-Vargas S, Trejos-Mendoza AE, Balbin-Ramon GJ, Dhama K, et al. Importance of the One
Health approach to study the SARS-CoV-2 in Latin America. One Health 2020;10:100147.
[104] Tiwari R, Dhama K, Sharun K, Iqbal Yatoo M, Malik YS, Singh R, et al. COVID-19: animals, veterinary and zoonotic links. Vet Q 2020;
40(1):169
82.
[105] Rodriguez-Morales AJ, Dhama K, Sharun K, Tiwari R, Bonilla-Aldana DK. Susceptibility of felids to coronaviruses. Vet Rec 2020;186(17):e21.
Chapter 3

Biology, prevention, and treatment of

SARS-CoV-2 (COVID-19)
Kalanghad P. Srinivas
Department of Microbiology, NYU School of Medicine, Alexandria Center for Life Sciences, New York, NY, United States

3.1 Introduction
In December 2019 a cluster of pneumonia cases were epidemiologically linked to an open-air live animal market in the
city of Wuhan (Hubei Province), China [1
3], which led local health officials to issue an epidemiological alert to the
Chinese Center for Disease Control and Prevention and the World Health Organization’s (WHO) China Country Office.
COVID-19 has rapidly spread across all continents and turned into a public health emergency that was ultimately
declared as a pandemic by the WHO in early 2020. In early January 2020 the etiological agent responsible for the pneu-
monia cases was found to be a coronavirus initially referred to as COVID-19 or novel Coronavirus (2019-nCoV) [4],
was subsequently named SARS-CoV-2 by an International Committee on Taxonomy of Viruses (ICTV) Study Group
[5]. As it is a new virus, scientists are learning more about it each day. The first available sequence data placed this
novel human pathogen in the Sarbecovirus subgenus of Coronaviridae, the same subgenus as the SARS virus that
caused a global outbreak of .8000 cases in 2002
03. Although most people who have COVID-19 have mild symp-
toms, some groups such as older adults and people who have certain underlying medical conditions are at increased risk
of severe illness and even death [6].
Coronaviruses, named for the crown-like spikes on their surfaces, are a large family of viruses that are common in
people and many different species of animals, including camels, cattle, cats, and bats. There are many types of human
coronaviruses, including some that commonly cause mild upper-respiratory tract illnesses. COVID-19 is a new disease,
caused by a novel (or new) coronavirus that has not previously been seen in humans.
As on January 5, 2021, the cumulative number of COVID-19 cases Worldwide tally over 83 million reported cases
and over 1.8 million deaths since the start of the pandemic. The total number of confirmed cases in India is about 10
million with more than 150,000 deaths. In the United States, the confirmed number of cases stands around 21 million
cases with more than 400,000 deaths, while in Europe the numbers stand around more than 26 million confirmed cases
with more than 500,000 deaths (CDC, 2021; ECDC, 2021; WHO, 2021). Recent reports of different variants of SARS-
CoV-2 have raised concern about and interest in the impact of viral changes. As of January 5, 2021, the VOC-202012/
01 variant initially detected in the United Kingdom has been detected in a small number of cases in 40 other countries/
territories/areas in five of the six WHO regions, and the 501Y.V2 variant initially detected in South African in six other
countries/territories/areas.

3.2 Origin of SARS-CoV-2

Since the first reports of novel coronavirus (COVID-19) from Wuhan, Hubei province, China [1,7
9], there has been
considerable debate and question on the origin and evolution of the causative virus, SARS-CoV-2.
Animal coronaviruses rarely infect people and then spread between people. But this occurred with two corona-
viruses, MERS-CoV and SARS-CoV in recent years. SARS-CoV-2 is a beta coronavirus, like MERS-CoV and SARS-
CoV. All three of these viruses have their origins in bats. The sequences reported from the US patients are quite like
the one initially reported from China, suggesting a likely single, recent emergence of the virus from a possible animal
reservoir. However, the exact source of this virus has not been identified. There are some outstanding evolutionary

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00008-2

© 2021 Elsevier Inc. All rights reserved. 25
26 Pandemic Outbreaks in the 21st Century

questions on the recent emergence of SARS-CoV-2 such as the role of reservoir species, the role of recombination, and
its time of divergence from animal viruses. Subgenus sarbecoviruses containing SARS-CoV and SARS-CoV-2 were
shown to undergo frequent recombination and also shown to exhibit structured genetic diversity on a regional scale in
China. Various scientific studies indicate that the SARS-CoV-2 has emerged from the same horseshoe bat subgenus
that harbors SARS-like coronaviruses. The similarity between SARS-CoV and SARS-CoV-2 is their divergence time
(40
70 years ago) from currently known extant bat virus lineages such as RaTG13 [10]. SARS-CoV-2’s receptor-
binding motif (RBD motif), vital for specificity to human angiotensin-converting enzyme 2 (ACE2) receptors, appears
to be an ancestral trait shared with bat viruses and not one acquired recently via recombination, and SARS-CoV-2 have
been shown to be circulating in horseshoe bats for many decades [10].
Strong purifying selection around the receptor-binding motif (RBD motif) in the spike and other genes among bat,
pangolin, and human coronaviruses was shown to have a similar evolutionary constraint in different host species.
Studies also claim that the SARS-CoV-2’s entire RBM was introduced through recombination with coronaviruses from
pangolins, a possible critical step in the evolution of SARS-CoV-2’s ability to infect humans. Similar selection in differ-
ent host species, together with frequent recombination among coronaviruses, suggests a common evolutionary mecha-
nism that could result in more new emerging human coronaviruses [11].

3.3 SARS-CoV-2 evolution and genomic analyses

The SARS-CoV-2 is in the same species as SARS-CoV and the two viruses are 94.6% similar at amino acid sequence
level (80% nucleotide sequence similarity) and B50% with MERS-CoV [12,13]. The Coronavirus Study Group (CSG)
of the ICTV officially named the novel coronavirus as SARS-CoV-2 in February 2020, after analyzing viral genomes
from several patients and assessing the phylogenetic (evolutionary) relationships between the new virus and already
known coronaviruses. The committee found that the genome of viruses isolated from patients was similar enough to
SARS genomes to be considered a variant of SARS, not an entirely novel virus. Even though the clinical manifesta-
tions, epidemiological pattern, and host range of SARS-CoV-2 differ from the SARS-CoV, it is the genetic similarity,
the reason they concluded these two viruses as severe acute respiratory syndrome-related coronaviruses and for this rea-
son, CSG named them as SARS-CoV and SARS-CoV-2, variants of the same species (SARS-CoV-2 Genetics, Johns
Hopkins Center for Health Security 2020, centerforhealthsecurity.org).
The mutation rate of SARS-CoV-2 has been estimated in various groups, ranging from about 1.05 3 10
3 to
1.26 3 10
3 substitutions per site per year, which is similar to some estimates of MERS-CoV mutation rates [14
16].
Analyzing 220 genomic sequences from the GISAID database revealed that the number and the occurrence of mutations
are significantly higher in Europe, and North America compared to Asia suggesting a probably different mutation pat-
tern. The distribution of SARS-CoV-2 mutation patterns shows a difference in time, geography, and age, but the simi-
larity in gender. However, a recent study on SARS-CoV-2 mutations in the United States revealed that the genome
samples isolated from women patients have shown a higher mutation rate compared to those isolated from men.
Likewise, a female-dominated pattern was seen in mutation 27964C . T (S24L) on the ORF8 protein. Considering the
role of mutation as one of the most important mechanism of evolution in RNA viruses, several studies have detected
genomic variations of SARS-CoV-2, which resulted in finding rich genetic variations of different types, including mis-
sense, synonymous, insertion, deletion, and noncoding mutations. Reportedly, missense and synonymous mutations
were the most frequent type of mutations along the SARS-CoV-2 genome [17]. Selection analysis of the SARS-CoV-2
genome suggests that two genes, S and N, are under episodic selection as the virus is transmitted between humans,
which is normal for emerging viruses and means that parts of the genome are undergoing positive selection [18,19].
Mutations and adaptation in the S and N genes could affect virus stability and pathogenicity [14]. Analysis of the
genome sequence diversity across samples has revealed the highest diversity occurring in the structural genes, espe-
cially the S protein, ORF3a, and ORF8 (SARS-CoV-2 Genetics, Johns Hopkins Center for Health Security 2020, center-
forhealthsecurity.org). Toyoshima et al. [20] comprehensively investigated 12,343 SARS-CoV-2 genome sequences
isolated from patients in six geographic areas and identified a total of 1234 mutations in comparison with the reference
SARS-CoV-2 sequence and through a hierarchical clustering based on the mutant frequencies, they classified the 28
countries into three clusters showing different fatality rates of COVID-19 and identified ORF1ab 4715L and S protein
614G variants showed significant positive correlations with fatality rates. The genomic analysis of the SARS-CoV-2 in
several studies has reported mutations in various genes, including ORF1ab, ORF3a, ORF6, ORF7, ORF8, ORF10, S,
M, E, and N [17]. Among them, nsp1, nsp2nsp3, nsp12, and nsp15of ORF1ab, S, as well as ORF8 genes were reported
to have remarkably more mutations than other genes [17,21,22].
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 27

The S gene of SARS-CoV-2 is highly variable from SARS-CoV, sharing ,75% nucleotide identity [12,13,23].
Comparative sequence analysis of the SARS-CoV-2 genome indicates striking similarities to the BAT-CoV, suggesting
a possible mammalian origin from bats in the Wuhan city of China [23]. However, there is evidence suggesting bats as
the natural reservoirs of SARS-like CoVs, including the SARS-CoV-2 [24
26]. SARS-CoV-2 is most closely related,
by average genetic distance, to two coronaviruses isolated from bats, RaTG13 and RmYN02. However, there is a seg-
ment of high amino acid similarity between human SARS-CoV-2 and a pangolin-isolated strain, GD410721, in the
receptor-binding domain (RBD) of the spike protein, a pattern that can be caused by either recombination or by conver-
gent amino acid evolution driven by natural selection [27,28].
A recent study proposed that SARS-CoV-2 can be divided into two major lineages (L and S) and intriguingly, the S
and L lineages can be clearly defined by just two tightly linked SNPs at positions 8782 (orf1ab: T8517C, synonymous)
and 28,144 (ORF8: C251T, S84L) that show complete linkage across SARS-CoV-2 strains. Orf1ab, which encodes rep-
licase/transcriptase, is required for viral genome replication and might also be important for viral pathogenesis.
Although the T8517C mutation in orf1ab does not change the protein sequence [it changes the codon AGT (Ser) to
AGC (Ser)], it may affect orf1ab translation since AGT is preferred than AGC. ORF8 promotes the expression of
ATF6, the endoplasmic reticulum (ER) unfolded protein response factor, in human cells. They found the L lineage
(B70%) to be more prevalent than the S lineage (B30%) and suggested the S lineage appeared to be more related to
coronaviruses in animals [27].

3.4 Molecular biology of SARS-CoV-2

SARS-CoV-2 is an enveloped positive-sense single-stranded RNA containing virus with a genome size of B30 kb. The
genome contains 38% of the GC content and the genetic arrangement of open reading frames (ORFs) highly resembles
the SARS-CoV and MERS-CoV. Its genome comprises 12
15 ORFs, two-thirds of which encode 16 nonstructural pro-
teins (nsp 1
16) (Table 3.1) such as 3-chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), helicase,
and RNA-dependent RNA polymerase (RdRp) and 15 of these nsps make up the replicase and transcription complex
(RTC) [13,23]. The remaining one-third encodes nine accessory proteins (ORF) and four structural proteins: spike (S),
envelope (E), membrane (M), and nucleocapsid (N), which are needed to produce a structurally complete viral particle.
Interspersed among the structural genes, the 30 -end of the genome contains nine putative ORFs for accessory factors
[7
9] (Fig. 3.1). The nucleotide content of the viral genome is held mainly by ORF1a and ORF1ab that encode poly-
proteins pp1a and pp1ab, where polyprotein pp1ab is encoded by the ribosomal frameshift mechanism of the gene 1b.
These polyproteins are further processed by virally encoded proteinases and produce 16 proteins, which are well con-
served in all CoVs belonging to the same family.
The spike protein mediates cell entry and it is highly variable in comparison to the SARS-CoV spike protein
[12,13,23]. The differential host cell tropism, replication kinetics, and transmission of SARS-CoV-2 might be deter-
mined by S protein and ACE2 binding affinities. The SARS-CoV-2 spike protein has a RBD that mediates direct con-
tact with a cellular receptor, ACE2, and an S1/S2 polybasic cleavage site that is proteolytically cleaved by cellular
cathepsin L/furin and the transmembrane protease serine 2 (TMPRSS2) [29
31]. A peculiar feature of the SARS-CoV-
2 S protein is the acquisition of a polybasic cleavage site (PRRAR) at the S1
S2 boundary, which permits efficient
cleavage by the prototype proprotein convertase furin. Cleavage results in enhanced infection and has been proposed to
be a key event in SARS-CoV-2 evolution as efficient S protein cleavage is required for successful infection and is the
main determinant in overcoming species barriers [32
34]. This preprocessing of the SARS-CoV-2 S protein by furin
may contribute to the expanded cell tropism, zoonotic potential, and might increase transmissibility. Such cleavage sites
have not been identified in other members of the Sarbecovirus genus. However, there are multiple instances of furin-
like cleavage site acquisitions during coronavirus evolution and similar cleavage sites are identified in other human
coronaviruses such as HCoV-HKU1, HCoV-OC43, and MERS-CoV49 [30]. Recently, an independent insertion of
amino acids (PAA) at the same region of the S protein has been identified in the bat coronavirus RmYN02. These inde-
pendent insertion events highlight the zoonotic potential of bat coronaviruses and may increase the possibility of future
outbreaks [30]. TMPRSS2 facilitates viral entry at the plasma membrane surface, whereas cathepsin L activates
SARS-CoV-2 Spike in endosomes and can compensate for entry into cells that lack TMPRSS2 [30]. Various single-cell
transcriptomic studies have also shown that ACE2 expression is highly correlated with the expression of alanyl amino-
peptidase and dipeptidyl peptidase-4 that are shown to be receptors for other human CoVs [35
37]. This suggests that
these peptidases may act as co-receptors or auxiliary SARS-CoV-2 receptors [38]. Furthermore, the identification of the
transmembrane glycoprotein CD147 [39,40], as well as the presence of furin-like cleavage sites in the spike (S) protein
 TABLE 3.1 Functional aspects of SARS-CoV-2 nonstructural proteins (Nsp’s).

SARS-CoV-2 Description Proposed function

Nsp’s
Nsp1 N-terminal product of the viral replicase Leader protein host translation inhibitor. Mediates RNA replication
and processing. Involved in mRNA degradation
Nsp2 Replicase product essential for Modulation of host cell survival signaling pathway by interacting with
proofreading viral replication host PHB and PHB2
Nsp3 Papain-like proteinase contains several Functions as a protease to separate the translated polyprotein into its
domains distinct proteins
Nsp4 A membrane-spanning protein contains Believed to anchor the viral replication
transcription complex to
transmembrane domain 2 (TM2) modified ER membranes
Nsp5 3C-like proteinase and main proteinase Involved in viral polyprotein processing during replication
Nsp6 Putative transmembrane domain Plays a role in the initial induction of autophagosomes from host
endoplasmic reticulum
Nsp7 Nsp7 is an RNA-dependent RNA Forms a hexadecameric super-complex with nsp8 that adopts a
polymerase hollow cylinder-like structure implicated in replication
Nsp8 Multimeric RNA polymerase; replicase Forms a hexadecameric super-complex with nsp7 that adopts a
hollow cylinder-like structure implicated in replication
Nsp9 Single-stranded RNA-binding viral protein Participate in viral replication by acting as an ssRNA-binding protein
Nsp10 Growth-factor-like protein containing two Plays an essential role in viral mRNAs cap methylation by stimulating
zinc-binding motifs both nsp14 30 -50 exoribonuclease and nsp16 20 -O-methyltransferase
activities
Nsp11 Made of 13 amino acids (sadaqsflngfav) Unknown
and identical to the first segment of
Nsp12.
Nsp12 RNA-dependent RNA polymerase (RdRp) Responsible for replication and transcription of the viral RNA genome
Nsp13 Zinc-binding domain, NTPase/helicase A helicase core domain that binds ATP. Zinc-binding domain is
domain, RNA 50 -triphosphatase involved in replication and transcription
Nsp14 Proofreading exoribonuclease domain Exoribonuclease activity acting in a 30 to 50 direction and N7-guanine
(ExoN/nsp14) methyltransferase activity
Nsp15 EndoRNAse; nsp15-A1 and nsp15B- Mn(2 1 )-dependent endoribonuclease activity
NendoU Uridylate-specific
endoribonuclease
Nsp16 20 -O-ribose methyltransferase Methyltransferase that mediates mRNA cap 20 -O-ribose methylation
to the 50 -cap structure of viral mRNAs

FIGURE 3.1 (A) Structure of SARS-CoV-2 virion. (B) Genome organization of SARS-CoV-2. The genome is a positive-sense ssRNA of B30 kb in
size and contains two large genes ORF1a and ORF1b that code for 16 nonstructural proteins (Nsp’s), most of which form a replication
transcription
complex (RTC), essential for replication and transcription. The remaining part of the genome contains genes for various subgenomic RNAs (sgRNAs)
that encode structural proteins of the virus.
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 29

(absent for other SARS-CoVs) [41], might be associated to viral
host mechanisms of invasion and pathogenicity of
COVID-19.
Once the genome is released into the host cytosol, ORF1a and ORF1b are translated into viral replicase proteins,
which are cleaved into individual nsps (via host and viral proteases: PLpro); these form the RdRp (nsp12 derived from
ORF1b). Here, the replicase components rearrange the ER into double-membrane vesicles (DMVs) that facilitate viral
replication of genomic and subgenomic RNAs (sgRNA); the latter are translated into accessory and viral structural pro-
teins that facilitate virus particle formation. The viral structural and accessory proteins are translated from a set of
nested sgRNAs, all terminating with the 30 -end of the full-length gRNA, and the generation of these sgRNAs starting
from negative-sense RNA intermediates is regulated by the transcriptional regulatory sequences (TRSs). During minus-
strand RNA synthesis, the viral RNA polymerase pauses at each TRS sequence and this pause is usually resolved either
by continuing the synthesis through the TRS into the adjacent gene or it can lead to the termination of transcription
with the generation of a sgRNA. The exact molecular mechanisms that determine either of the possible outcomes are
yet to be fully revealed [42,43].
Coronaviral RNA is synthesized by the RTC, associated with a complex vesicular network. Such membranous struc-
tures comprise convoluted membranes (CVs) and DMVs, originating from the ER of the infected host cell. These differ-
ent membranous elements correlate with a precise spatial distribution of the different components of the RTC. The viral
replication machinery is anchored to the CVs, thanks to the viral transmembrane proteins Nsp3, Nsp4, and Nsp6, while
the dsRNA originating from the replication
transcription process is mainly contained within the DMVs, they may act
as protective structures to avoid detection of dsRNA by innate immunity sensors, and subsequent degradation [44].
SARS-CoV-2 RTC machinery involves an array of functional proteins from the N- to C-termini of the polyprotein,
PP1ab (Fig. 3.2). These include the essential RNA-dependent RNA polymerase (RdRp, Nsp12), the zinc-binding heli-
case (HEL, Nsp13), and a number of other enzymatic functions related to viral RNA modification, such as messenger
RNA (mRNA) capping (Nsp14, Nsp16), RNA proofreading (Nsp14), and uridylate-specific endoribonuclease activity
(NendoU, Nsp15), which has been shown to counteract double-strand RNA sensing. The activity of these enzymes is

FIGURE 3.2 Mechanism of SARS-CoV-2 induced tissue damage. Once SARS-CoV-2 enters into the respiratory tract, the virus traverses deep into
the lower lung, where it infects a broad range of cells expressing ACE2 including alveolar airway epithelial cells, vascular endothelial cells, and alveo-
lar macrophages. Upon entry, the viral RNA is likely detected by cytosolic innate immune sensors, as well as endosomal TLRs that signal downstream
to produce type-I/III IFNs and pro-inflammatory mediators. The high concentration of inflammatory cytokines/chemokines amplifies the tissue dam-
age via endothelial dysfunction and vasodilation, allowing the recruitment of immune cells such as macrophages and neutrophils. Vascular leakage
and compromised barrier function promote endotheliitis and lung edema, limiting gas exchange that then facilitates a hypoxic environment leading to
respiratory/organ failure. Hyperinflammation in the lung further induces transcriptional changes in macrophages and neutrophils that perpetuate tissue
damage that ultimately leads to irreversible lung damage. The viral infection may also result in systemic inflammation, probably resulting in damage
to other tissues including heart tissues. BALF, Bronchoalveolar lavage fluid; IRF-3, interferon regulatory factor 3; NF-κB, nuclear factor-κB; RIG-I,
retinoic acid-inducible gene I; STAT1/2, signal transducer and activator of transcription 1/2; STING, Stimulator of interferon genes. Adapted from
Harrison AG, Lin T, Wang P. Mechanisms of SARS-CoV-2 transmission and pathogenesis. Trends Immunol 2020; 41(12):100
1115.
30 Pandemic Outbreaks in the 21st Century

further regulated by the association with other nonstructural proteins (Nsp7
Nsp10) that are likely necessary to achieve
all the replication and transcription processes. As observed with other nidoviruses, all of these protein subunits likely
associate in a replication
transcription enzyme complex anchored to membranes derived from the host cell ER [45],
which drives the synthesis of new genome molecules and also subgenomic (sg) mRNAs [46,47].
In addition to the RNA replication functions, these viral proteins are also involved in other activities that are impor-
tant to enhance the efficiency of the whole machinery. Nsp1, a major CoV virulence factor, is involved in the suppres-
sion of host gene expression and in the blockage of innate immune responses in infected host cells [48,49]. The primary
role of the nucleocapsid N protein is to protect the viral genome by packing it into a helical ribonucleocapsid.
Accordingly, the N protein must tightly bind the RNA, even though it is exposed during viral infection, to make it
accessible to the replication machinery. N protein with its C terminus interacts with viral envelope protein M and aids
in the genomic condensation and packaging of the viral particle [46].

3.5 Tissue tropism and molecular pathogenesis of SARS-CoV-2

The establishment of viral tropism depends on the susceptibility and permissiveness of a specific host cell. During the
SARS epidemic, patients often presented with respiratory-like illnesses that progressed to severe pneumonia, observa-
tions mirroring the disease course of COVID-19, suggesting that the lung is the primary tropism for SARS-CoV-2 (JSM
[50]). Both CoVs were then found to bind the same entry receptor, ACE2 [1,51]. The key mutations within the RBD
domain of SARS-CoV-2 Spike protein correlate with its higher binding affinity toward ACE2, and perhaps increased
infectivity [1,51]. The presence of a unique furin cleavage site at the S1/S2 junction of SARS-CoV-2 Spike is also pre-
sumed to enhance human transmission events, although this remains to be further investigated [29
31]. The currently
predominant SARS-CoV-2 isolate circulating worldwide has a D614G mutation that is absent from its presumptive com-
mon ancestor, and is more infectious, likely underlying, in part, an increased human-to-human transmission efficiency
[52,53]. Although associated with an increased viral load in the upper-respiratory tract (URT) of patients with COVID-
19, the D614G variant does not correlate with disease severity, suggesting that pathogenesis of severe COVID-19 is
linked to mechanisms that are more than just SARS-CoV-2 infectivity [54]. The expression of ACE2 has been identified
in the lungs, intestines, cardiovascular (CV) tissues, brain, and kidneys [55]; these organs have been coincidentally tar-
geted by SARS-CoV-2 to further activate intracellular signaling pathways leading to cytokine release syndrome (CRS).

3.6 Pulmonary system

The main clinical complication due to COVID-19, which also leads to a high fatality rate and affects B42% of the
patients, is attributed to the acute respiratory distress syndrome (ARDS), which is a life-threatening lung injury, that
allows fluid to leak into the lungs as a result of which breathing becomes difficult and oxygen cannot get into the body
because of severe hypoxemia. Pathophysiological features include (1) the infiltration and aberrantly enrichment of
active immune cells (especially neutrophils and mononuclear cells) and platelets, leading to a hyperinflammatory state
(generating vascular permeability and diffuse alveolar damage) and (2) hypercoagulation, resulting in disseminated
microvascular coagulation. In COVID-19-associated ARDS the cytokine storm consisting of high serum levels of IL-
1β, TNF, and IL-6 is possibly responsible for severe plasma leakage, vascular permeability, and disseminated coagula-
tion and thrombosis.
Once SARS-CoVs enter the host via the respiratory tract, the airway and alveolar epithelial cells, vascular endothe-
lial cells and alveolar macrophages are their first targets of viral entry and are probably “ground-zero” for early infec-
tion and subsequent replication due to their expression of ACE2 [56
59]. Although ACE2 mRNA is detected in human
and many mammalian (bat, ferret, cat, dog, etc.) lungs, their expression is rather low compared with extrapulmonary tis-
sues. Thus, the permissiveness of these cells to SARS-CoVs may depend on additional, unappreciated cell-intrinsic fac-
tors that aid in an efficient infection. First, viral entry may heavily depend on the expression of TMPRSS2, because
cells with nearly undetectable amounts of ACE2 still support SARS-CoV entry as long as TMPRSS2 is present, and
second, the mRNA expression of cellular genes, such as endosomal sorting complex required for transport machinery
gene members (including CHMP3, CHMP5, CHMP1A, and VPS37B) related to a pro-SARS-CoV-2 lifecycle is higher
in a small population of human type II alveolar cells with abundant ACE2, relative to ACE2-deficient cells [60,61].
This suggests that SARS-CoV-2 hijacks a small population of type II alveolar cells with high expression of ACE2 and
other proviral genes for its productive replication. Third, the lung, as the main tropism of SARS-CoVs, may be contin-
gent on the regulation of ACE2 at the transcriptional and protein levels [57,59,62,63]. For example, in human airway
epithelial cells, ACE2 gene expression is upregulated by type-I and -II interferons (IFNs) during viral infection. Lastly,
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 31

compared with other SARS-CoVs, SARS-CoV-2 Spike contains a unique insertion of RRAR at the S1/S2 cleavage site
and this site can be precleaved by furin, thus reducing the dependence of SARS-CoV-2 on target cell proteases
(TMPRSS2/cathepsin L) for its entry and potentially extending its cellular tropism, given that proteolytically active fur-
in is abundantly expressed in human bronchial epithelial cells [54,64]. These cell types express molecules capable of
recognizing pathogen-associated molecular patterns such as TLR 3 and TLR 7/8 that recognize viral nucleic acids and
result in induced expression of type-I IFN genes. TLR 3 and TLR 7/8 overlay their activation through the adaptor pro-
teins TRIF and MyD88, which lead to the recruitment and nuclear import of IFN regulatory factors (IRFs) 3/7. IRFs
3/7 act as transcription factors to induce expression of IFNs (subtypes α, β, and γ), which are major inducers of antivi-
ral response. The recruitment of TRIF and MyD88 by TLR 3 and TLR 7/8 also activates TRAF6 and TAK1, which
results in downstream activation of the IKK complex, further phosphorylation and degradation of IκB, and finally,
enabling NF-kB nuclear translocation. The great importance of NF-κB toward pro-inflammatory gene expression in the
lungs is highlighted in a few studies exploring models of coronavirus infection. The NF-κB activation in Calu-3 human
bronchial epithelial cells, in response to SARS infection, can mediate an antiviral gene response after 48 h postinfec-
tion, as indicated by the upregulation of IFN genes and pro-inflammatory cytokines and chemokines, including IL-6,
IL-8, and TNF-α [54,65].
The relevance of TLRs to stimulate inflammation in the lungs has been well established. During acute lung injury,
the presence of TLR4 in macrophages acts as a key factor mediating the severity of the inflammation as well as tissue
damage. TLR4 polymorphisms have been also correlated to poor ARDS prognosis. Many viruses, such as influenza and
rhinoviruses, rely on strategies to silently surpass TLR-based warning signal in the airway epithelium, via inhibition of
proteins such as RIG-1 and MDA5, components of TLR4 signaling and IRF-3, to drive IFN production. Interestingly,
TLR3 or TL4 knockout (KO) mice are more susceptible to SARS-CoV infection, while TRIF KO mice present a greatly
exacerbated inflammatory influx. The lack of TRIF hampers IFN expression and leads to clinical features that largely
resemble severe ARDS patients. Even though TLRs are crucial for correct viral defense, the constitutive TLR signaling,
particularly due to TLR4, might contribute to an excessive inflammation in COVID-19-associated ARDS. Studies
involving Ebola virus-like particles have shown that TLR4 also impairs protective antiviral cytokine production in
fibroblasts via suppressor of cytokine signaling 3 and could be involved in the disease initiation. TLR4 polymorphism
and dysregulation of associated pathway could be potentially correlated with COVID-19-mediated suppression of IFN
antiviral response in severe patients and then contribute to excessive lung inflammation at later stages [54,65].
One of the distinctive features of SARS-CoV-2 in comparison to SARS-CoV is its ability to efficiently infect the URT,
such as nasopharyngeal and/or oropharyngeal tissues, possibly due to higher expression of ACE2 in these tissues
[56,57,66
68]. The readily detectable titers of SARS-CoV-2 in the URT mucus of patients with COVID-19 during prodro-
mal periods might help explain the more rapid and effective transmissibility of SARS-CoV-2 relative to SARS-CoV [69].

3.7 Gastrointestinal system

Human CoVs were known to cause enteric infections, with variable degrees of pathogenicity [70]. Gastrointestinal ill-
ness has been frequently reported in patients with COVID-19 [71
73] as ACE2 and TMPRSS2 are known to be abun-
dantly expressed within the human and many other mammalian intestinal tracts, specifically the brush borders of
intestinal enterocytes [54]. Furthermore, furin, a serine protease that acts similar to TMPRSS2 by unbounding S1 and
S2 domains of the viral spike (S), is abundant in the small intestine enabling coronavirus infection [74]. Consistent with
this, SARS-CoV has been recovered from stool samples of patients with SARS, suggesting a potential feco
oral route
of transmission for these CoVs. Of note, B20% of patients with COVID-19 examined have had detectable SARS-CoV-
2 RNA in their feces, even after respiratory symptoms subsided, suggesting that SARS-CoV-2 titers may be prolonged
in the intestinal tract. Although further studies are needed, these data suggest the possibility that fecal
oral transmis-
sion of SARS-CoV-2 occurs. Evidently, robust epidemiological studies are needed to conclusively demonstrate whether
patients with COVID-19 recovering from respiratory illness can spread SARS-CoV-2 [54,65]. The systemic inflamma-
tory (i.e., cytokine storm) response promoted by SARS-CoV-2 infection may directly or indirectly damage the digestive
tract and its resident microbiota, which could synergistically act with the viremia to promote respective symptoms [65].

3.8 Nervous system

Among the identified coronaviruses, at least three—229E, OC43 [75
78], and SARS-CoV24,25—are neuroinvasive
and neurotropic. Both SARS-CoV-2 and SARS-CoV enter human cells via the ACE2 receptor. Therefore, like SARS-
CoV, SARS-CoV-2 might also be able to invade the central nervous systems (CNS). In addition, the structure and
32 Pandemic Outbreaks in the 21st Century

mode of replication of SARS-CoV-2 are quite similar to those of neuroinvasive animal coronaviruses, such as porcine
hemagglutinating encephalitis virus, feline coronavirus, and mouse hepatitis virus (MHV) [79]. MHV has been shown
to persist in the mouse CNS after acute infection and to induce an immune-mediated, chronic demyelinating disease,
similar to multiple sclerosis in humans [80]. Taken together, these observations have led to speculation about the possi-
ble involvement of SARS-CoV-2 in neurological diseases; however, definitive conclusions about neuroinvasivity are
missing. Consistent with this, SARS-CoV-2 has also been reported to be associated with certain neurological manifesta-
tions [7
9,81]. Symptoms affecting both peripheral nervous systems (PNS) and CNS have been reported in B36% of
COVID-19 patients, such as headache, acute cerebral diseases, impaired consciousness, seizure, smell/taste impairment,
muscle injury, and neuralgia. Although mechanistic analyses are still in progress, current literature strongly suggests the
potential ability of coronaviruses to reach brain-related tissues and cause neurological damage [65]. However, an inter-
esting aspect that remains controversial is the presence of SARS-CoV-2 in the cerebrospinal fluid (CSF) of patients
developing neurological symptoms, such as encephalitis/meningitis. Since some studies have reported the absence of
virus in the CSF while others have fully detected it, some concerns have been raised about any direct neuroinvasive
potential of the novel coronavirus, as also described for SARS-CoV and MERS-CoV [65].
The mechanisms of how coronaviruses may affect the CNS, theories have been proposed to better explain how
SARS-CoV-2 may reach the CNS and cause brain damage. Although the neuroinvasivity of SARS-CoV-2 has not yet
been confirmed, multiple lines of evidence suggest that other human coronaviruses can use both the hematogenous route
and neuronal dissemination to penetrate the CNS46
48. Baig et al. (2020) have described that SARS-CoV-2 may reach
the CNS via bloodstream passing into the cerebral circulation, reaching the cerebral capillary endothelium which
expresses ACE2 with which spike protein of virus interacts and lead to the release of virus particles by damaging the
endothelial cells of the blood
brain barrier. This effect promotes the viral entry and the consequent activation of ACE2
receptors also expressed in neurons, thus leading to local inflammation and demyelination [65,79]. Virus-driven demye-
lination in the CNS has been reported in several viral infections, including coronaviruses, in humans and animal models
[82
85]. Furthermore, evidence suggests that coronaviruses can infect leukocytes. Once activated by infection, these leu-
kocytes disseminate toward other tissues and cross the blood
brain barrier to access the CNS, the process has been
referred to as a Trojan horse mechanism [86]. In the CNS, leukocytes produce pro-inflammatory cytokines such as TNF
that can damage oligodendrocytes and/or neurons, and chemokines such as CCL5, CXCL10, and CXCL11 that induce
chemoattraction of activated T cells and/or other leukocytes [86]. After sensing infection, astrocytes can also produce
chemokines, including CCL2, CCL5, and CXCL12, which participate in the recruitment of more infected leukocytes.
SARS-CoV-2 might, therefore, initiate an aberrant neuroinflammatory loop, which results in neuropathology48.
Alternatively, coronaviruses have been shown to infect vascular endothelial cells, which then spread the virus directly to
glial cells in the CNS [65]. The second possibility is the neuronal retrograde route, which is associated with the virus pen-
etration upon nasal infection (using cribriform plate and olfactory bulb as entry routes). Nasal infection may primarily
lead to damage of the olfactory epithelium that expresses both ACE2 and TRPMSS2 [65]. The consequent damage on
the olfactory endothelium is known to be a part of the clinical symptoms presented by COVID-19 patients, particularly
related to the PNS, such as anosmia or hyposmia. The third route that could promote the entry of the SARS-CoV-2 virus
into the brain is the glymphatic system, a physiological route located in the CNS that shows perivascular tunnels consist-
ing of astroglial cells, connected to the cervical and olfactory lymphatic vessels, that enable the waste elimination and
promote the wide distribution of several compounds in the brain [65]. Moreover, brain damage may occur upon distur-
bance of the drainage system due to viral infection, thus leading to the entrance of viruses into the CSF [1
3].
Accordingly, some COVID-19 patients have been reported to present paranasal sinusitis with the presence of lymph endo-
thelial cells infected by SARS-CoV-2. A recent study has presented neurochemical evidence of brain injury in severe pos-
itive COVID-19 patients, consistent with the development of neurological manifestations, indication of brain injury in
postmortem analysis and the controversial aspect of SARS-CoV-2 detection in CSF [65,79]. Acute cerebral diseases,
such as ischemic stroke and cerebral hemorrhage were also detected in positive COVID-19 patients. In addition to the
neurological manifestations described for the CNS, the PNS also appears to be affected by COVID-19, as patients were
presented with smell and/or taste impairments, as well as complications due to the large amount of cytokines released
systemically in the face of infection [65,79]. Koralnik and Tyler[87] described that COVID-19 positive patients devel-
oped Guillain
Barre syndrome after the onset of viral infection, as an immune-mediated complication of SARS-CoV-2.

3.9 Cardiovascular system

Preexisting conditions such as cardiovascular disease (CVD), diabetes, hypertension, and obesity are correlated with
higher severity and a significant increase in the fatality rate of COVID-19. COVID-19 induces multiple CV
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 33

complexities, such as cardiac arrest, myocarditis, acute myocardial injury, stress-induced cardiomyopathy, cardiogenic
shock, arrhythmias, and, subsequently heart failure (HF). In certain cases, some patients with underlying CVDs might
have an increased risk of death [88]. Various recent patient data originating from different geographical locations showed
that preexisting CVD is associated with a more severe COVID-19 infection [7
9,89
91]. SARS-CoV-2 appears to
affect the myocardium and cause myocarditis [66,67]. Sporadic autopsy cases suggest infiltration of myocardium by
interstitial mononuclear inflammatory cells [66,67]. Various cases of severe myocarditis with reduced systolic function
have been reported after COVID-19 [92,93] Studies involving cardiac biomarker reveal a high prevalence of cardiac
injury in hospitalized patients. Myocardial injury is likely associated with infection-related myocarditis and/or ischemia
and is an important prognostic factor in COVID-19 [94]. The precise mechanisms of how SARS-CoV-2 may cause myo-
cardial complications are not clearly understood. ACE2 is highly expressed in the CV system tissues, possibly playing a
major role in the regulation of the ACE2-Ang (1
7) signaling in proliferation, inflammation, and vascular fibrosis and
remodeling. In healthy subjects, the levels of ACE2 in the plasma are very low, in contrast with the high levels found in
the plasma of CV disease patients. SARS-CoV-2 was shown to infect cardiomyocytes in vitro in an ACE2 and
cathepsin-dependent manner and the infection of cardiomyocytes was inhibited by the antiviral drug remdesivir [95].
Thus, the CV system can be also affected by SARS-CoV-2 infection and, as such, it may potentially be a key for ill-
ness severity. In fact, patients with CV conditions have presented a case fatality rate of 10.5%, which is higher than the
overall COVID-19 cases (i.e., fatality rate of 2.3%) [65].
Troponin, a major regulatory protein complex involved in muscle contraction, is typically released during myocar-
dial damage, so the detection of troponin levels in the serum has served as a sensitive and specific test for the diagnosis
of CV diseases. According to a report from the National Health Commission of China, B12% of patients hospitalized
due to COVID-19, without history of CV diseases, have presented elevated troponin levels and a high incidence of car-
diac arrest during hospitalization, indicating that not only CV diseases could be a risk factor for COVID-19 but the
presence of SARS-CoV-2 could also promote myocardial injury [96].
The impact of the CV diseases in severe COVID-19 patients has been clearly demonstrated by a recent study that
compared nonsurviving and surviving COVID-19 patients [1], in which 52% of the deceased patients presented HF,
whereas only 12% of the survivors presented the same symptoms. Furthermore, 59%of the nonsurvival cases (vs only
1% of the survivors) were affected by cardiac injury. Another study has shown that out of 68 patients who died from
COVID-19, 13 had previous CV diseases while none of the 82 patients who survived presented a history of CV condi-
tion [97]. Despite the evidence, it is still unclear why CVDs are so prevalent among the fatalities from COVID-19. One
potential explanation relates to the ACE-mediated infection of cardiomyocytes, pericytes, and fibroblasts, thus leading
to myocardial injury (Hendren et al., 2020). Another hypothesis considers the impact of cytokine storm, triggered by an
imbalanced response of T-helper cells and elevated levels of intracellular calcium, which can also promote extensive
damage to myocardial cells [65].

3.10 Renal system

The kidneys have also been indicated as major targets of SARS-CoV-2 infection. Studies also suggest that SARS-CoV-
2 has kidney tropism including the ability of the virus to replicate in kidney cells, and that kidney transduction by
SARS-CoV-2 is associated with shorter survival time and increased incidence of acute kidney injury (AKI). Kidney tis-
sue in patients with COVID-19 contains the infective SARS-CoV-2 virus [98]. Although an early study has not identi-
fied any cases of AKI in a cohort of 116 COVID-19 patients from the Wuhan area, clinical reports have largely
supported the association of SARS-CoV-2 infection with kidney conditions. Victor et al. [99] reported SARS-CoV-2
viral load in autopsy tissue samples obtained from 22 patients who had died from COVID-19 and 17 patients (77%)
had more than two coexisting conditions, and a greater number of coexisting conditions was associated with SARS-
CoV-2 tropism for the kidneys, even in patients without a history of chronic kidney disease. This study concluded that
renal tropism is a potential explanation of commonly reported new clinical signs of kidney injury in patients with
COVID-19, even in patients with SARS-CoV-2 infection who are not critically ill. According to a large study including
701 COVID-19 patients, the most frequent finding related to kidney dysfunction was mild to moderate proteinuria
(43.9%), possibly due to the disruption of glomerular filtration, while 26.7% of patients exhibited hematuria [100].
Interestingly, a retrospective study has shown that AKI was predominantly found in critically ill patients [101]. Another
report focusing on 113 nonsurviving COVID-19 patients pointed out that AKI was highly associated with increased
mortality [100]. Another study that reviewed records from 13 academic and community hospitals in metropolitan New
York found that AKI was reported in 36.6% of the admitted COVID-19 patients, particularly in patients with respiratory
failure who required mechanical ventilation (89% of the cases) [102].
34 Pandemic Outbreaks in the 21st Century

Several pathophysiological mechanisms have been proposed for the renal injuries observed in COVID-19, including
organ-crosstalk and systemic-wide effects. One of them is the lung
kidney crosstalk. In a retrospective study including
357 patients, AKI has been shown to be secondary to pneumonia in 68% of ARDS patients [103], and the damage in
the lung
kidney axis may be bidirectional, as shown by the association of IL-6 cytokine released in the serum due to
injured renal tubular epithelium with higher alveolar-capillary permeability and pulmonary hemorrhage [104].
Similarly, a heart
kidney crosstalk may also be considered as a contributor of AKI in COVID-19 patients, since cardio-
myopathy and acute viral myocarditis equally contribute to renal hypoperfusion that leads to a reduction in the glomeru-
lar filtration rate. Systemic effects such as CRS have also been proposed for the etiology of AKI [100]. Sustained
elevation of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α in the circulation can induce extensive endo-
thelial dysfunction and disseminated intravascular coagulation, ultimately leading to multiple organ dysfunction syn-
drome [105], which can be directly responsible for renal damage. In fact, TNF-α has been demonstrated to bind
directly to TNF receptor-1 in renal tubular cells, triggering apoptosis [106], and IL-6 has been extensively reported to
be associated with the onset and severity of AKI in patients and animal models, including ischemic AKI, nephrotoxin-
induced AKI, and sepsis-induced AKI, promoting renal injury via binding to sIL6R and downstream signaling through
STAT3 in tubular epithelial cells [107]. Studies showing septic shock syndrome in COVID-19 patients raise the possi-
bility that intrarenal inflammation may be partially responsible for the association of AKI in severe COVID-19 cases
[108]. Direct SARS-CoV-2 infection has also been shown to be an important underlying cause of renal injury. Direct
evidence of SARS-CoV-2 infection in the renal system has been provided by autopsy reports identifying virus particles
and vacuoles characteristic of SARS-CoV-2 in the proximal tubular epithelium and podocytes, using electron micros-
copy [109]. These reports support a direct pathophysiological mechanism for the kidney damage due to COVID-19 fol-
lowing SARS-CoV-2 entry via ACE2 receptor [88].
SARS-CoV-2 organotropism beyond the respiratory tract, includes the kidneys, liver, heart, and brain, and it can be
speculated that this varied organotropism of SARS-CoV-2 influences the course of COVID-19 disease and, possibly,
aggravates preexisting conditions [99].

3.11 Transmission
SARS-CoV-2 is a new virus and scientists are still learning the behavior and different modes of its transmission.
Current evidence suggests that SARS-CoV-2 is predominantly spread from person to person. Understanding how,
when, and in what types of settings SARS-CoV-2 spreads is critical to develop effective public health and infection pre-
vention and control measures to break chains of transmission. Respiratory viruses are generally transmitted in three
main ways. First, contact transmission, where someone comes into direct contact with an infected person or touches a
surface that has been contaminated. Second, through droplet transmission of both large and small respiratory droplets
that contain the virus and third, through airborne transmission of smaller droplets and particles that are suspended in the
air over longer distances and time than droplet transmission (The Lancet Respiratory Medicine, https://doi.org/10.1016/
S2213-2600(20)30514-2).

3.11.1 Contact and droplet transmission

Transmission of SARS-CoV-2 can occur through direct, indirect, or close contact with infected people through infected
secretions such as saliva and respiratory secretions or their respiratory droplets, which are expelled when an infected
person coughs, sneezes, talks, or sings (2
10). Respiratory droplets are .5
10 μm in diameter, whereas droplets
,5 μm in diameter are referred to as droplet nuclei or aerosols (WHO; https://www.who.int/news-room/commentaries/
detail/transmission-of-sars-cov-2-implications-for-infection-prevention-precautions). Size threshold of these droplets is
also confusing. A recent study indicates a terminology to distinguish between aerosols and droplets using a size thresh-
old of 100 μm, not the historical 5 μm (Kimberly et al., 2020) and this size more effectively separates their aerodynamic
behavior, ability to be inhaled, and efficacy of interventions. Viruses in droplets (larger than 100 μm) typically fall to
the ground in seconds within 2 m of the source and can be sprayed like tiny cannonballs onto nearby individuals.
Because of their limited travel range, physical distancing reduces exposure to these droplets. Viruses in aerosols (smal-
ler than 100 μm) can remain suspended in the air for many seconds to hours, like smoke, and be inhaled. They are
highly concentrated near an infected person, so they can infect people most easily in proximity. But aerosols containing
infectious viruses can also travel more than 2 m and accumulate in poorly ventilated indoor air, leading to super spread-
ing events (Kimberly et al., 2020). Indirect contact transmission involving contact of a susceptible host with a contami-
nated object or surface (fomite transmission) may also be possible.
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 35

3.11.2 Airborne transmission

Airborne transmission is defined as the spread of an infectious agent caused by the dissemination of droplet nuclei
(aerosols) that remain infectious when suspended in air over long distances and time. WHO, together with the scientific
community, has been actively discussing and evaluating whether SARS-CoV-2 may also spread through aerosols in the
absence of aerosol-generating procedures, particularly in indoor settings with poor ventilation?
The physics of exhaled air and flow physics have generated hypotheses about possible mechanisms of SARS-CoV-2
transmission through aerosols. These theories suggest that (1) a number of respiratory droplets generate microscopic
aerosols (,5 μm) by evaporating, and (2) normal breathing and talking results in exhaled aerosols. Thus, a susceptible
person could inhale aerosols and could become infected if the aerosols contain the virus in sufficient quantity to cause
infection within the recipient. However, the proportion of exhaled droplet nuclei or of respiratory droplets that evapo-
rate to generate aerosols, and the infectious dose of viable SARS-CoV-2 required to cause infection in another person
are not known, but it has been studied for other respiratory viruses. Recent clinical reports of health workers exposed to
COVID-19 index cases, not in the presence of aerosol-generating procedures, found no nosocomial transmission when
contact and droplet precautions were appropriately used, including the wearing of medical masks as a component of the
personal protective equipment (PPE). These observations suggest that aerosol transmission did not occur in this context.
Further studies are needed to determine whether it is possible to detect viable SARS-CoV-2 in air samples from settings
where no procedures that generate aerosols are performed and what role aerosols might play in transmission.
Outside of medical facilities, some outbreak reports related to indoor crowded spaces have suggested the possibility
of aerosol transmission, combined with droplet transmission, for example, during choir practice, in restaurants, or fit-
ness classes. In these events, short-range aerosol transmission, particularly in specific indoor locations, such as crowded
and inadequately ventilated spaces over a prolonged period with infected persons cannot be ruled out. However, the
detailed investigations of these clusters suggest that droplet and fomite transmission could also explain human-to-
human transmission within these clusters.

3.11.3 Fomite transmission

Respiratory secretions or droplets expelled by infected individuals can contaminate surfaces and objects, creating
fomites or contaminated surfaces. Viable SARS-CoV-2 virus and/or RNA detected by RT-PCR can be found on those
surfaces for extending periods ranging from hours to days, depending on the ambient environment (including tempera-
ture and humidity) and the type of surface, in particular at high concentration in health care facilities where COVID-19
patients were being treated. Therefore transmission may also occur indirectly through touching surfaces or objects con-
taminated with viruses from an infected person, followed by touching the mouth, nose, or eyes.
There are no specific reports that demonstrated direct fomite transmission. People who encounter potentially infec-
tious surfaces often also have close contact with the infectious person, making the distinction between respiratory drop-
let and fomite transmission difficult to discern. However, fomite transmission is considered a likely mode of
transmission for SARS-CoV-2, given consistent findings about environmental contamination in the vicinity of infected
cases and the fact that other coronaviruses and respiratory viruses can transmit by this way.

3.11.4 Fecal
oral transmission

SARS-CoV has been recovered from stool samples of patients with COVID-19, suggesting a potential fecal
oral route
of transmission for these CoVs. Of note, B20% of patients with COVID-19 examined have had detectable SARS-CoV-
2 RNA in their feces, even after respiratory symptoms subsided, suggesting that SARS-CoV-2 titers may be prolonged
in the intestinal tract. Although further studies are needed, these data suggest the possibility that fecal
oral transmis-
sion of SARS-CoV-2 occurs [54,65]. One study found viable SARS-CoV-2 in the urine of one patient [110]. Three
studies have cultured SARS-CoV-2 from stool specimens [39,40,71,72,111]. To date, however, there have been no pub-
lished reports of transmission of SARS-CoV-2 through feces or urine.

3.11.5 Other modes of transmission

Studies have reported detection of SARS-CoV-2 RNA in either plasma or serum, and the virus can replicate in blood
cells. However, the role of blood-borne transmission remains uncertain; and low viral titers in plasma and serum sug-
gest that the risk of transmission through this route may be low [39,40,112]. Currently, there is no evidence for
36 Pandemic Outbreaks in the 21st Century

intrauterine transmission of SARS-CoV-2 from infected pregnant women to their fetuses, although data remain limited.
WHO has recently published a scientific brief on breastfeeding and COVID-19 [113]. This brief explains that viral
RNA fragments have been found by RT-PCR testing in a few breast milk samples of mothers infected with SARS-
CoV-2, but studies investigating whether the virus could be isolated, have found no viable virus. Transmission of
SARS-CoV-2 from mother to child would necessitate replicative and infectious virus in breast milk being able to reach
target sites in the infant and to overcome infant defense systems. WHO recommends that mothers with suspected or
confirmed COVID-19 should be encouraged to initiate or continue to breastfeed [113].
To date, SARS-CoV-2 has not been reported to be sexually transmitted. But there are reports which detected the
presence of SARS-CoV-2 RNA by RT-qPCR in semen samples of COVID-19 patients [114]. But nothing is known if
viable viruses can be found in those samples. As discussed, earlier SARS-CoV-2 RNA was also detected in feces sam-
ples but again nothing much is known if viable virus is present in those sources or not. If there is viable virus in these
sources, it could raise the possibility of sexual transmission depending on the persons’ sexual practices but again there
are no confirmed reports to date that point toward the sexual transmission of SARS-CoV-2.
Evidence to date shows that SARS-CoV-2 is most closely related to known beta coronaviruses in bats and the role
of an intermediate host in facilitating transmission in the earliest known human cases remains unclear [1,115]. In addi-
tion to investigations on the possible intermediate host(s) of SARS-CoV-2, there are also several studies underway to
better understand the susceptibility of SARS-CoV-2 in different animal species. Current evidence suggests that humans
infected with SARS-CoV-2 can infect other mammals, including dogs, cats, and farmed mink. However, it remains
unclear if these infected mammals pose a significant risk for transmission to humans.

3.12 Prevention and treatment

Since the transmission of SARS-CoV-2 is thought to mainly occur through respiratory droplets transmitted from an
infectious person, SARS-CoV-2 infection via airborne transmission of small particles tends to occur after prolonged
exposure (.30 min) to an infectious person who is in an enclosed space with poor ventilation. Three most important
ways to slow the spread of SARS-CoV-2 are as follows: (1) wear a mask to protect yourself and others and to stop the
spread of the virus; (2) stay at least 6 feet (about two arm lengths) from others who do not live with you; and (3) avoid
crowded places. The more people you are in contact with, the more likely you are about to be exposed to COVID-19.
The risk of SARS-CoV-2 transmission can be reduced by covering coughs and sneezes and maintaining a distance from
others. When consistent distancing is not possible, face coverings in the form of face masks may further reduce the
spread of infectious droplets from individuals with SARS-CoV-2 infection to others. Frequent handwashing is also
effective in reducing the risk of infection. Health care providers should follow the CDC/WHO recommendations for
infection control and appropriate use of PPE.

3.12.1 Vaccines
Since vaccines are the best preventive strategy for protecting the large population from this pandemic, the development
of vaccines for SARS-CoV-2 is aggressively being pursued. Vaccine development is typically a lengthy process, often
requiring multiple candidates before one proves to be safe and effective. To address the current pandemic, several plat-
forms such as inactivated vaccines, live-attenuated vaccines, and protein subunit vaccines are being pursued. Some
novel approaches are being investigated, including DNA-based and RNA-based strategies and replicating and nonrepli-
cating vector strategies [116], with the hope of identifying a safe and effective SARS-CoV-2 vaccine. The Food and
Drug Administration (FDA) has issued an Emergency Use Authorization (EUA) for two vaccines BNT162b2 (Pfizer-
BioNTech) and mRNA-1273 (Moderna) that are mRNA-based vaccines and as of January 2021, several countries
including the United States, United Kingdom, Canada, and some European and Middle East countries have already
started the vaccination regimes against SARS-CoV-2 using these two vaccines. The list of some of the vaccine plat-
forms and their preclinical/clinical status were detailed in Table 3.2.

3.12.2 Antiviral therapy

Because SARS-CoV-2 replication leads to many of the clinical manifestations of COVID-19, antiviral therapies are
being investigated for the treatment of COVID-19. These drugs inhibit viral entry (via ACE2 receptor and TMPRSS2),
viral membrane fusion and endocytosis, or the activity of the SARS-CoV-2 3CLpro and the RdRP [117]. Because viral
replication may be particularly active early in the course of COVID-19, antiviral therapy may have the greatest impact
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 37

TABLE 3.2 List of vaccine candidates against SARS-CoV-2.

Vaccine Type of vaccine Developer Current stage of evaluation

platform
RNA vaccine mRNA-Ad5-nCoV Pfizer-BioNTech (United States/ FDA-approved & recommended vaccine.
BNT162b2 targeting Germany) Authorized in the United States, Canada,
viral spike protein Israel, United Kingdom, EU, and many other
countries
RNA vaccine mRNA-1273 targeting Moderna (United States) FDA-approved & recommended vaccine.
viral spike protein Authorized in the United States, Canada,
Israel, United Kingdom, EU
Replication- AZD1222 (ChAdOx1 University of Oxford/AstraZeneca/ Authorized in India
deficient viral expressing SARS-CoV-2 SII, India
vectored spike protein)
Recombinant Ad5-nCoV CanSino Biologics; Beijing Institute Phase III, Approved in China
adenovirus of Biotechnology of the Academy of
type 5 vector Military Medical Sciences
Inactivated Covaxin (BBV152)— Bharat Biotech; Indian Council of Authorized in India
Inactivated SRAS-CoV-2 Medical Research
Inactivated BBIBP-CorV— Sinopharm; Beijing Institute of Authorized in UAE, Bahrain, China. EUA in
Inactivated SARS-CoV-2 Biological Products, Wuhan Egypt and Jordan
(vero cells) Institute of Biological Products
Inactivated CoronaVac—Inactivate- Sinovac, China EUA in China, Bolivia
CoV-2d SARS
Nonreplicating Sputnik V— Gamaleya Research Institute of EUA in Russia, Belarus, Argentina, Bolivia,
viral vector Nonreplicating Epidemiology and Microbiology, Venezuela, Serbia, Guinea
adenoviral vector Russia
Nonreplicating Ad26.COV2.S Janssen Pharmaceuticals, United EUA pending in Canada, EU, South Africa
viral vector nonreplicating vector States
(Adenovirus serotype
26)
DNA ZyCoV-D-DNA plasmid Cadila Healthcare, India Phase III
expressing SARS-CoV-2
S protein
Recombinant NVX-CoV2373- Novavax, CEPI Phase III in the United States, India, United
nanoparticle recombinant spike Kingdom, Mexico. EUA pending in Mexico
with adjuvant protein
Virus-like CoVLP—Recombinant, Medicago, GSK Phase II/III
particles (VLPs) plant-based VLPs with
adjuvant
RNA Zorecimeran CureVac, CEPI Phase2b/III; Argentina, Belgium, Colombia,
(CVnCoV)—modRNA Dominican Republic, France, Germany,
Mexico, Netherlands, Panama, Peru, Spain
Inactivated Formalin- and UV- Baxter Vaccines, Austria Preclinical
virus inactivated virus
vaccine
Nonreplicating Recombinant International Vaccine Institute (IVI) Preclinical
viral vector adenovirus expressing
truncated S protein
(rADV-S)
Replicating Recombinant measles University Health Network, Preclinical
viral vector virus expressing SARS- Canada;CDC
CoV-2 spike protein
Replicating MV-SARS recombinant Institute Pasteur Phase III
viral vector measles virus vaccine
expressing SARS-CoV
antigen

(Continued )
38 Pandemic Outbreaks in the 21st Century

TABLE 3.2 (Continued)

Vaccine Type of vaccine Developer Current stage of evaluation

platform
Protein subunit Receptor-binding Baylor College Medicine; Sabin; Preclinical
domain (RBD) of the New York Blood Center (NYBC);
SARS-CoV-2 spike University of Texas Medical Branch
protein (UTMB);
Walter Reed Army Institute of
Research
(WRAIR);
National Institute of Allergy and
Infectious Diseases (NIAID)
Protein subunit Recombinant peptide Saitama Medical University; Preclinical
N223 on liposomes Josai University; Nippon Oil and
Fat Corporation;
National Institute of Infectious
Diseases, Japan
Inactivated Inactivated whole virus Sanofi Preclinical
virus

before the illness progresses into the hyperinflammatory state that can characterize the later stages of the disease, including
critical illness [118]. For this reason, it is necessary to understand the role of antivirals in treating mild, moderate, severe, and
critical illnesses in order to optimize treatment for people with COVID-19. Several drugs are being tested for their antiviral
activities against SARS-CoV-2. Remdesivir is the only FDA-approved drug for the treatment of COVID-19. Remdesivir
(GS-5734), an inhibitor of the viral RdRP with in vitro inhibitory activity against SARS-CoV-1 and the MERS-CoV was
identified as a promising therapeutic candidate for COVID-19 because of its ability to inhibit SARS-CoV-2 in vitro [119].
Other drugs such as Chloroquine/Hydroxychloroquine, Lopinavir/Ritonavir (HIV Protease Inhibitors), Ivermectin,
Favipiravir, and others are shown to be promising in various studies, most of these are in various stages of clinical trials and
not recommended by FDA for their use as therapeutics in treating COVID-19 patients. In fact, CDC/WHO recommends
against the use of Chloroquine/Hydroxychloroquine. Baricitinib, an orally administered Janus Kinase (JAK) inhibitor that is
selective for JAK1 and JAK2. Baricitinib is approved by the FDA to treat moderate to severe rheumatoid arthritis. In
November 2020 the FDA issued a EUA for the use of baricitinib in combination with remdesivir in hospitalized adults and
children aged $ 2 years with COVID-19 who require supplemental oxygen, invasive mechanical ventilation, or extracorpo-
real membrane oxygenation (https://www.covid19treatmentguidelines.nih.gov/).

3.12.3 Immune-based therapy

Given the hyperactive inflammatory effects of SARS-CoV-2, agents that modulate the immune response are being
explored as adjunctive treatments for the management of moderate to critical COVID-19. These agents include human
blood
derived products and immunomodulatory therapies. Some human blood
derived products such as convalescent
plasma, immunoglobulin products are obtained from individuals who have recovered from SARS-CoV-2 infection and
these products are postulated to have either direct antiviral properties, such as with convalescent plasma, and/or immu-
nomodulatory effects. Additionally, several neutralizing monoclonal antibodies directed against SARS-CoV-2 have
been developed and are under investigation in clinical trials. In November 2020 FDA has issued a EUA for
Casirivimab and Imdevimab (Regeneron), which are two recombinant human monoclonal antibodies that bind to non-
overlapping epitopes of the spike protein RBD of SARS-CoV-2. The Casirivimab/Imdevimab combination blocks the
binding of the RBD to the host cell and these monoclonal antibodies can be used for the treatment of nonhospitalized
patients with mild to moderate COVID-19 who are at high risk for progressing to severe disease and/or hospitalization.
Bamlanivimab (LY-CoV555; Lilly) is another SARS-CoV-2 neutralizing antibody that got EUA recently from FDA
(https://www.covid19treatmentguidelines.nih.gov/statement-on-baricitinib-eua/). Other agents in this group include ther-
apeutics currently approved for the treatment of other immune and/or inflammatory syndromes. These agents include
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 39

corticosteroids (e.g., glucocorticoids), which abrogate systemic inflammation, and more targeted antiinflammatory treat-
ments such as interleukin inhibitors, IFNs, kinase inhibitors, and others.

3.12.4 Adjunctive therapy

In addition to the antiviral medications and the immune-based therapies for the treatment of COVID-19, adjunctive
therapies are frequently used in patients with COVID-19 to prevent and/or treat the infection or its complications. Some
of these agents are being studied in clinical trials. Infection with SARS-CoV-2 is associated with a prothrombotic state
and an increased incidence of thromboembolic disease. Hence, antithrombotic therapy in patients with COVID-19 is
also being studied (https://www.covid19treatmentguidelines.nih.gov/).

References
[1] Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan,
China: a retrospective cohort study. Lancet 2020;395:1054
62.
[2] Li Q, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med 2020;382:1199
207.
[3] Li Z, Liu T, Yang N, Han D, Mi X, Li Y, et al. Neurological manifestations of patients with COVID-19: potential routes of SARS-CoV-2 neu-
roinvasion from the periphery to the brain. Front Med 2020;14:533
41.
[4] World Health Organization. Novel Coronavirus (2019-nCoV) situation report 1; 2020.
[5] Gorbalenya AE, et al. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.
Nat Microbiol 2020;5:536
44.
[6] CDC. 2019. Available from: https://www.cdc.gov/coronavirus/2019-ncov/cdcresponse/about-COVID-19.html.
[7] Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature
2020;579:265
9.
[8] Wu Y, Xu X, Chen Z, Duan J, Hashimoto K, Yang L, et al. Nervous system involvement after infection with COVID-19 and other corona-
viruses. Brain Behav Immun 2020;87:18
22.
[9] Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of
a report of 72314 cases from the Chinese Center for Disease Control and Prevention. JAMA 2020;323:1239
42.
[10] Boni MF, Lemey P, Jiang X, et al. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. Nat
Microbiol 2020;5:1408
17.
[11] Xiaojun L, Giorgi EE, Marichannegowda MH, et al. Emergence of SARS-CoV-2 through recombination and strong purifying selection. Sci
Adv 2020;6:eabb9153.
[12] Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat ori-
gin. Nature 2020;579(7798):270
3.
[13] Lu R, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet
2020;395:565
74.
[14] Baric RS, Yount B, Hensley L, Peel SA, Chen W. Episodic evolution mediates interspecies transfer of a murine coronavirus. J Virol 1997;71
(3):1946
55.
[15] Dudas G, Carvalho LM, Rambaut A, Bedford T. MERS-CoV spillover at the camel-human interface. eLife 2018;7:e31257.
[16] Rambaut A. Phylodynamic analysis of SARS-CoV-2 genomes—27-Jan-2020. Virological January 27, 2020. ,http://virological.org/c/novel-
2019-coronavirus/33/l/latest.; 2020 [accessed 28.01.20].
[17] Azadeh R, Mirzazadeh A, Tavakolpour S. Genetics and genomics of SARS-CoV-2: a review of the literature with the special focus on genetic
diversity and SARS-CoV-2 genome detection. Genomics 2020;113(1 Pt 2):1221
32.
[18] Nextstrain/ncov. nextstrain. ,https://nextstrain.org/ncov?m 5 num_date.; 2020.
[19] Sironi M, Cagliani R, Forni D, Clerici M. Evolutionary insights into host
pathogen interactions from mammalian sequence data. Nat Rev
Genet 2015;16(4):224
36.
[20] Toyoshima NK, Matsumoto S, Nakamura Y, Kiyotani K. SARS-CoV-2 genomic variations associated with mortality rate of COVID-19. J Hum
Genet 2020;65:1075
82.
[21] Chan F, Kok KH, Zhu Z, Chu H, To KK, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated
from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 2020;9:221
36.
[22] Feng W, Hai Y, Jinyue G, et al. Identification of the hyper-variable genomic hotspot for the novel coronavirus SARS-CoV-2. J Infect 2020;80
(6):671
93.
[23] Zhang YZ, Holmes EC. A genomic perspective on the origin and emergence of SARS-CoV-2. Cell 2020;181:223
7.
[24] Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, Cheung CL, et al. Isolation and characterization of viruses related to the SARS coronavirus
from animals in southern China. Science 2003;302:276
8.
[25] Lau SK, Woo PC, Li KS, Huang Y, Tsoi H, Wong H, et al. Severe acute respiratory syndrome coronavirus like virus in Chinese horseshoe bats.
Proc Natl Acad Sci USA 2005;102:14040
5.
[26] Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 2005;310:676
9.
40 Pandemic Outbreaks in the 21st Century

[27] Xiaolu T, Wu C, Li X, Song Y, Yao X, Wu X, et al. On the origin and continuing evolution of SARS-CoV-2. Natl Sci Rev 2020;7
(6):1012
23.
[28] Hongru W, Lenore P, Rasmus N. Synonymous mutations and the molecular evolution of SARS-CoV-2 origins. Virus Evol 2020;7(1):veaa098.
[29] Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2
and is blocked by a clinically proven protease inhibitor. Cell 2020;181:271
80.
[30] V’kovski P, Kratzel A, Steiner S, et al. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiology
2020;19:155
70.
[31] Walls AC, et al. Structure, function, and antigenicity of the SARS- CoV-2 spike glycoprotein. Cell 2020;181:281
92.
[32] Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS- CoV-2 and other lineage B betacoronaviruses.
Nat Microbiol 2020;5:562
9.
[33] Li F. Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol 2016;3:237
61.
[34] Tortorici MA, Veesler D. Structural insights into coronavirus entry. Adv Virus Res 2019;105:93
116.
[35] Li F. Receptor recognition mechanisms of coronaviruses: a decade of structural studies. J Virol 2015;89:1954
64.
[36] Peck KM, Scobey T, Swanstrom J, Jensen KL, Burch CL, Baric RS, et al. Permissivity of dipeptidyl peptidase 4 orthologs to Middle East respi-
ratory syndrome coronavirus is governed by glycosylation and other complex determinants. J Virol 2017;91 e00534-17.
[37] Raj VS, Mou H, Smits SL, Dekkers DHW, Muller MA, Dijkman R, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging
human coronavirus-EMC. Nature 2013;495:251
4.
[38] Qi F, Qian S, Zhang S, Zhang Z. Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses.
Biochem Biophys Res Commun 2020;526:135
40.
[39] Wang K, Chen W, Zhang Z, et al. CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells. Signal Transduct Target Ther
2020;5:283.
[40] Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA 2020;323
(18):1843
4.
[41] Coutard B, Valle C, de Lamballerie X, Canard B, Seidah NG, Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a
furin-like cleavage site absent in CoV of the same clade. Antivir Res 2020;176:104742.
[42] Dufour D, Mateos-Gomez PA, Enjuanes L, Gallego J, Sola I. Structure and functional relevance of a transcription-regulating sequence involved
in coronavirus discontinuous RNA synthesis. J Virol 2011;85:4963
73.
[43] Mateos-Gomez PA, Morales L, Zuniga S, Enjuanes L, Sola I. Long-distance RNA-RNA interactions in the coronavirus genome form high-
order structures promoting discontinuous RNA synthesis during transcription. J Virol 2013;87:177
86.
[44] Knoops K, Kikkert M, Worm SH, Zevenhoven-Dobbe JC, van der Meer Y, Koster AJ, et al. SARS-coronavirus replication is supported by a
reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 2008;6:e226.
[45] Angelini MM, Akhlaghpour M, Neuman BW, Buchmeier MJ. Severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6
induce double-membrane vesicles. mBio 2013;4:e00524 13.
[46] Maria R, Ruggiero A, Squeglia F, Maga G, Berisio R. A structural view of SARS-CoV-2 RNA replication machinery: RNA synthesis, proof-
reading and final capping. Cells 2020;9:1267.
[47] Sawicki SG, Sawicki DL, Siddell SG. A contemporary view of coronavirus transcription. J Virol 2007;81:20
9.
[48] Kamitani W, Huang C, Narayanan K, Lokugamage KG, Makino SA. Two-pronged strategy to suppress host protein synthesis by SARS corona-
virus Nsp1 protein. Nat Struct Mol Biol 2009;16:1134
40.
[49] Narayanan K, Huang C, Lokugamage K, Kamitani W, Ikegami T, Tseng CT, et al. Severe acute respiratory syndrome coronavirus nsp1 sup-
presses host gene expression, including that of type I interferon, in infected cells. J Virol 2008;82:4471
9.
[50] Peiris JSM, Chu CM, Cheng VCC, Chan KS, Hung IFN, Poon LLM, et al. Clinical progression and viral load in a community outbreak of
coronavirus-associated SARS pneumonia: a prospective study. Lancet 2003;361(9371):1767
72.
[51] Wrapp D, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020;367:1260
3.
[52] Yurkovetskiy L, et al. Structural and functional analysis of the D614G SARS-CoV-2 spike protein variant. Cell 2020;183:739
51.
[53] Korber B, et al. Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus. Cell
2020;182:812
27.
[54] Andrew GH, Lin T, Wang P. Mechanisms of SARS-CoV-2 transmission and pathogenesis. Trends Immunol 2020;41(12):100
1115.
[55] Harmer D, Gilbert M, Borman R, Clark KL. Quantitative mRNA expression profiling of ACE 2, a novel homologue of angiotensin converting
enzyme. FEBS Lett 2002;532:107
10.
[56] Ziegler CGK, et al. SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific
cell subsets across tissues. Cell 2020;181:1016
35.
[57] Hamming I, Timens W, Bulthuis MLC, Lely AT, Navis GJ, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS
coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004;203:631
7.
[58] Jia HP, et al. ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway
epithelia. J Virol 2005;79:14614
21.
[59] Kuba K, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 2005;11:875
9.
[60] Zhao Y, et al. Single-cell RNA expression profiling of ACE2, the receptor of SARS-CoV-2. Am J Respir Crit Care Med 2020;202:756
9.
 Biology, prevention, and treatment of SARS-CoV-2 (COVID-19) Chapter | 3 41

[61] Shulla A, et al. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus
entry. J Virol 2011;85:873
82.
[62] Imai Y, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 2005;2005(436):112
16.
[63] Smith JC, et al. Cigarette smoke exposure and inflammatory signaling increase the expression of the SARSCoV-2 receptor ACE2 in the respira-
tory tract. Dev Cell 2020;53:514
29.
[64] Lukassen S, et al. SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J 2020;39:
e105114.
[65] Daniella SB, Dragunas G, Klein MO, Ayub ALP, Velloso FJ, Correa RG. Unpuzzling COVID-19: tissue-related signaling pathways associated
with SARS-CoV-2 infection and transmission. Clin Sci 2020;134:2137
60.
[66] Xu H, et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 2020;12:8.
[67] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of COVID-19 associated with acute respiratory distress syn-
drome. Lancet Respir Med 2020;8(4):P420
2.
[68] COVID-19 Investigation Team. Clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (COVID-19) in the
United States. Nat Med 2020;26:861
8.
[69] Wölfel R, et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020;581:465
9.
[70] Su S, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 2016;24:490
502.
[71] Xiao F, et al. Evidence for gastrointestinal infection of SARS-CoV-2. Gastroenterology 2020;158:1831
3.
[72] Xiao F, Sun J, Xu Y, Li F, Huang X, Li H, et al. Infectious SARS-CoV-2 in feces of patient with severe COVID-19. Emerg Infect Dis 2020;26
(8):1920
2.
[73] Cholankeril G, Podboy A, Aivaliotis VI, Tarlow B, Pham EA, Spencer SP, et al. High prevalence of concurrent gastrointestinal manifestations
in patients with SARS-CoV-2: early experience from California. Gastroenterology 2020;159:775
7.
[74] Monkemuller K, Fry L, Rickes S. Covid-19, Coronavirus, SARS-CoV-2 and the small bowel. Rev Esp Enferm Dig 2020;112:383
8.
[75] Bonavia A, Arbour N, Yong VW, Talbot PJ. Infection of primary cultures of human neural cells by human coronaviruses 229E and OC43. J
Virol 1997;71:800
6.
[76] Arbour N, Côté G, Lachance C, Tardieu M, Cashman NR, Talbot PJ. Acute and persistent infection of human neural cell lines by human coro-
navirus OC43. J Virol 1999;73:3338
50.
[77] Arbour N, Ekandé S, Côté G, Lachance C, Chagnon F, Tardieu M, et al. Persistent infection of human oligodendrocytic and neuroglial cell lines
by human Coronavirus 229E. J Virol 1999;73(4):3326
37.
[78] Arbour N, Day R, Newcombe J, Talbot PJ. Neuroinvasion by human respiratory coronaviruses. J Virol 2000;74:8913
21.
[79] Alessandro P, Alessandro P. Lifting the mask on neurological manifestations of COVID-19. Nat Rev Neurol 2020;16:636
44.
[80] Hosking MP, Lane TE. The pathogenesis of murine coronavirus infection of the central nervous system. Crit Rev Immunol 2010;30:119
30.
[81] Mao L, Jin H, Wang M, Hu Y, Chen S, He Q, et al. Neurologic manifestations of hospitalized patients with coronavirus disease 2019 in
Wuhan. China JAMA Neurol 2020;77:1
9.
[82] Fazakerley JK, Walker R. Virus demyelination. J Neurovirol 2003;9:148
64.
[83] Lane TE, Buchmeier MJ. Murine coronavirus infection: a paradigm for virus-induced demyelinating disease. Trends Microbiol 1997;5:9
14.
[84] Murray RS, Cai GY, Hoel K, Zhang JY, Soike KF, Cabirac GF. Coronavirus infects and causes demyelination in primate central nervous sys-
tem. Virology 1992;188:274
84.
[85] Stohlman SA, Hinton DR. Viral induced demyelination. Brain Pathol 2001;11:92
106.
[86] Desforges M, Coupanec AL, Dubeau P, Bourgouin A, Lajoie L, Dubé M, et al. Human coronaviruses and other respiratory viruses: underesti-
mated opportunistic pathogens of the central nervous system? Viruses 2019;12(1):14.
[87] Koralnik IJ, Tyler KL. COVID-19: a global threat to the nervous system. Ann Neurol 2020;88:1
11.
[88] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Lancet 2020;395:497
506.
[89] Ajit M, Raj K. Cardiovascular manifestations of COVID-19 infection. Cells 2020;9(11):2508.
[90] Driggin E, Madhavan MV, Bikdeli B, Chuich T, Laracy J, Biondi-Zoccai G, et al. Cardiovascular considerations for patients, health care work-
ers, and health systems during the COVID-19 pandemic. J Am Coll Cardiol 2020;75:2352
71.
[91] Murthy S, Gomersall CD, Fowler RA. Care for critically ill patients with COVID-19. JAMA 2020;323:1499.
[92] Hu H, Ma F, Wei X, Fang Y. Coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin. Eur Heart J 2020;
ehaa190.
[93] Inciardi RM, Lupi L, Zaccone G, et al. Cardiac involvement 1 with coronavirus 2019 (COVID-19) infection. JAMA Cardiol 2020;5(7):819
24.
[94] Mohammad M, Safavi-Naeini P, Solomon SD, Vardeny O. Potential effects of coronaviruses on the cardiovascular system. JAMA Cardiol
2020;5(7):831
40.
[95] Denisa B, Julian UG, Wagner M, Shumliakivska GS, Aslan U, Saleem A, Hansen G, et al. SARS-CoV-2 infects and induces cytotoxic effects
in human cardiomyocytes. Cardiovasc Res 2020;116(14):2207
15.
[96] Zheng YY, Ma YT, Zhang JY, Xie X. COVID-19 and the cardiovascular system. Nat Rev Cardiol 2020;17:259
60.
[97] Ruan Q, Yang K, Wang W, Jiang L, Song J. Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients
from Wuhan, China. Intensive Care Med 2020;46:846
8.
[98] Anitha V, Humphreys BD. SARS-CoV-2 in the kidney: bystander or culprit? Nat Rev Nephrol 2020;16:703
4.
42 Pandemic Outbreaks in the 21st Century

[99] Victor GP, Lütgehetmann M, Lindenmeyer MT, Sperhake JP, Wong MN, Allweiss L, et al. Multiorgan and renal tropism of SARS-CoV-2. N
Engl J Med 2020;383:6.
[100] Cheng Y, Luo R, Wang K, Zhang M, Wang Z, Dong L, et al. Kidney disease is associated with in-hospital death of patients with COVID-19.
Kidney Int 2020;97:829
38.
[101] Yang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in
Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med 2020;8:475
81.
[102] Hirsch JS, Ng JH, Ross DW, Sharma P, Shah HH, Barnett RL, et al. Acute kidney injury in patients hospitalized with COVID-19. Kidney Int
2020;98:209
18.
[103] Panitchote A, Mehkri O, Hastings A, Hanane T, Demirjian S, Torbic H, et al. Factors associated with acute kidney injury in acute respiratory
distress syndrome. Ann Intensive Care 2019;9:74.
[104] Husain-Syed F, Slutsky AS, Ronco C. Lung-kidney cross-talk in the critically ill patient. Am J Respir Crit Care Med 2016;194(402):414.
[105] Teijaro JR, Walsh KB, Cahalan, Fremgen DM, Roberts E, Scott F, et al. Endothelial cells are central orchestrators of cytokine amplification
during influenza virus infection. Cell 2011;146:980
91.
[106] Cunningham PN, Dyanov HM, Park P, Wang J, Newell KA, Quigg RJ. Acute renal failure in endotoxemia is caused by TNF acting directly
on TNF receptor-1 in kidney. J Immunol 2002;168:5817
23.
[107] Nechemia-Arbely Y, Barkan D, Pizov G, Shriki A, Rose-John S, Galun E, et al. IL-6/IL-6R axis plays a critical role in acute kidney injury. J
Am Soc Nephrol 2008;19:1106
15.
[108] Martinez-Rojas MA, Vega-Vega O, Bobadilla NA. Is the kidney a target of SARS-CoV-2? Am J Physiol Ren Physiol 2020;318:F1454
62.
[109] Farkash EA, Wilson AM, Jentzen JM. Ultrastructural evidence for direct renal infection with SARS-CoV-2. J Am Soc Nephrol
2020;31:1683
7.
[110] Sun J, Zhu A, Li H, Zheng K, Zhuang Z, Chen Z, et al. Isolation of infectious SARS-CoV-2 from urine of a COVID-19 patient. Emerg
Microbes Infect 2020;9:991
3.
[111] Zhang Y, Chen C, Zhu S, Shu C, Wang D, Song J, et al. Isolation of 2019-nCoV from a stool specimen of a laboratory-confirmed case of the
coronavirus disease 2019 (COVID-19). China CDC Wkly 2020;2:123
4.
[112] Chang L, Zhao L, Gong H, Wang L, Wang L. Severe acute respiratory syndrome Coronavirus 2 RNA detected in blood donations. Emerg
Infect Dis 2020;26:1631
3.
[113] World Health Organization. Breastfeeding and COVID-19. Geneva: World Health Organization; 2020. Available from: https://www.who.int/
news-room/commentaries/detail/breastfeeding-and-covid-19.
[114] Diangeng L, Meiling J, Pengtao B, Weiguo Z, Shixi Z. Clinical characteristics and results of semen tests among men with Coronavirus disease
2019. JAMA Netw Open 2020;3(5):e208292.
[115] Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med 2020;26(4):450
2.
[116] Srinivas KP. Recent developments in vaccines strategies against human viral pathogens. In: Viswanath B, editor. Recent developments in
applied microbiology and biochemistry. Academic Press; 2021. p. 3
12.
[117] Sanders JM, Monogue ML, Jodlowski TZ, Cutrell JB. Pharmacologic treatments for coronavirus disease 2019 (COVID-19): a review. JAMA
2020;323(18):1824
36.
[118] Siddiqi HK, Mehra MR. COVID-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal. J Heart Lung
Transpl 2020;39(5):405
7.
[119] John HB, Tomashek KM, Dodd LE, Mehta AK, Zingman BS, Kalil AC, et al. Remdesivir for the treatment of Covid-19—final report. N Engl
J Med 2020;83:1813
26.
Chapter 4

Avian influenza A virus infections in

humans: current knowledge to enhance
host innate immunity to control Avian
influenza
Bosetty Anjana1,2, Buddolla Viswanath2 and Soumya Dakshinamurthy3
1
Department of Biotechnology, Sri Padmavathi Mahila Visvavidyalayam (Women’s University), Tirupati, India, 2Dr. Buddolla’s Institute of Life
Sciences, A Unit of Dr. Buddolla’s Educational Society, Tirupati, India, 3Department of Microbiology, Sri Venkateswara Institute of Medical Sciences
(SVIMS), Tirupati, India

4.1 Introduction
Influenza is a virus that causes disease, in both humans and animals, a virus (IAVs) that causes immense morbidity.
There are four types of influenza viruses (A, B, C, and D), with the influenza D virus being most recently discovered
(IDV). Among these four A and B causes seasonal epidemics especially B virus that is known as flu causing virus.
IAVs evolve through mutation, recombination, and reassortment, posing a persistent threat to their human and animal
host’s immune systems. Influenza viruses are members of the Orthomyxoviridae family, as well as negative-sense RNA
viruses with segmented single-stranded genomes. Although aquatic birds are the natural reservoir for all influenza A
viruses (IAVs), these viruses also infect and cause disease in domestic poultry and several mammal species, including
humans [1]. There are three subtypes of influenza viruses that are endemic to humans: H1N1, H2N2, and H3N2. As
seasonal IAVs, influenza A subtypes H1N1 and H3N2 circulate in humans. Avian influenza A (H7N9) is a type of
influenza virus previously discovered in birds. Influenza A (H7N9) virus had never been seen in either animals or
humans until March 2013, when it was discovered in China. Avian IAVs are RNA viruses with segmented genomes
that are divided into subtypes 16H and 9N based on two surface glycoproteins: hemagglutinin (H) viruses and neur-
aminidase viruses (N). There are 18 types of IAVs based on hemagglutinin (HA) glycoproteins and 11 types based on
neuraminidase (NA) glycoproteins.
Additional virus subtypes have recently been discovered in bats, but their significance in humans is unknown. IAVs are
naturally found in a wide variety of avian and mammalian species, including humans. The greatest diversity of virus subtypes
has been discovered in aquatic waterfowl, which is thought to be a natural reservoir of IAVs. In 2009 the H1N1 virus of
“swine origin” caused the most recent pandemic. Swine and poultry are two important IAV reservoirs, as well as two rapidly
growing livestock industries. Because livestock industry farms frequently house tens of thousands of birds or pigs infected
with a variety of IAV subtypes, they are frequently regarded as high-risk areas for the spread of novel IAVs. The routine
introduction of immunologically naive animals into large livestock populations aids in IAV conservation in livestock. New
IAV strains may be introduced into flocks or herds by animal workers or other animal species [2]. These same animal work-
ers may serve as a bridging population in moving IAVs circulating among livestock to other humans.

4.2 Major IAV lineages

IAVs are classified into subtypes based on two proteins found on their surface: hemagglutinin (H) and neuraminidase
(N). There are 18 different subtypes of HA and 11 different subtypes of NA (H1 through H18 and N1 through N11,

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00018-5

© 2021 Elsevier Inc. All rights reserved. 43
44 Pandemic Outbreaks in the 21st Century

respectively). While there are potentially 198 different influenza A subtype combinations, only 131 have been found in
nature. A(H1N1) and A(H3N2) are the current subtypes of IAVs that circulate in humans (H3N2) [3]. Influenza A sub-
types can be further classified into genetic “clades” and “subclades.” Clades and subclades can also be referred to as
“groups” and “sub-groups,” respectively. An influenza clade or group is a subset of influenza viruses that differ from
subtypes or lineages based on the similarity of their HA gene sequences. The influenza A (H1N1) viruses that are cur-
rently circulating are related to the pandemic 2009 H1N1 virus, which emerged in the spring of 2009 and caused a flu
pandemic (CDC 2009 H1N1 Flu website). Since then, this virus, known scientifically as the “A (H1N1)pdm09 virus”
and colloquially as the “2009 H1N1,” has circulated seasonally [4]. Over time, these H1N1 viruses have undergone rel-
atively minor genetic changes as well as changes in their antigenic properties (i.e., the properties of the virus that affect
immunity). Of all the influenza viruses that routinely circulate and cause illness in humans, influenza A(H3N2) viruses
change at a faster rate, both genetically and antigenically. In recent years, influenza A(H3N2) viruses have formed sev-
eral genetically distinct clades that continue to circulate.

4.3 Epidemiology
According to the World Health Organization (WHO), human cases of novel avian influenza A H7N9 infection were
found in late March and April 2013 in eastern China. However, the first case was identified on February 19, 2013
[5,6,7]. Furthermore, the bulk of the cases have occurred on China’s mainland. On December 1, 2013, in mainland
China, total of 139 laboratory-confirmed cases and one suspected case were determined in 10 provinces and two munic-
ipalities [8]. Among the confirmed cases in mainland China, 50 cases were found in Zhejiang, 33 cases in Shanghai, 28
cases in Jiangsu, six cases in Jiangxi, five cases in Fujian, four cases in both Anhui and Henan, two cases in Hunan,
Beijing, Shandong, and Guangdong, respectively, and one case in Hebei and Jilin [9]. Since 1997, sporadic human
infections have been increasingly detected with avian influenza viruses, partly because molecular analysis surveillance
and laboratory capability have improved worldwide, and also because changes in poultry production marketing prac-
tices have increased the opportunities for potential zoonotic viruses to emerge and distribute them.
Avian IAV classification is based on specific molecular and bird-pathogenic criteria and is considered as highly
pathogenic avian influenza (HPAI) or low pathogenic avian influenza (LPAI) viruses. LPAI viruses have been the cause
of past pandemics. While HPAI viruses have important economic and agricultural consequences, HPAI and LPAI infec-
tions have caused numerous mild to fatal diseases in human life. Therefore, the emphasis on the subtype of virus rather
than the pathogenicity of viruses in birds is on public health impact.
The first instance of a zoonotic avian IAV causing severe disease was in 1997 in Hong Kong when 18 cases of
H5N1 virus disease were detected leading to six deaths [10]. Outbreaks in poultry preceded human cases. The Hong
Kong outbreak stopped in December 1997 when all poultry were slaughtered on markets and farms in Hong Kong. In
the wider region, H5N1 viruses continue to circulate and develop among poultry. Zoonotic disease was again observed
in early 2003 with two deaths among two confirmed and one probable case [11] In 2005 wild migratory birds also
became infected with this virus and spread over central Asia, South Asia, the Middle East, and parts of Africa via bird
migration. Although these poultry outbreaks were stamped out successfully and repeatedly in some countries (e.g.,
Japan, Malaysia), they became enzootic within poultry in others, evolving into antigenically distinct and genetic diverse
clades leading to zoonotic disease [12]. As of May 2019, 861 human cases of H5N1 virus infection and 455 deaths had
been reported from 17 countries since November 2003, and the cumulative case fatality proportion among reported
H5N1 cases has remained greater than 50%, although few cases have been reported worldwide since 2016 [13,14].
H5N1 Clade 2.3.4.4 viruses have been ratings for H5N6, H5N8, and the other related subtypes with other avian IAVs
since 2013. More recently, H5N6 has become the dominant H5 lineage virus circulating in China, sometimes causing
zoonotic disease [15]. In 2003 an outbreak of HPAI H7N7 virus in poultry was associated with zoonotic disease affect-
ing 89 people in the Netherlands, who mostly have conjunctivitis, others had influenza-like illness, and one fatal pneu-
monia in a veterinarian. There was evidence of limited human-to-human transmission to family members of persons
directly exposed to infected poultry [16]. Six epidemics of human cases of H7N9 virus infection (1564 laboratory-
confirmed cases and 612 deaths) occurred in China through September 2017, typically during the fall, winter, and
spring months, including a very large fifth epidemic during 2016 to 2017 [17] Since 2013, 1568 laboratory-confirmed
H7N9 virus infections have been acquired in China, until May 2019. H7N9, as an LPAI virus, caused little to no illness
in poultry and spread across China. The seasonal increase in human cases was followed by an increase in virus circula-
tion among poultry. The cumulative case fatality proportion among reported H7N9 cases has remained approximately
40% since 2013 [17]. In 2016 the H7N9 virus acquired HPAI virus properties, causing disease in poultry. Since 2017,
China has introduced a bivalent H5N1/H7N9 vaccination program in poultry, which has resulted in a decrease in virus
 IAV infections in humans Chapter | 4 45

activity in poultry and a major reduction in zoonotic H7N9 disease. In 2018 only two H7N9 cases were registered, with
one more in the early spring of 2019. The recognition of clinically mild infections comes from sporadic cases identified
through routine influenza surveillance among outpatients with influenza-like illness [18], testing of ill persons with poul-
try exposures during large outbreaks of avian influenza follow-up of close contacts of confirmed cases (H7N9), and sero-
epidemiologic studies [19,20]. Therefore, asymptomatic and clinically mild illness cases of infections with avian IAVs
are likely underestimated, the true denominator of all infections is unknown, and the case fatality proportions for hospital-
ized patients are likely a substantial overestimate of the overall case fatalities for different virus infections [21,22].
In the recent years between April 9 and April 15, 2021, no new cases were reported on humans with H5N1 accord-
ing to WHO in the Western Pacific Region. In total from January 2003 to April 15, 2021 by cumulating all the positive
cases from the countries of Western Pacific Region, WHO reported 239 humans were prone to (H5N1) and from these
139 were death cases, resulting in a case fatality rate of 56%. The last case was reported from Lao PDR, with an onset
date of October 13, 2020 (one case, no death). From Russia seven human cases were infected with H5N8, a strain of
Avian influenza A and were first reported according to WHO on February 18, 2021, and all these cases were asymp-
tomatic. In the year 2007, 20 cases were positive to H5N1 and were reported from Pakistan [23]. Nasreen et al. [24]
reported seropositive from the workers who are working in Live Bird Market, in these studies they have taken samples
from 404 suspected workers among these only nine were responded to be seropositive and the remaining were seroneg-
ative. In spite of regular exposure to H5N1 strain these workers remained intermittent to H5N1 viral infection. From
Egypt, Fasina et al. [25] reported 90 human cases which were positive to H5N1, and among these 27 were death cases
from 2006 to 2007. Among these cases, women were exposed more to H5N1 when compared to men. During the out-
break of Avian influenza A, between 2005 and 2009 in Indonesia, it has been reported by Aditama et al. [26] that
H5N1 has tendency to increase transmission from human to human. In 2019 Potdar et al. [27] from India reported that
Avian influenza A H9N2 which is termed as low pathogenic has caused a severe acute respiratory infection in a 17-
month-old boy. There was the first report from Thailand that transmission of H5N1 is possible from human to human
within a family cluster in which three of the family were positive for H5N1, two were expired and the autopsy stated
that both were H5N1-infected cases and when the samples were sequenced there was no change in the receptor binding
of HA [28]. In 2003, in Hong Kong, another strain of Avian influenza A H9N2 was reported and the lineage was
closely related to poultry birds of Hong Kong [29].

4.4 Exposure risk factors to humans

When it came to exposure studies to different potential sources of A(H9N2) infection (Table. 4.1), most participants
(95%) had direct contact with live birds and (89%) had contact with dead birds. Only 4% of those respondents said

TABLE 4.1 The risk factors for human infections with avian influenza A viruses.

S. Risk factors Viruses

No.
1 Direct contact with infected well-appearing poultry or poultry products LPAI A viruses
2 Direct contact with infected sick or dead poultry or poultry products HPAI A viruses (e.g., H5N1, H5N6,
H7N7, H7N9)
3 Close exposure to infected well-appearing poultry LPAI A viruses
4 Close exposure to infected sick or dead poultry HPAI A viruses (e.g., H5N1, H5N6,
H7N7, H7N9)
5 Visiting a live poultry market LPAI A viruses (e.g., H7N9); HPAI A
viruses (e.g., H5N1)
6 Raising backyard poultry LPAI A viruses (e.g., H7N9); HPAI A
viruses (e.g., H5N1, H5N6)
7 Close, unprotected, prolonged exposure to an ill person with avian influenza A virus LPAI A viruses (e.g., H7N9); HPAI A
infection and respiratory illness viruses (e.g., H5N1)
8 Contact with live birds H9N2
9 Contact with virus/antigen H9N2
10 Vaccinator H9N2
46 Pandemic Outbreaks in the 21st Century

they had treated a live virus or antigen. Fourteen percent of participants reported contact with blood, while 23%
reported handling or processing animal tissue. The most important exposure was interaction with virus/antigen, as 92%
of exposure positive participants had seropositivity, compared to 47% in the exposure negative category. Most avian
IAV infections in humans have been sporadic and have been linked to recent direct interaction or near exposure with
domestic poultry, such as raising backyard poultry or attending a live poultry market. However, the source of exposure
is not always determined for some cases of human infection with avian IAVs [30]. Although cooking destroys virus
infectivity, contamination from the carcass before cooking may contribute to some of the cases of zoonotic avian IAV
infection with no history of direct exposure to live poultry [31]. Human exposure to avian IAVs such as H5N1, H5N6,
and H7N9 has been common in areas where enzootic poultry infections have occurred, but zoonotic disease is
unpredictable and uncommon. It is unclear that why there is a discrepancy between exposure and disease. One of the
study that carried out in Canada has been reported that the patient who had traveled from China to Canada has been
severely affected by H5N1 and led to death. After examining the postmortem report it has been revealed that most of
the sites are infected with H5N1 and by neuroimaging they reported that the severity leads to meningoencephalitis [32].

4.5 Pathophysiology
4.5.1 Viral replication in host
Avian influenza A H7N9 viruses isolated during the first four waves contain a cleavage site of HA with a single basic
amino acid, indicating that these are low pathogenic influenza virus strains. This allows the virus to spread silently
among poultry causing no or mild illness to indicate its presence [33]. The HA of the virus binds to cell receptors,
which then bind and fuse with the host endosomal membrane’s envelope. This HA should be cleaved by a host protease
after translation to allow fusion. Transmembrane protease S2 is the host protease that cleaves and activates the HA
influenza virus in the human respiratory tract. After the fusion process, the viral protein is released into the host cell by
the HA. Haemagglutinin precursor molecule (HAO) is the uncleaved precursor for known RNA. HAO is made up of
two subtypes: HA1 and HA2. These two proteins are known as fusion proteins. The amino acid sequence of HA recep-
tors affects the receptors either avian (-2,3 linked galactose) or human (-2,4 linked galactose). Recent studies have
shown that recombinant avian influenza A H7N9 virus with Q2261 (or Q226L) mutations is particularly associated with
human receptors. However, some recombinant virus lacking these mutations bind equally to both avian and human-type
receptors. Human upper air sacs are mainly exposed to alpha-galactose receptors, while human lung tissue contains
both alpha-2,3-linked galactose and alpha-2,6-linked galactose receptors. The H7N9 virus binds to human-type recep-
tors indicating that it displays tropism for both upper and lower airway cells and the virus transmission is also possible
from human to human. However, few studies have shown that H7 HA binds avian-type receptors (α2
3) to human-
type receptors. Analysis of infected human tracheal sections and tissues has shown that H7N9 can invade epithelial cells
in the lower respiratory tract and type-2 alveolar cells through the expression of viral nucleoprotein.

4.5.1.1 Role of chemokines and cytokines

Pattern recognition receptors (PRRs) and inflammasomes, which detect conserved viral pathogen-associated molecular
patterns (PAMPs), are important components of the antiviral innate immune system. The initial activation of PRRs by
viruses, primarily viral DNA and RNA, is crucial for a successful host response to viral infections. The activation of
PRR signaling pathways results in the expression of pro-inflammatory cytokines, which recruit immune cells, as well as
type I and type III interferons (IFNs), which induce IFN-stimulated genes (ISGs), powerful virus restriction factors that
maintain the “antiviral state.” Despite the fact that cytokine storms are a host defense response to pathogens, the highly
elevated levels of inflammatory mediators can compromise immunity and contribute to infection severity. However, the
underlying immunopathological mechanism of H7N9 infection is still remains unclear [34]. In a study that measured
the serum concentrations of chemokines and cytokines in patients infected with avian influenza A H7N9, the levels of
several chemokines and cytokines [i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1 beta,
IFN-inducible protein-10 (IP-10), interleukin (IL)-6, IL-8] were elevated compared to that of healthy individuals [35].
In another study serum concentrations of IP-10, IL-6, IL-17, and IL-2 were increased in avian influenza A H7N9-
infected patients, with severely ill patients exhibiting significantly higher levels of IL-6 and IP-10 [36]. When IAV gets
across the mucus that covers the respiratory epithelium, it first invades and infects respiratory epithelial cells, from
where it spreads to other nonimmune and immune cells (e.g., macrophages and dendritic cells). In these cells, the virus
can be sensed by the PRRs, triggering the production of type I IFNs which induce the expression of hundreds of ISGs
that block viral replication and further virus spread. Simultaneously, activation of PRRs also leads to production of
 IAV infections in humans Chapter | 4 47

pro-inflammatory cytokines [IL-6, IL-1β, IL-18, tumor necrosis factor (TNF), etc.] and chemokines. Pro-inflammatory
cytokines induce topical and systemic inflammation, cause fever and anorexia, and direct the adaptive immune response
against the virus. Chemokines, on the other hand, recruit innate immune cells [neutrophils, monocytes, and natural killer
(NK) cells] which engulf and inactivate the virus, kill virally infected cells, and guide subsequent innate and adaptive
immune responses that mediate ultimate viral clearance [37]. Fig. 4.1 schematically represents the host
pathogen inter-
action between humans and influenza A virus.
G IP-10 is a pro-inflammatory cytokine generated by endothelial cells, monocytes, fibroblasts, and hepatocytes, among
other cells. IP-10 belongs to the CXC chemokine family and is capable of recruiting and activating T-cells, NK
cells, monocytes/macrophages, dendritic cells, and eosinophils [38].
G γ d T-cells, Th17 lymphocytes, CD8 1 T-cells, and NK T-cells may all produce IL-17 (NKT). Pro-inflammatory
chemokines are activated by IL-17, and neutrophils are recruited into the respiratory tract. IL-17A plays an impor-
tant protective role in host defense in various infections, including asthma [38].
G Regulated on activation, normal T cell expressed and secreted, also known as CCL5 is a small 68-amino acid protein
that belongs to a rapidly growing chemokine family. It can be strongly induced by viral and bacterial infections and
recruits T-cells, dendritic cells, eosinophils, NK cells, mast cells, and basophils to sites of inflammation and infec-
tion [34].
G EOTAXIN is a potent chemokine that acts as a chemoattractant for eosinophils to sites of inflammation by selec-
tively stimulating the agonist activity for CC chemokine receptor 3 [34].
Increased levels of pro-inflammatory mediators in human lung endothelial cells during virus infection indicate that
H7N9 viruses can cause elevated levels of pro-inflammatory mediators in these cells, even though these cells may not
promote efficient viral replication. Endothelial and epithelium cells are the main cell structures in the human lungs
[38,39]. Infection of epithelial cells with H7N9 resulted in the release of a large amount of virus with cleaved HA and
damage to the epithelial monolayer due to cell death, especially necrosis. As a result of the release of H7N9 virus from
adjacent epithelial cells, pulmonary endothelial cells become vulnerable to infection. As a result, the viruses release
high levels of cytokines and chemokines, which draw lymphocytes to the lungs and aid viral clearance. However, a
cytokine cascade can result in enhanced lymphocyte infiltration and lung inflammation and damage, leading to pneumo-
nia and acute respiratory distress syndrome (ARDS) [40].
H5N1 influenza is a relatively new disease with limited knowledge of its anatomy and pathogenesis. Just a small
number of studies reporting pathological findings in human H5N1 cases have been reported in the time since the first
documented outbreak nearly a decade ago. Despite these challenges, recent research, coupled with early results, has
contributed to a deeper understanding of the H5N1 virus’s cell and organ pathology, as well as viral tissue tropism.

FIGURE 4.1 The host
pathogen

interaction between humans and
influenza A virus.
48 Pandemic Outbreaks in the 21st Century

These results, along with animal and in vitro studies, have helped to develop a basic understanding of the disease’s
pathogenesis. Several viral genes and gene products have been identified at the molecular level that may be responsible
for the H5N1 influenza virus’s high pathogenicity.
H9N2 infection is termed to be rare and majority of the studies were carried out in the mice to study the pathophysi-
ology and its survival in vivo, major histopathological changes in infected lungs included diffuse pneumonia and alveo-
lar damage, with neutrophil-dominant inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage,
and severe bronchiolitis/peribronchiolitis. In addition, H9N2 viral infection resulted in severe progressive hypoxemia,
lymphopenia, and a significant increase in neutrophils, TNF-α, and IL-6 in BALF. The features described above satisfy
the criteria for ARDS. Our data show that H9N2 viral infection resulted in ARDS in mice, and this may facilitate stud-
ies of the pathogenesis of future potential H9N2 disease in humans [41].

4.6 Innate immunity and adaptive immunity

The risk of the avian influenza virus infecting humans is a cause for concern. If three requirements are met: The popula-
tion lacks previous immunity to the novel virus, the virus causes illness and the virus can spread efficiently from person
to person via sustained chains of transmission then the virus can cause community-wide outbreaks. Even though influ-
enza A and B viruses cause seasonal influenza epidemics, influenza B viruses do not pose a pandemic danger because
there is no nonhuman reservoir in nature from which novel influenza B viruses can be introduced into humans.
Influenza viruses are enveloped viruses with an eight-gene segmented negative-sense RNA genome that each encodes
one or more proteins. Point mutations, which occur because the error-prone RNA-dependent RNA polymerase lacks
proofreading function, and genetic reassortment, in which a virus can acquire a novel genotype by deriving entire gene
segments from two separate parent viruses that coinfect a cell, account for the tremendous genetic diversity of IAVs.
Both occurrences are common in nature. Pathogen recognition receptors (PRRs) in infected cells, including dendritic
cells, detect PAMPs from the invading microorganism as the first step in the initiation of immune responses (DCs).
This recognition initiates a series of signaling events that result in the secretion of inflammatory cytokines, type I IFN,
chemokines, and antimicrobial peptides, and induces maturation of DCs [42]. PRRs are found in a variety of subcellular
locations. As a result, some of them, such as Toll-like receptors (TLRs) like TLR2, TLR4, TLR5, TLR6, and some C-
type lectins, are found in the plasma membrane. Additionally, the cell presents cytoplasmic receptors, as RNA heli-
cases, that are capable of sensing intracellular viral RNAs, like the retinoic acid-inducible gene I (RIG-I) and the mela-
noma differentiation antigen 5, or the nucleotide oligomerization domain-like receptors [43]. Since pathogens are
constantly present in the upper and lower airways, viral interactions with PRRs in these tissues are critical for eliciting
innate immune responses in the host against those pathogens. Epithelial cells in the lungs are the key cell targets for
influenza viruses, which express TLR3 and RIG-I, both of which are needed for the induction of type I IFN in response
to the detection of influenza virus replicative and genomic RNA. Immune cells such as macrophages and DCs are found
in the respiratory system, and they are important components of the innate immune response to pathogens because of
their high PRR expression and efficiency in generating pro-inflammatory cytokines (which also induce antiviral
responses in neighboring cells) when activated.
Influenza A viruses primarily target and infect airway and alveolar epithelial cells, which contain the salicylic acid
glycans as receptors, thus causing alveolar epithelial injury and eventually failure of gas exchange [44]. As a result,
human IAV infection can cause ARDS and even death. The innate immune response, which is quick but nonspecific, is
the first line of defense against viral infection. During IAV infection, host-PRRs, such as RIG-I and TLR, recognize
viral conserved components called PAMPs, resulting in innate immune signaling activation. As a result, the synthesis of
various cytokines and antiviral molecules is induced. PRRs can tell the difference between self and nonself molecules
in infected cells. RIG-I is the key receptor in infected host cells that recognizes intracellular ssRNA and transcriptional
intermediates of IAVs. Nonself RNA and transcriptional products of IAVs in the cytoplasm are also sensed by mela-
noma differentiation-associated gene 5 [45] RIG-I is activated and its caspase activation and recruitment domains
(CARDs) are revealed after PAMPs are recognized. Then the CARD is modulated by dephosphorylation or ubiquitina-
tion by E3 ligases, such as TRIM-containing protein 25 [46]. Thus, CARD-dependent association of RIG-I and MAVS
triggers the downstream transduction signaling at the outer mitochondrial membrane [47]. Following that, transcription
factors such as IFN regulatory factor 3 and IRF7, as well as nuclear factor kappa-light-chain-enhancer of activated B
cells (NF-B), become activated, resulting in the development of a variety of IFNs and cytokines [48]. TLR3 detects
virus-infected cells, while RIG-I and NLRP3 detect viral RNA that has been taken up into the endosomes of sentinel
cells (cell-intrinsic recognition), and TLR7 (and TLR8 in humans) detect viral RNA that has been taken up into the
endosomes of sentinel cells (cell-extrinsic recognition). During an IAV infection, dendritic cells, which are specialized
 IAV infections in humans Chapter | 4 49

antigen-presenting cells, serve as a link between the innate and adaptive immune responses. When naive and memory T
lymphocytes identify viral antigens provided by DCs, the adaptive immune response begins.
Enhancement of innate immunity against Avian influenza: During viral infections, the first line of protection is the
innate immune response, which is made up of both cellular and soluble mediators. Indeed, during HPAI infection of
birds, the protective function of innate immunity is clearly illustrated, with ducks exhibiting asymptomatic or mild dis-
ease while chickens become ill. The ability of CD81 T-cells to provide protection against antigenically novel influenza
viruses is based on their ability to recognize small, virus-derived peptides presented by major histocompatibility com-
plex (MHC) class I glycoprotein (HLA-I in humans) on the surface of infected cells. These peptides are most often
derived from the internal proteins of the virus, which show considerably less antigenic diversity than the surface glyco-
proteins. The innate immune system’s defense mechanisms provide a substantial barrier to the influenza virus. To com-
bat pathogen invasion, different mucosal surfaces have their own immune system. Various PRRs recognize the viral
RNA present within infected cells as foreign, resulting in the secretion of IFNs, pro-inflammatory cytokines,
eicosanoids, and chemokines. IFNs released by macrophages, pneumocytes, DCs, and plasmacytoid DCs stimulate the
expression of hundreds of genes in neighboring cells, collectively known as ISGs, resulting in an antiviral state. Pro-
inflammatory cytokines and eicosanoids cause local and systemic inflammation, as well as causing fever and anorexia
and instructing the adaptive immune response to the influenza virus. Chemokines generated at the site of infection
attract additional immune cells to the airways, such as neutrophils, monocytes, and NK cells. Virally infected epithelial
cells become the targets of NK cells, which mediate viral clearance [49]. Monocytes and neutrophils are quickly allo-
cated to the infected lung by the influenza virus and assist in the clearing of infected dead cells. Together with alveolar
macrophages, phagocytic clearance of virus-infected cells by recruited phagocytes provides an important mechanism of
viral clearance [50]. In humans, immune-histological analyses of lung tissues in five fatal cases of H5N1 showed an
influx of neutrophils into the alveolar spaces, while TNF and IL-6 were detected in monocytes/macrophages in situ [51].
In both cancer and chronic viral infections, NK cells, which make up to 10% of the lymphocyte compartment, play a
role in immune homeostasis and viral immunity. Their activation is regulated by the interaction of combinations of acti-
vating and inhibitory receptors with self and altered-self ligands, for example, killer Ig-like receptors, CD16 (Fcγ recep-
tor), NKp46 [52,53]. The host organism’s health can be restored by reducing pathogen burden (antiviral resistance) or
reducing the negative effect of infection on host fitness (disease tolerance). A unique host defense mechanism is a
host’s ability to withstand the presence of a pathogen. Although most acute infections necessitate resistance for the host
to survive, disease tolerance is a defense mechanism that protects the host from certain acute and chronic infections.
For example, infection of the African green monkey and sooty mangabey with simian immunodeficiency virus results
in high viral burden but is nonpathogenic [54]. The functions of these sensors and pathways in both innate resistance
and host tolerance have been revealed in studies using high or lethal doses of influenza A viruses in mice deficient in
innate sensors and signaling pathways, as the host frequently succumbs to the infection before they have had a chance
to produce robust adaptive immune responses. Simultaneously, challenging the host with sublethal doses of influenza A
virus or inactive virus allows the host to survive the infection by staging a defensive adaptive immune response, and
such studies have revealed which viral sensors link innate recognition to adaptive immunity. Increased innate resistance
or increased host tolerance to infection may provide host defense against any infection. High-pathogenic influenza
viruses, on the other hand, cause excessive inflammatory responses that reduce host health. Protective innate immune
responses minimize disease burden in infected hosts by raising antiviral resistance and/or increasing disease tolerance,
while pathogenic innate immune responses have a negative effect on host health by causing tissue damage in the
attempt to reduce pathogen burden.
The immune cross-reactivity between human and avian influenza (H5N1) strains in healthy donors vaccinated for
seasonal influenza A (H1N1)/(H3N2). A small frequency of CD4 T-cells specific for subtype H5N1 was detected in
several persons at baseline, and seasonal vaccine administration enhanced the frequency of such reactive CD4 T-cells.
It has been observed that seasonal vaccination has the ability to raise neutralizing immunity against influenza (H5N1)
in a large number of donors and it was also observed that there is no correlation between influenza-specific CD4
T-cells and humoral responses. There is a possible way to get cross-type cellular and humoral immunity by seasonal
vaccination against Avian Influenza A (H5N1) [55]. Innate immunity is most critical during the early phase of IAV
infection before getting into adaptive immune responses. Pulmonary surfactant collectins (especially surfactant protein
D) in both immune responses are restricting the extent of viral replication and limiting potentially damaging inflamma-
tion in the first few days of IAV infection. The other type of inhibitors against IAV that were included are type I IFNs
and alveolar macrophages. Soluble scavenger receptor
rich glycoprotein 340 and defensins also have anti-IAV activity.
Neutrophils also contribute in complex ways to host response to IAV, and increased neutrophil influx characterizes
severe IAV infection in which the usual innate responses are ineffective. Some innate mechanisms strongly affect
50 Pandemic Outbreaks in the 21st Century

elaboration and intensity of the adaptive immune response to IAV. A key conclusion of this review is that the innate
immune system not only plays a role in nonspecific restriction of viral replication but also in downregulating nonspe-
cific and antigen-specific immune injury. Excessive inflammatory responses are now implicated in many severe out-
comes of IAV infection. Better understanding of innate immunity to IAV should clarify why some subjects are at
greater risk for severe IAV infection, and why certain IAV strains are more virulent, and also suggest novel antiviral
and antiinflammatory therapeutic approaches [56].

4.7 Diagnosis
There are currently only a few diagnostic tests available to identify H7N9. As a result, reverse transcription-polymerase
chain reaction (RT-PCR) has emerged as the gold standard for avian influenza detection. Because the signs, symptoms,
and clinical results of H7N9 are nonspecific, clinical suspicion of avian influenza exists. A virus infection (all subtypes
with at least one human infection) is focused on eliciting a history of recent poultry exposure in a virus enzootic area,
specifically visiting a market where live poultry are sold or slaughtered, small farms, or inside/outside homes (where
poultry are confined) (viruses in which limited human-to-human transmission has been reported, namely, H5N1, H7N7,
and H7N9). The best respiratory specimens to collect are determined by the time between illness onset and appearance
of symptoms, the suspected site of major pathology, and the nature of the patient’s disease. For example, a nasopharyn-
geal specimen may be appropriate for detecting certain avian influenza A viruses associated with upper respiratory
symptoms, but a throat swab specimen has a higher yield for detecting H5N1 virus in patients without extreme lower
respiratory tract disease. The collection of respiratory specimens from several respiratory sites, including sputum, will
improve the chances of detecting avian influenza A virus infection in hospitalized patients. An endotracheal aspirate or
bronchoalveolar lavage specimen should be collected for testing in critically ill patients with respiratory failure who are
receiving invasive mechanical ventilation. Commercially available influenza tests, including molecular assays, detect
influenza A and B viruses in clinical settings but do not distinguish between seasonal influenza A viruses circulating
worldwide and zoonotic avian influenza A viruses. As a result, respiratory specimens must be sent to a public health
laboratory for specific testing for avian influenza A virus subtypes (e.g., H5, H7, H9) and additional analyses using RT-
PCR. For detecting avian influenza A virus infection, antigen detection tests are less sensitive than RT-PCR assays.
Serologic examination of paired acute and convalescent sera may be used to render a retrospective diagnosis, but it
must be conducted in a specialized public health or research facility.

4.8 Clinical findings in H5N1 infection

Most of the laboratory’s confirmed cases of H5N1 infection were hospitalized patients with severe illness complicated
by ARDS and multiorgan failure, although some milder illnesses have been reported. Among H5N1-infected patients,
high viral load, lymphopenia, unusually high serum levels of IP-10, and high serum levels of inflammatory cytokines
and chemokines were associated with a fatal outcome [57].

4.9 Detection of Avian influenza

Infection diagnosis or understanding that a pandemic has occurred is only possible if technology is available for rapid
detection and analysis of the influenza virus. Individual diseases, epidemics, pandemics, and so on all need fast and
responsive sensors and diagnostics to react and prevent them. Worldwide, health organizations, government depart-
ments, academia, and independent laboratories have made significant efforts in influenza detection and identification.
These activities have led to a change in strategy away from culture-based serological assays and toward genetic charac-
terization methods and modern optical and electrical biosensors in recent decades. For detecting viruses and measuring
influenza virus gene expression, the PCR and related techniques such as RT-PCR and real-time PCR have been com-
monly used in biomedical laboratories. In addition, whole-genome sequencing methods have been used to expand infor-
mation on viral genetics [58]. These molecular detection methods are sensitive, allowing for the rapid and precise
detection of genetic variations in a wide range of influenza viruses. In the context of viral surveillance for vaccine
development, although thousands of influenza virus samples from patients are collected and analyzed during year-round
surveillance for influenza [59,60,61]. Antigenicity and evolution of influenza viruses are not always fully understood
using genetic knowledge. This is because genetic mutations may alter virus recognition by components of the immune
system in ways that are challenging to predict [62,63]. As a result, selection of the appropriate vaccine candidates
frequently results in suboptimal protection against circulating viral strains (Center for Disease Control, 2005
18).
 IAV infections in humans Chapter | 4 51

For example, overall vaccine effectiveness was reported to be only 19.8% for the influenza A H3N2 subtypes circulat-
ing in the 2014
15 season [64]. Efficacy during the 2017
18 season was even worse, dropping as low as 13% and
resulting in the highest spike in influenza-associated deaths and hospitalizations across the United States in recent years
[65]. As a result of these problems, alternative high-throughput methods for testing influenza virus antigenicity that do
not rely on sequencing are important. Assays based on immunosensors are being developed as fast, low-cost, and multi-
plex tools. These have a lot of potential for studying the host immune response as well as the antigenicity of the influ-
enza virus. As a result, these assays will make a valuable contribution to the ongoing process of influenza surveillance
and vaccine development.
Viruses use the cellular machinery to complete their replication cycle as intracellular parasites. The basic structure
of virus is comprised of genome (DNA or RNA), protein capsid (for nucleic acid protection) and in some a lipidic enve-
lope that covers the capsid [66,67]. Influenza virus is a member of the Orthomyxoviridae family, with (2)ssRNA
nucleic acid, and the species A and B are the most commonly associated with human infection and disease [68]. The
influenza virus exhaust path consists of the alteration in the HA antigens (1
18) and the NA (1
11), responsible for
the attachment and penetration in the host cell, respectively [69].
Immunosensors (antibody
antigen interaction), enzymatic biosensors (enzyme
target analyte interaction), DNA
biosensors (hybridization), and whole-cell biosensors are all types of biosensors that detect analytes or reactions.

4.9.1 Biosensors
A biosensor is an analytical device defined as biological (or)bioinspired receptor with corresponding analyte. The ana-
lytes are often biological origin like DNA of bacteria/virus/protein. It determines the presence and concentration of a
specific substance in any test solution. Currently, standard influenza antigenicity immunoassays depend on serological
methods involving live cells, viruses, and unique serum reagents. The most popular immunoassays for detecting influ-
enza viruses and determining antigenicity are hemagglutination and hemagglutination inhibition assays, which quantify
the precise interaction of HA with the host cell surface glycan.

4.9.2 Electrical biosensors

Electrochemical biosensors detect analytes by measuring changes in electrical properties such as voltage and current
induced by biochemical interactions. A standard electrochemical biosensor device has a sensing electrode and a refer-
ence electrode separated by an aqueous electrolyte, which completes the current flow circuit. This conceptually simple
design can be implemented in principle at low cost and with high energy efficiency, because inexpensive electrodes can
be easily integrated with low power electronic systems to perform rapid and real-time measurements in miniaturized
formats [70]. These characteristics make electrochemical biosensors highly attractive for applications in medical diag-
nosis and infectious disease monitoring [71]. For many years, most electrochemical biosensors for influenza virus detec-
tion have focused on nucleic acid detection [72]. On the electrode surface, the hybridization phase results in the
formation of a duplex structure of nucleic acid probes and targets, which changes the thickness of the layer between the
electrode and the electrolyte.

4.9.3 Immunosensors
Immunosensors are biological sensors that rely on antibodies and antigens interacting in a particular way. When lym-
phocyte B comes into contact with an antigen, it releases antibodies and goes through clonal expansion and differentia-
tion. After, the antigens are eliminated, followed by the apoptosis of effector lymphocytes and remaining of memory B
cells [73]. It has been developed for continuous monitoring of analytes through point-of-care devices, which provide
low cost, full automation, portability, fast response, high sensitivity, accuracy, and precision [74]. The application of
immunosensors in clinical diagnosis and monitoring of diseases has been emphasized in recent works and it is mainly
reported for the detection of biomarkers, hormones, bacteria, and toxins.

4.9.4 Enzymatic biosensors

Enzymatic biosensors take advantage of enzymes’ catalytic properties, which are biorecognition molecules with high
chemical specificity and excellent selectivity for their target substrate. In enzymatic sensing, the device is combined
with a transducer, which reacts selectively with its analyte, generating an electrochemical [75], optical biosensor.
52 Pandemic Outbreaks in the 21st Century

This signal is proportional to the analyte concentration in the sample. Enzyme electrodes are used in a variety of point-
of-care and clinical applications for a wide range of analytes since they provide many distinct advantages.

4.9.5 Geno sensors

Geno sensors, or deoxyribonucleic acid biosensors, have been used to detect and diagnose various diseases due to their
inherent physicochemical stability and suitability. DNA is the genetic information carrier and is found in any living
organism, virus, and pathogen. As a result of their unique nucleic acid sequences, DNA biosensors can distinguish
between species and diagnose a variety of diseases and human pathogens. The detection theory of a DNA biosensor is
based on the immobilization of a DNA or RNA strand (probe) on the surface of a physical transducer in order to detect
its complementary (target) sequence through hybridization. The duplex formation can be detected following the associa-
tion of an appropriate hybridization indicator or through other changes accrued from the binding event [76,77,78].
Through the efforts of researchers, many new Geno sensors have emerged in recent years into clinical applications to
detect various diseases and pathogens [5,79].

4.9.6 Whole-cell biosensors

As detection components, whole cells can be used. Biorecognition can be triggered by a number of surface antigens
found on cell envelopes, such as proteins, glycoproteins, lipopolysaccharides, and peptidoglycan. Biosensors for entire
microorganisms are difficult to produce because they must detect analytes that are much larger (micrometer scale) than
standard molecular analytes like proteins (nanometer scale). Nonspecific interactions with the sensor surface can be
caused by a variety of surface epitopes. Nevertheless, organisms used to develop whole-cell biosensors are generally
experimentally modified to incorporate transducer capacity or increase their sensitivity [80]. The whole cell-based bio-
sensor is increasingly being reported in the literature, and these reports have shown high selectivity, sensitivity, and
great potential for their use in biomedical diagnostics [81,82].

4.10 Conclusion
The most likely candidate for the next influenza pandemic appears to be avian influenza. Vaccines are likely the most
effective means of specific protection, but their ability to produce effective vaccines in a timely manner will limit their
role in the early stages of the pandemic. Efforts to avert or mitigate the effects of the impending pandemic should not
be limited to therapeutic measures. Simple community infection control and personal hygiene measures are important
for respiratory infections, as demonstrated by the 2003 SARS epidemic. Currently, human avian infection is limited to
close contact with infected animals. Avoiding close contact with poultry or wild birds could significantly reduce the
risk of infection for travelers to avian influenza endemic areas. Furthermore, assays that are as sensitive as PCR for
viral DNA and RNA detection and at the same time require minimal resources and swift in generating the results are
needed. Biosensors meet these criteria, and they have been designed for a variety of applications, including health care.
We suggest taking advantage of the recent advances in biosensors for respiratory viruses, with a focus on influenza, cor-
onaviruses, and respiratory syncytial virus, these sensors could be utilized for real-time monitoring, and rapid on-site
pathogen detection aiding in the early detection and control of the future pandemics of influenza A viruses.

Acknowledgment
We are very thankful to Dr. Buddolla’s Institute of Life Sciences for supporting us in writing this chapter.

Conflict of interest
The authors declare that they have no conflict of interest.

References
[1] Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EPJ, Chen L, et al. Transmission of equine influenza virus to dogs. Science
2005;310(5747):482
5.
[2] Rajao DS, Vincent AL, Perez DR. Adaptation of human influenza viruses to swine. Front Vet Sci 2019;5:347.
[3] Chen R, Holmes EC. The evolutionary dynamics of human influenza B virus. J Mol Evol 2008;66(6):655.
 IAV infections in humans Chapter | 4 53

[4] Steel J, Lowen AC. Influenza A virus reassortment. Curr Top Microbiol Immunol 2014;385:377
401.
[5] Shen Y, Lu H. Global concern regarding the fifth epidemic of human infection with avian influenza A (H7N9) virus in China. Biosci Trends
2017;11(1):120
1.
[6] Zhou L, Ren R, Yang L, Bao C, Wu J, Wang D, et al. Sudden increase in human infection with avian influenza A (H7N9) virus in China,
September
December 2016. WPSAR 2017;8(1):6.
[7] Bae JY, Kim JI, Park S, Yoo K, Kim IH, Joo W, et al. Effects of Lactobacillus plantarum and Leuconostoc mesenteroides probiotics on human
seasonal and avian influenza viruses. J Microbiol Biotechnol 2018;28(6):893
901.
[8] Poovorawan Y. Epidemic of avian influenza A (H7N9) virus in China. Pathog Global Health 2014;108(4):169.
[9] Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Preliminary report: epidemiology of the avian influenza A (H7N9) outbreak in China. N
Engl J Med 2013;10.
[10] Chan PK. Outbreak of avian influenza A (H5N1) virus infection in Hong Kong in 1997. Clin Infect Dis 2002;34(Suppl. 2):S58
64.
[11] Peiris JSM, Yu WC, Leung CW, Cheung CY, Ng WF, Nicholls JA, et al. Re-emergence of fatal human influenza A subtype H5N1 disease.
Lancet 2004;363(9409):617
19.
[12] Webster RG, Peiris M, Chen H, et al. H5N1 outbreaks and enzootic influenza. Emerg Infect Dis 2006;12(1):3
8.
[13] Lai S, Qin Y, Cowling BJ, Ren X, Wardrop NA, Gilbert M, et al. Global epidemiology of avian influenza A H5N1 virus infection in humans,
1997
2015: a systematic review of individual case data. Lancet Infect Dis 2016;16(7):e108
18.
[14] World Health Organization. Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to WHO, ,http://www.who.
int/csr/di-sease/avianinfluenza/country/en/.; 2009.
[15] Claes F, Morzaria SP, Donis RO. Emergence and dissemination of clade 2.3. 4.4 H5Nx influenza viruses—how is the Asian HPAI H5 lineage
maintained. Curr Opin Virol 2016;16:158
63.
[16] Koopmans M, Wilbrink B, Conyn M, Natrop G, van der Nat H, Vennema H, et al. Transmission of H7N7 avian influenza A virus to human
beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 2004;363(9409):587
93.
[17] Wang X, Jiang H, Wu P, Uyeki TM, Feng L, Lai S, et al. Epidemiology of avian influenza A H7N9 virus in human beings across five epi-
demics in mainland China, 2013
17: an epidemiological study of laboratory-confirmed case series. Lancet Infect Dis 2017;17(8):822
32.
[18] Xu C, Havers F, Wang L, Chen T, Shi J, Wang D, et al. Monitoring avian influenza A (H7N9) virus through national influenza-like illness sur-
veillance, China. Emerg Infect Dis 2013;19(8):1289.
[19] Lopez-Martinez I, Balish A, Barrera-Badillo G, Jones J, Nuñez-Garcı́a TE, Jang Y, et al. Highly pathogenic avian influenza A(H7N3) virus in
poultry workers, Mexico, 2012. Emerg Infect Dis 2013;19(9):1531
4. Available from: https://doi.org/10.3201/eid1909.130087.
[20] Chen Z, Liu H, Lu J, Luo L, Li K, Liu Y, et al. Asymptomatic, mild, and severe influenza A (H7N9) virus infection in humans, Guangzhou,
China. Emerg Infect Dis 2014;20(9):1535.
[21] Yu H, Cowling BJ, Feng L, Lau EH, Liao Q, Tsang TK, et al. Human infection with avian influenza A H7N9 virus: an assessment of clinical
severity. Lancet 2013;13:138
45. Available from: https://doi.org/10.1016/S0140-6736(13)61207-6.
[22] Feng L, Wu JT, Liu X, Yang P, Tsang TK, Jiang H, et al. Clinical severity of human infections with avian influenza A (H7N9) virus, China,
2013/14. Eurosurveillance 2014;19(49):20984.
[23] Zaman M, Ashraf S, Dreyer NA, Toovey S. Human infection with avian influenza virus, Pakistan, 2007. Emerg Infect Dis 2011;17(6):1056.
[24] Nasreen S, Khan SU, Luby SP, Gurley ES, Abedin J, Zaman RU, et al. Highly pathogenic avian influenza A (H5N1) virus infection among
workers at live bird markets, Bangladesh, 2009
2010. Emerg Infect Dis 2015;21(4):629.
[25] Fasina FO, Ifende VI, Ajibade AA. Avian influenza A (H5N1) in humans: lessons from Egypt. Eurosurveillance 2010;15(4):19473.
[26] Aditama TY, Samaan G, Kusriastuti R, Sampurno OD, Purba W, Santoso H, et al. Avian influenza H5N1 transmission in households,
Indonesia. PLoS One 2012;7(1):e29971.
[27] Potdar V, Hinge D, Satav A, Simões EA, Yadav PD, Chadha MS. Laboratory-confirmed avian influenza A (H9N2) virus infection, India, 2019.
Emerg Infect Dis 2019;25(12):2328.
[28] Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, Puthavathana P, et al. Probable person-to-person transmission of avian influ-
enza A (H5N1). N Engl J Med 2005;352(4):333
40.
[29] Butt KM, Smith GJ, Chen H, Zhang LJ, Leung YC, Xu KM, et al. Human infection with an avian H9N2 influenza A virus in Hong Kong in
2003. J Clin Microbiol 2005;43(11):5760
7.
[30] Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of human infections with avian influenza A (H7N9) virus in China. N Engl J
Med 2014;370(6):520
32.
[31] Mao X, Wu J, Lau EH, Cheng KL, Zhong Z, Song Y, et al. Monitoring avian influenza viruses from chicken carcasses sold at markets, China,
2016. Emerg Infect Dis 2017;23(10):1714.
[32] Rajabali N, Lim T, Sokolowski C, Prevost JD, Lee EZ. Avian influenza A (H5N1) infection with respiratory failure and meningoencephalitis in
a Canadian traveller. Can J Infect Dis Med Microbiol 2015;26(4):221
3.
[33] Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, et al. Genetic analysis of novel avian A (H7N9) influenza viruses isolated
from patients in China, February to April 2013. Eurosurveillance 2013;18(15):20453.
[34] Wu W, Shi D, Fang D, Guo F, Guo J, Huang F, et al. A new perspective on C-reactive protein in H7N9 infections. Int J Infect Dis
2016;44:31
6.
[35] Zhou J, Wang D, Gao R, Zhao B, Song J, Qi X, et al. Biological features of novel avian influenza A (H7N9) virus. Nature 2013;499
(7459):500
3.
54 Pandemic Outbreaks in the 21st Century

[36] Guo J, Huang F, Liu J, Chen Y, Wang W, Cao B, et al. The serum profile of hypercytokinemia factors identified in H7N9-infected patients can
predict fatal outcomes. Sci Rep 2015;5(1):1
10.
[37] Iwasaki A, Pillai PS. Innate immunity to influenza virus infection. Nat Rev Immunol 2014;14(5):315
28.
[38] Tenforde MW, Gupte N, Dowdy DW, Asmuth DM, Balagopal A, Pollard RB, et al. C-reactive protein (CRP), interferon gamma-inducible pro-
tein 10 (IP-10), and lipopolysaccharide (LPS) are associated with risk of tuberculosis after initiation of antiretroviral therapy in resource-limited
settings. PLoS One 2015;10(2):e0117424.
[39] Driscoll KE. Macrophage inflammatory proteins: biology and role in pulmonary inflammation. Exp Lung Res 1994;20(6):473
90.
[40] Zeng H, Belser JA, Goldsmith CS, Gustin KM, Veguilla V, Katz JM, et al. A (H7N9) virus results in early induction of proinflammatory cyto-
kine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with avian H5N1 virus. J
Virol 2015;89(8):4655
67.
[41] Deng G, Bi J, Kong F, Li X, Xu Q, Dong J, et al. Acute respiratory distress syndrome induced by H9N2 virus in mice. Arch Virol 2010;155(2):187
95.
[42] Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 2010;11(5):373.
[43] Kanneganti TD. Central roles of NLRs and inflammasomes in viral infection. Nat Rev Immunol 2010;10(10):688
98.
[44] van Riel D, den Bakker MA, Leijten LM, Chutinimitkul S, Munster VJ, de Wit E, et al. Seasonal and pandemic human influenza viruses attach
better to human upper respiratory tract epithelium than avian influenza viruses. Am J Pathol 2010;176(4):1614
18.
[45] Pichlmair A, Schulz O, Tan CP, Näslund TI, Liljeström P, Weber F, et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing
5’-phosphates. Science 2006;314(5801):997
1001. Available from: https://doi.org/10.1126/science.1132998.
[46] Munir I, Yaqub T, Mukhtar N, Shabbir MZ, McCauley JW. Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild
terrestrial birds. Veterinary research 2013;44(1):1
10.
[47] Yoneyama M, Onomoto K, Jogi M, Akaboshi T, Fujita T. Viral RNA detection by RIG-I-like receptors. Curr Opin Immunol 2015;32:48
53.
Available from: https://doi.org/10.1016/j.coi.2014.12.012.
[48] Hiscott J. Triggering the innate antiviral response through IRF-3 activation. J Biol Chem 2007;282:15325
9.
[49] Gazit R, Gruda R, Elboim M, Arnon TI, Katz G, Achdout H, et al. Lethal influenza infection in the absence of the natural killer cell receptor
gene Ncr1. Nat Immunol 2006;7(5):517
23.
[50] Hashimoto Y, Moki T, Takizawa T, Shiratsuchi A, Nakanishi Y. Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neu-
trophils and macrophages in mice. J Immunol 2007;178(4):2448
57.
[51] Nakajima N, Van Tin N, Sato Y, Thach HN, Katano H, Diep PH, et al. Pathological study of archival lung tissues from five fatal cases of avian
H5N1 influenza in Vietnam. Mod Pathol 2013;26(3):357
69.
[52] Jegaskanda S, Reading PC, Kent SJ. Influenza-specific antibody-dependent cellular cytotoxicity: toward a universal influenza vaccine. J
Immunol 2014;193(2):469
75.
[53] Parham P, Norman PJ, Abi-Rached L, Guethlein LA. Human-specific evolution of killer cell immunoglobulin-like receptor recognition of major
histocompatibility complex class I molecules. Philos Trans R Soc B Biol Sci 2012;367(1590):800
11.
[54] Paiardini M, Pandrea I, Apetrei C, Silvestri G. Lessons learned from the natural hosts of HIV-related viruses. Annu Rev Med 2009;60:485
95.
[55] Gioia C, Castilletti C, Tempestilli M, Piacentini P, Bordi L, Chiappini R, et al. Cross-subtype immunity against avian influenza in persons
recently vaccinated for influenza. Emerg Infect Dis 2008;14(1):121.
[56] Tecle T, White MR, Hartshorn KL. Innate immunity to influenza A virus infection. Curr Respir Med Rev 2005;1(2):127
45.
[57] de Jong MF, Rolán HG, Tsolis RM. Innate immune encounters of the (Type) 4th kind: Brucella. Cell Microbiol 2010;1:1195
202. Available
from: https://doi.org/10.1111/j.1462-5822.2010.01498.x.
[58] McGinnis J, Laplante J, Shudt M, George KS. Next generation sequencing for whole genome analysis and surveillance of influenza A viruses. J
Clin Virol 2016;79:44
50.
[59] Ginsberg J, Mohebbi MH, Patel RS, Brammer L, Smolinski MS, Brilliant L. Detecting influenza epidemics using search engine query data.
Nature 2009;457(7232):1012
14.
[60] Nelson MI, Edelman L, Spiro DJ, Boyne AR, Bera J, Halpin R, et al. Molecular epidemiology of A/H3N2 and A/H1N1 influenza virus during
a single epidemic season in the United States. PLoS Pathog 2008;4(8):e1000133.
[61] Ghedin E, Laplante J, DePasse J, Wentworth DE, Santos RP, Lepow ML, et al. Deep sequencing reveals mixed infection with 2009 pandemic
influenza A (H1N1) virus strains and the emergence of oseltamivir resistance. J Infect Dis 2011;203(2):168
74.
[62] Glaser L, Stevens J, Zamarin D, Wilson IA, Garcı́a-Sastre A, Tumpey TM, et al. A single amino acid substitution in 1918 influenza virus hem-
agglutinin changes receptor binding specificity. J Virol 2005;79(17):11533
6.
[63] Smith DJ, Lapedes AS, De Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, et al. Mapping the antigenic and genetic evolution of
influenza virus. Science 2004;305(5682):371
6.
[64] Zimmerman RK, Nowalk MP, Chung J, Jackson ML, Jackson LA, Petrie JG, et al. 2014
2015 influenza vaccine effectiveness in the United
States by vaccine type. Clin Infect Dis 2016;63(12):1564
73.
[65] Flannery B, Chung JR, Belongia EA, McLean HQ, Gaglani M, Murthy K, et al. Interim estimates of 2017
18 seasonal influenza vaccine effec-
tiveness—United States, February 2018. Morb Mortal Wkly Rep 2018;67(6):180.
[66] Wessner DR. The origins of viruses. Nat Educ 2010;3. Available from: https://www.nature.com/scitable/topicpage/the-origins-of-viruses-
14398218/ [accessed 01.04.20].
[67] Tortora GJ, Funke BR, Case CL. Vı́rus, Viróides e Prions. In: Laser House, editor. Microbiologia. 8th ed. Porto Alegre: Artmed; 2006, p.
376
408.
 IAV infections in humans Chapter | 4 55

[68] Ribeiro BV, Cordeiro TAR, e Freitas GRO, Ferreira LF, Franco DL. Biosensors for the detection of respiratory viruses: a review. Talanta Open
2020;2:100007.
[69] Yang JM, Kim KR, Kim CS. Biosensor for rapid and sensitive detection of influenza virus. Biotechnol Bioprocess Eng 2018;23(4):371
82.
[70] Hammond JL, Formisano N, Estrela P, Carrara S, Tkac J. Electrochemical biosensors and nanobiosensors. Essays Biochem 2016;60(1):69
80.
[71] Campuzano S, Yáñez-Sedeño P, Pingarrón JM. Electrochemical biosensing for the diagnosis of viral infections and tropical diseases.
ChemElectroChem 2017;4(4):753
77.
[72] Grabowska I, Malecka K, Jarocka U, Radecki J, Radecka H. Electrochemical biosensors for detection of avian influenza virus
current status
and future trends. Acta Biochim Pol 2014;61(3):471
8.
[73] Abbas AK, Lichtman AH, Pillai S. Cellular and molecular immunology E-book. Elsevier Health Sciences; 2014.
[74] Bahadır EB, Sezgintürk MK. Applications of electrochemical immunosensors for early clinical diagnostics. Talanta 2015;132:162
74.
[75] Liu A, Lang Q, Liang B, Shi J. Sensitive detection of maltose and glucose based on dual enzyme-displayed bacteria electrochemical biosensor.
Biosens Bioelectron 2017;87:25
30.
[76] Kavita V. DNA biosensors—a review. J Bioeng Biomed Sci 2017;7(2):222.
[77] Rasheed PA, Sandhyarani N. Electrochemical DNA sensors based on the use of gold nanoparticles: a review on recent developments.
Microchim Acta 2017;184(4):981
1000.
[78] Rashid JIA, Yusof NA. The strategies of DNA immobilization and hybridization detection mechanism in the construction of electrochemical
DNA sensor: a review. Sens Bio-sens Res 2017;16:19
31.
[79] Kaur G, Paliwal A, Tomar M, Gupta V. Detection of Neisseria meningitidis using surface plasmon resonance-based DNA biosensor. Biosens
Bioelectron 2016;78:106
10.
[80] Gui Q, Lawson T, Shan S, Yan L, Liu Y. The Application of Whole Cell-Based Biosensors for Use in Environmental Analysis and in Medical
Diagnostics. Sensors (Basel) 2017;17(7):1623. Available from: https://doi.org/10.3390/s17071623.
[81] Riangrungroj P, Bever CS, Hammock BD, Polizzi KM. A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid
insecticide exposure. Sci Rep 2019;9(1):1
9.
[82] Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM. Whole-cell Escherichia coli lactate biosensor for monitoring mam-
malian cell cultures during biopharmaceutical production. Biotechnol Bioeng 2017;114(6):1290
300.
This page intentionally left blank
Chapter 5

Swine-origin influenza A (H1N1) virus:

current status, threats, and challenges
Praveen Belagal1, Hemanth Naick Banavath2 and Buddolla Viswanath3
1
DST PURSE Centre, Sri Venkateswara University, Tirupati, India, 2Department of Sports Bio-Sciences, Central University of Rajasthan, Ajmer,
India, 3Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr. Buddolla’s Educational Society, Tirupati, India

5.1 Introduction
Viral infections are the major concern to public health and scientific communities. Regular influenza virus outbreaks do
happen, but the severity of outbreaks may differ due to point mutations and reassortment of genetic segments that con-
tribute to the emergence of new variants. Influenza viruses belong to the family Orthomyxoviridae and are grouped into
four genera, that is, type A (IAV), B (IBV), C (ICV) [1], and the type D (IDV) recently identified [2] and characterized
[3]. The genomes of IAVs are highly dynamic causing outbreaks with epidemic or pandemic potential. Primarily, influ-
enza types A and B are responsible for major outbreaks in humans. IAVs are identified as both seasonal epidemics (dur-
ing winter) and global pandemics, whereas IBV is associated with seasonal epidemics only but do not cause pandemics
[4]. Types C and D are less virulent, genetically stable, and infect only animals. Influenza pandemics are
unpredictable but recurring events can have severe consequences on human health and economic development. Until
recently, epidemiological studies of influenza were limited to resource-rich countries. There are three principal events
all-inclusive must be met for any influenza pandemic in humans to breakout.
G A novel virus strain to which the vast majority of the population lacking immunity has to appear.
G The new virus has to cause severe disease in humans.
G The virus has to be able to easily acquire a sustained human
human transmissibility.
The flu, in general, is an infection in the nose, throat, and lungs that is caused by a virus and is contagious through
droplets in the air released from coughing or sneezing, or through touching surfaces (fomites) that has the virus and
then touching the mouth, nose, or eyes through which the infection starts. There are different kinds of flu, like the one
that people get every winter season, for which one can get a flu shot as a preventive measure. Bird flu and swine flu are
other types that primarily infect animals.
Influenza A virus (IAV) though was reported in the history, reliable data of H1N1 strain, most likely originating
from an avian species were reported during the 1918 pandemic, also known as “Spanish flu” [5]. Post-1918 pandemic,
it was confirmed in the United States in 1930 that the same H1N1 can also infect pigs as classical swine H1N1 [6].
Pigs have receptors for both human influenza virus as well as avian influenza viruses and act as a site for genetic reas-
sortment of different segments of genes and responsible for the potential antigenic shift. Thus, pig acts as a natural mix-
ing vessel and responsible for interspecies transmission [7,8]. However, infection in humans by avian IAVs is also
possible [9] but the barrier is bit high for avian preferable receptors (SA α-2,3) as they are less abundant that too con-
fined to lower respiratory tract (LRT) [10,11]. Although various strains of avian influenza strains have been recognized
for decades, the lethality and mutability of the H1N1 subtype of the IAV served as the source of the human influenza
pandemic via zoonotic transfer from swine to humans and then acquiring human
human transmissibility.
Swine flu in pigs worldwide is caused by type A influenza viruses, principally the subtypes H1N1, H1N2, H2N1,
H3N1, H3N2, and H2N3. So far, only the subtypes H1N1, H2N2, and H3N2 have caused pandemics through sustained
human-to-human transmission. Similar to seasonal influenzas, swine influenza leads to respiratory disease which can
potentially affect the respiratory tract of pigs which is why it was originally called Swine flu. One cannot contract this

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00015-X

© 2021 Elsevier Inc. All rights reserved. 57
58 Pandemic Outbreaks in the 21st Century

swine flu infection by consuming pork, bacon, sausages, or any other pork products but people in close proximity with
pigs have developed swine flu (zoonotic transmission) with moderate to severe illness in humans of all age groups.
Although this does not always lead to human influenza, a novel H1N1 (swine flu) which is highly lethal but human-to-
human transferable influenza A variant, emerged in 2009, in the Mexico-American borders after preparing the world
for 2008 pandemic avian influenza, that is, H5N1 [12]. This novel H1N1 (swine flu) virus strain is popularly called
2009 Swine Flu. In the literature, it is also designated as H1N1/09, A/(H1N1) pdm09, or S-OIV (swine-origin influenza
virus). In this chapter we adapt to the nomenclature A(H1N1) p09 or more generally “2009 swine flu.” Pandemic caus-
ing A(H1N1) p09 virus contains two prominent surface epitopes, HA-1 and NA-1, and causes typical respiratory tract
infections and other complications such as bronchitis and pneumonia in humans. However, this novel virus was not a
zoonotic one as it was not transferred from pigs to humans latest. Instead, it was the progeny strain that evolved as one
of the descendants of the 1918 H1N1 strain due to quadruple reassortment in triply assorted virus with Eurasian
(Europe and Asia) swine virus [13]. The A(H1N1) p09 swine flu infection caused symptoms similar to those seen in
pigs, probably due to reassortment, that permitted human-to-human transfer ability [14,15]. The transmission of this A
(H1N1) p09 swine flu strain among the humans seems to be higher than that observed with seasonal influenza [16].

5.2 Genome, structure, and functions

Swine flu strain belongs to type A (IAV) and most of the pandemics so far are caused by type A virus. Influenza A
(and B viruses) genome size is B13.5 kb and contains eight negative-sense, single-stranded viral RNA (vRNA) gene
segments, encoding transcripts for a total of 11 essential viral proteins, including several strain-dependent accessory
proteins [17]. The eight segments of influenza A (and B) viruses (1
8) are numbered in decreasing order of their
lengths.
Eight RNA segments of the swine flu genome encode for 11 proteins, namely:
G two envelope proteins: hemagglutinin (HA) and neuraminidase (NA),
G three vRNA polymerases: polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), and a
nucleoprotein (NP),
G a nonstructural protein NS2 (NEP), required for viral replication,
G two virulence factors: (1) a nonstructural protein, NS1 essential for pathogenesis (host interferon antagonist), (2)
PB1-F2 (proapoptotic factor), and
G matrix protein (M1) and membrane protein (M2).
In IAVs, segments 1, 2, 3, 4, 5, and 6 encode just one protein per segment, namely, the PB2, PB1, PA, HA NP, and
NA proteins, respectively. However,
G Segment 2 in some strains of IAV (e.g., 2009 swine flu strain), in addition to PB1, encodes for an accessory protein
PB1-F2 (proapoptotic factor) which is a truncated 87 amino acid protein in 11 alternate reading frame [18].
G Segment 7 of IAV, in addition to M1 matrix protein it also expresses, an M2 ion channel protein by mRNA splicing
[19].
G Segment 8 of IAV (and IBV) codes for two proteins, namely, the NS1 protein, an interferon-antagonist [20], and the
NEP/NS2 by mRNA splicing mediating viral RNP export from the host cell nucleus to the cytoplasm [21].
In IBVs, similar to IAVs, segments 1, 2, 3, 4, and 5 (but not 6) encode one protein per segment, namely, the PB2,
PB1, PA, HA, and NP proteins, respectively.
G Segment 6 of IBVs encodes both the NA protein and, the NB matrix protein in a 21 alternate reading frame, which
is an integral membrane protein, corresponding to the M2 protein of IAV [22].
G Segment 7 of IBV codes for the M1 matrix protein and BM2 membrane protein in a 12 alternate reading frame
[23] thus differing antigenically with IAV.
G Influenza B viruses have a single subtype with two lineages, Victoria and Yamagata. Though influenza B viruses
have been identified in seals, they are mostly confined to humans [24].
ICV, IDV viruses have each seven RNA segments and encode nine proteins, and are not yet identified to cause
significant disease in humans. However, influenza C infection can cause flu-like illness in some cases, especially in
children [25]. The genomic organization of influenza C viruses is generally similar to that of influenza A and B
viruses [26]; however, in ICVs, HA and NA proteins are replaced by only one major surface glycoprotein, the
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 59

hemagglutinin-esterase-fusion (HEF) protein [27] which functionally corresponds to the HA and NA of IAV and IBV.
Thus, the influenza C genome has only 7 segments while influenza A or B viruses have 8.

5.2.1 Gene functions

Despite their antigenic differences, the genes encoded by the influenza A and B virus genomes have similar functions.
The HA protein (encoded from segment 4) is critical for binding to sialic acid (SA)
containing receptors and fusion of
the viral and endosomal membranes facilitating the viral entry. The NA protein is accountable for the virus release
from infected cells and (those bound to nonfunctional receptors) through removing SAs from cellular and viral HA and
NA proteins and helping in the viral spread. RNA segment 5 encodes NP through which the RNA genome is bound.
Replication and transcription of vRNAs are processed by the trimeric polymerase complex comprising PB2, PB1, and
PA proteins and the NP. The M1 protein furnishes a scaffold supporting the structure of the virion and together with
NEP (NEP, formerly called NS2) regulates the trafficking of the nascent viral ribonucleoprotein (vRNP) complexes into
cytoplasm and subsequently are assembled into virions at the plasma membrane. The M2 is a proton ion channel neces-
sary for viral entry and exit. The NS1 protein is a virulence factor essential for pathogenesis, which inhibits host antivi-
ral responses [25,28]. PB1-F2 is a truncated protein with an antiapoptotic activity. More about PB-F2 is described in
the later section.
RNA genome and other viral proteins are held in the central core of the viral particle while the other viral proteins
function as the protector of the genome RNA. At their ends, each RNA segment forms a helical hairpin, bound by het-
erotrimeric RNA polymerase complex and the remaining part of the segment is coated with positively charged arginine-
rich NP, which binds to the negatively charged phosphate backbone of the vRNA [29]. vRNAs contain at both 30 and 50
ends noncoding regions, of varying lengths. But, the extreme terminal ends of segments are highly conserved among all
influenza virus segments; these partially complementary termini, base-pair to function as the promoter for vRNA repli-
cation and transcription by the viral polymerase complex. The noncoding regions also include the mRNA polyadenyla-
tion signal and part of the packaging signals for virus assembly [28].

5.2.2 Virion structure

By electron microscopy, IAV and IBV viruses are virtually indistinguishable. Morphologically, IAVs can be either
spherical with a diameter of B100 nm or filamentous reaching up to 20 μm in length [30]. Upon passaging in MDCK
cells or, eggs, the filamentous form is usually lost [31]. Various groups have attributed the morphological changes to
M1 matrix protein and M2 membranous protein, apparently by their function in supporting the envelope [32]. The IAV
is studded with glycoprotein spikes of HA and NA, in a ratio of B4:1, projecting from a host cell
derived lipid mem-
brane [27]. A fewer number of matrix (M2) ion channels traverse the lipid membrane, with M2:HA ratio in the order of
one M2 ion channel per 10
100 HA molecules [33]. The membrane and its three integral membrane proteins HA, NA,
and M2 overlay a matrix of M1 protein, enclosing the virion core. Irrespective of the shape (spherical or filamentous),
HA protein in the envelope is the most abundant, followed by NA and M2 [34]. Interior to the M1 matrix, there are
nuclear export protein (NEP; also called nonstructural protein 2, NS2) and the ribonucleoprotein (RNP) complex, which
consists of vRNA segments coated with NPs and the hetero-trimeric RNA-dependent RNA polymerase, comprising of
two “polymerase basic” and one “polymerase acidic” subunits (PB1, PB2, and PA) [27,28].
The organization of the IBV virion is similar to IAV, with four envelope proteins: HA, NA, and, instead of M2, it
has NB and BM2. The ICVs are structurally distinct from those of the IAVs and IBVs; on infected cell surfaces, they
form long cordlike structures on the order of 500 μm. However, ICVs are compositionally similar, with a glycoprotein-
studded lipid envelope overlying a protein matrix and RNP complex. The influenza C viruses have only one major sur-
face glycoprotein, the HEF protein, which functionally corresponding to the HA and NA of influenza A and B viruses,
and one minor envelope protein, CM2 [27,28].

5.2.3 Viral attachment

The influenza virus has an outer lipid layer membrane, which is taken from the host cell. The IAV envelope consists of
glycoproteins (proteins linked to sugars), named hemagglutinin (HA) and NA, and M2 protein. The HA and the NA are
major determinants of virus pathogenicity that play a crucial role in virus binding and release, respectively. HA and NA
surface glycoproteins are anchored in a lipid membrane from the infected cell through specific receptors on the host
cell surface. The HA is a lectin, mediating the binding of the virus to target cells and entry of viral genome into the
60 Pandemic Outbreaks in the 21st Century

target cells by endocytosis. HA helps in attachment of virus through SA of glycoprotein on the surface receptors of
upper respiratory tract or erythrocytes of host, whereas enzyme NA cleaves α-ketosidic linkage between the SA (N-
acetylneuraminic) and an adjacent sugar residue and free virus progeny from infected patient cells [35]. The amino acid
sequence of NA is coded by the sixth RNA segment and the polypeptide chain and NA comprises of 470 amino acid
residues.
The infection process is initiated via HA spikes present on the viral envelope. Upon reaching a potential host cell,
the receptor-binding site of HA attaches the virus to surface glycoprotein conjugates that contain terminal SA residues
[36]. IAVs then scan the cell surface for the proper “sialylated-receptor” by using the sialidase function of NA to
remove local SAs and liberate nonproductive HA associations [37]. SAs are nine-carbon acidic monosaccharides gener-
ally found at the termini of several glycoconjugates with wide tissue tropism in several animal species. The C-2 of the
terminal SA can bind to either the C-3 or C-6 of galactose, forming α-2,3- or α-2,6-linkages resulting in unique steric
configurations of the terminal SA. The receptors on host cell surface, that is, SA moieties are recognized and bound by
the HA spikes from the envelope of influenza viruses, which have a preferential specificity for either α-2,3- or α-2,6-
linkages. The human IAVs usually prefer α-2,6-linked SASA, whereas the avian influenza viruses bind to α-2,3-linked
SA. On the contrary, swine cells harbor and prefer both α-2,3- or α-2,6-linked SAs [38]. While α-2,6-linkages predomi-
nate in human tracheal epithelial cells, α-2,3-linkages are more common in duck gut epithelium. SA moieties with ter-
minal α-2,3-linkages also exist in humans though in less abundance than those SA moieties with α-2,6-linkages, that
too confined to LRT [10,11]; as a consequence, humans and other primates can be infected by avian IAVs but with
overall less efficiency than by human or swine strains [39].

5.2.4 Fusion and entry

HA-mediated binding to the receptor triggers endocytosis of the virion which can either occur in a clathrin-dependent
manner, mediating dynamin and the Epsin-1, an adaptor protein or by macropinocytosis [40]. Once inside the cell, the
IAV is trafficked to the endosome, where the low pH activates the M2 ion channel [41], and triggers a large conforma-
tional change in HA exposing the fusion peptide [42]. Opening of the M2 ion channel acidifies (H 1 ions from the
endosomes are propelled into the virions through the M2 ion channel) the interior of virion, releasing the packaged
vRNPs from M1 protein, which enables the transfer of the vRNPs to the host cytoplasm following HA-mediated fusion
[43
45]. The M2 transmembrane ion channels are the target of the amantadine class of antiinfluenza drugs, which can
block the ion channel activity thus preventing the virus from uncoating [41,46]. Besides, since it is an envelope protein,
M2 has been proposed as a potential vaccine component [47]. Internal acidification of virion through the M2 channel
disrupts internal protein
protein interactions, thus permitting the viral RNPs to be released into the cytoplasm [43,44].

5.2.5 Trafficking to the host cell nucleus

As soon as the vRNPs are released from the virions, viral proteins’ nuclear localization signals direct the host cellular
proteins to traffic the vRNPs and other viral proteins to import into the host nucleus [48] where RNA synthesis, cap-
ping, polyadenylation of mRNAs takes place. Recent studies support the evidence that vRNP trafficking to the nucleus
depends highly on the host cell machinery and transport pathways [49]. Currently, it is believed that vRNPs use the
importin-α
importin-β nuclear import pathway to gain entry to the host cell nucleoplasm [17]. Latest imaging and
RNA labeling techniques using many immortalized cell lines reveal that trafficking of vRNPS to nucleus takes bulk of
the time, that is, in B1 h, while the entry and fusion occurring rather quickly (B10 min) [40]. Another study highlights
the importance of NP adaptation to the importin-α isoforms of a particular species is critical for potential IAV infec-
tions [50].

5.2.6 Replication and transcription

Inside the nucleus, the heterotrimeric vRNA-dependent RNA polymerase carries out the transcription and replication of
the vRNAs [51,52]. The replication of the influenza genome is carried out by RNA-dependent RNA polymerase which
uses the negative-sense vRNA as a template to synthesize 2 positive-sense RNA species: (1) mRNA templates required
for host-cell translation of viral proteins, and (2) complementary RNA (cRNA) intermediates from which the RNA
polymerase subsequently transcribes more copies of negative sense, genomic vRNAs for packaging that form progeny
virus [17]. Upon exiting the polymerase, the cRNAs complex with newly synthesized NPs and a single copy of the viral
polymerase assemble into a cRNP which can generate multiple vRNA copies similar to cRNA transcription. During the
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 61

course of infection, mRNA synthesis occurs prior to cRNA and vRNA transcription, and is much more abundant
because the use of primers significantly increases the initiation efficiency [53].
A positive sense poly (A) tail of IAV mRNA transcripts is encoded when viral polymerase encounters a short stretch
of five to seven uracil residues present in vRNA segments [54]. Capping occurs in a unique process called “cap snatch-
ing,” where PB1 and PB2 and PA proteins snatch 50 capped primers from host pre-mRNA transcripts to initiate viral
mRNA synthesis [55]. After polyadenylation and capping, viral mRNAs are exported and translated, similar way host
mRNAs are treated. Viral proteins, M1 and NEP/NS2 mediate the export of vRNA segments from nucleus to cytoplasm
[48]. M1 matrix protein brings both vRNA and NP within the RNP complex; M1 also connects with the NEP, thus
mediating the export of M1-RNPs through nucleoporins into the cytoplasm.

5.2.7 Host-cell translation of vmRNAs

The membrane proteins HA, NA, and M2 are synthesized, on membrane-bound ribosomes into the endoplasmic reticu-
lum, where they are folded and trafficked to the Golgi apparatus for posttranslational modification. All three proteins
contain sorting signals that direct them to the cell membrane for packaging and assembly of virions. Although not
much is known about the translation and sorting of the nonenvelope proteins, M1 is supposed to play a key role in
delivering the vRNP-NEP complex into contact with the envelope-bound HA, NA, and M2 proteins for packaging and
assembly to undergo at the host cell membrane [48].

5.2.8 Packaging of RNA and assembly of virus

Proper packaging of virions with full genome of all eight RNA segments is crucial for them to become potentially
infectious. It is now believed that packaging is a more selective process with discrete packaging signals on all vRNA
segments rather than random incorporation segments [28]. These researchers also look at vRNP assembly and traffick-
ing, as well as ER targeting and maturation of IAV membrane proteins.

5.2.9 Virus budding and release

The NA is a mushroom-shaped tetramer, firmly embedded by its transmembrane domain in the viral envelope [56]. NA
cleaves α-ketosidic linkage between the SA (N-acetylneuraminic) and an adjacent sugar residue and free virus progeny
from infected patient cells. Influenza virus budding can be initiated by an accumulation of M1 matrix protein at the cyto-
plasmic side of the lipid bilayer. Influenza progeny virus continues to attach to SA moieties via HA spikes until virions
from the host cell are actively released by NA activity of NA to cleave the terminal SA moiety from cell-surface glyco-
proteins and gangliosides. The NA also cleaves SA moieties from the virus envelope itself, which prevents viral particle
aggregation to enhance infectivity. Lack or inactive NA, or neuraminidase inhibitors (NAIs) reduce the infectivity of
virus particles due to cell surface clumping [57]. In addition to cleaving activities of SAs, NA enhances virus infection
by breaking down the mucins in respiratory tract secretions and facilitates virus entry into respiratory epithelial cells.
Anti NA antibodies and NAIs prevent releasing of virus from infected cells and thereby inhibit viral replication.
Two mechanisms, namely, reassortment and interspecies transmission, which are not mutually exclusive, yield in
the emergence of novel viruses with new HA and/or NA subtypes into human populations. These different segments
make new combinations, responsible for novel outbreaks of influenza with high mortality and morbidity in a population
that has no immunity, thus past flu vaccinations may not work effectively [58]. Hence, attachment and entry processes
differ with each pandemic of IAVs. Thus, the glycoproteins (HA and NA) are the targets for antiviral drugs and or anti-
gens (or vaccine candidates) to which antibodies can be raised [59,60].

5.3 Epidemiology
5.3.1 Origin of “2009 swine flu” or “A(H1N1) p09”
The pandemic 1918 deadly influenza also known as “Spanish Influenza” caused by the H1N1 influenza virus infected
approximately 500 million people worldwide, killing roughly 50
100 million people. The 2009 H1N1 swine flu variant
was the descendent of the strain that caused the 1918 flu pandemic. The descendant variants of the 1918 virus, although
persisting in pigs, have also known to infect humans contributing to seasonal outbreaks. However, this is uncommon
and does not always lead to human influenza but often resulting only in the generation of antibodies. Zoonotic transmis-
sion of the virus from pigs to humans has been a rare occurrence, with only a dozen documented incidents in the
62 Pandemic Outbreaks in the 21st Century

United States since 2005 [8]. These strains have disappeared in the human population but the precursors of A(H1N1)
p09 strains persisted in swine, thus essentially making pigs a “mixing vessel” or “reservoir” and subsequently emerged
as a potential variant to reinfect humans in 2009 [7,8].
Classical swine flu strain was first isolated from pigs in the United States in the 1930s and was the predominant
swine flu strain as a cause of flu infections in pigs worldwide for the next 60 years. Cross-species transmission may
have occurred through close association of humans with pigs and vice versa with their respective flu but this did not
cause endemics or pandemics up until 2009. The A(H1N1)p09 strain, responsible for the 2009 swine flu pandemic, was
first identified at the border between Mexico and the United States in 2009. The CDC, on April 15
17, 2009, con-
firmed the first two cases of human infection with the A(H1N1)p09 virus in San Diego, California. The two first cases
were diagnosed in the two children who did not seem to have any exposure to swine, indicating a potential human-to-
human transmission [61
67]. Within a span of few weeks, the disease had spread across many countries to become the
first pandemic of the 21st century. Due to the global trade and travel network, the “2009 swine flu” had spread across
122 countries in 6 weeks, while the previous pandemics had spread in 6 months [68]. However, it was not found to be a
zoonotic transmission (see above). The H1N1 swine influenza viruses can potentially cause infections in humans if anti-
genic characteristics of the virus change. This A(H1N1)p09 was found to be mainly transmitting among humans and
exhibits two surface antigens, hemagglutinin type 1 (HA1) and neuraminidase type 1 (NA1).
In 2009 the pandemic which started in Mexico with the H1N1 strain displayed a combination of segments of four
different influenza viruses (quadruple genetic reassortment):
1. pig-origin flu North American avian (comprising 34.4%),
2. bird-origin flu of the human influenza strain (comprising 17.5%),
3. North American swine (comprising 30.6%), and
4. Eurasian swine (comprising 17.5%).
Coinfection with several influenza viruses from diverse species permits the genomes to interact, mutate, and inter-
mix to form new strains that might acquire variable immunity. Although it had originated in pigs, it was able to spread
from human to human. When the flu spreads from human-to-human, rather than from animals-to-humans, there can be
further mutations, making it harder to treat because people have no natural immunity [69]. Among the eight RNA seg-
ments of genome of novel H1N1 flu, due to reassortment, one segment came from human flu strain, two derived from
avian (bird) strain, and five derived from swine strain [13,70] as given below.
Each segment was originated from a different lineage.
1. Segments PB2, PB1, PA, NP, and NS were derived from a triple reassortant H3N2 swine virus that originated in
North American swine during the mid-1990s.
2. The HA (H1) segment derived from the classical swine H1N1 lineage that has circulated in North American swine
since the 1918 pandemic.
3. The NA (N1) and MP segments (M1 and M2) were derived from avian-like Eurasian swine lineage that emerged in
European pigs in the late 1970s.
Whole genomic analysis of the 2009 influenza A (H1N1) strain revealed a close resemblance to classical swine and
avian IAVs isolated in North America, Europe, and Asia [71]; The novel 2009 Influenza A (H1N1) swine flu virus
genome reassortment contains
G three classical swine genes (H1, NP, and NS),
G one human gene (H3N2 PB1),
G two North American avian genes (PB2 and PA), and
G two Eurasian avian-like swine genes (N1 and M).

5.3.2 Incidence and mortality

In April 2009 an H1N1 variant of human IAV was identified in borders of Mexico-United States but with far less fatal-
ity consequences than the Spanish flu strain (1918). Recently, it was proposed to originally start in Asia but not Mexico
[72]. Colloquially termed “swine flu,” it was suspected as a re-assortment of bird, swine, and human flu viruses. The
disease has raged through South-East Asian countries, Egypt, and other countries within weeks to reach pandemic pro-
portions [73]. In 2009 swine flu had spread to 43 countries, with 12,515 cases and 91 deaths reported, and it was
declared a pandemic problem [74].
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 63

The epidemiology of the 2009 swine flu pandemic is not unlike previous pandemics but not the same as seasonal
influenza epidemics. Unlike, seasonal influenzas, young healthy people were disproportionately affected both in terms
of hospitalization and mortality [75] than the elder population. Similarly, pregnant women have been at higher risk of
swine flu than seasonal influenza [76]. Although the ultimate case fatality rate (CFR) of swine flu was way lower than
1918 Spanish flu death rates, it was perceived at the time as “lethal” because novel swine flu strain disproportionately
affected young adults who were previously healthy and often rapidly caused severe respiratory troubles. When com-
pared with the previous pandemics, the infectivity of “2009 swine flu” was higher among children than adults. A possi-
ble explanation for this could be older adults (in addition to cytokine storm applicable to the 1918 H1N1 outbreak)
were attributed to having developed immunity due to a similar H1N1 outbreak that occurred in the 1970s [77].
CFRs of “2009 swine flu” were ranging from 2.9% to 9.1% [78], whereas fatality rates from developed countries
with high-income varied three to nine times among the research studies conducted. In one of the studies, 80% of the
deaths were attributed to people below 64 years of age. Southeast Asia and Africa had the highest mortality compared
to other continents [79]. In-hospital mortality can be key indicator. In the case of high-income countries, where infra-
structure, preparedness, and quality of treatments are higher, the estimated in-hospital death rate varied between 1%
and 3%, which did not rely on the quality of hospital and hospital burden in all countries was still higher among chil-
dren and younger adults. Despite lower risk of infection in older patients, they had higher fatality rates than younger
patients in the event of hospitalization, largely because they often had comorbidities [80].
In Mexico, where the maximum fatalities were seen, complications such as severe pneumonia with multifocal infil-
trates and rapid progression to acute respiratory distress syndrome (ARDS) and multiorgan failure have been reported
[16]. CFRs in Mexico were estimated to be 0.4% [81], whereas in other countries quite lower fatality rates have been
reported in virologically confirmed cases. The lower CFR of swine flu (2009) is attributed to a few pathogenic markers.
PB2 gene lacking lysine at residue 627, a PDZ domain in NS1 protein, and the nonfunctional (truncated) PB1-F2 pro-
tein may be implicated for the lower pathogenicity of A(H1N1)p09 virus when compared with Spanish 1918 H1N1
virus [82]. Another reason for low fatality rate could be, human H1N1 IAVs have been circulated in the population
since 1977 and are already included in the vaccines. Thus, prior infection or vaccination can bring partial immunity in
humans. WHO could not alert health officials, as the unpredicted lower severity of the “2009 swine flu” was not among
six-point-phasing for pandemics.
According to a study by CDC, among the dead, 29% had reported bacterial superinfection with pneumococci as the
predominant pathogen [61
67]. This suggests that early prescription of antibiotic therapy and pneumococcal vaccine
may reduce the mortality rate [83]. In India the very first case of swine flu was reported in 2009 at Hyderabad. In 2015
the swine flu epidemic again hit India causing significant morbidity and mortality. In an attempt to identify predictors
of mortality of 2009 swine flu (during the 2015 swine flu epidemic), mean procalcitonin was found to be on the lower
side, along with normal range of WBC count, tachypnea (elevated respiratory rate), and hypoxia (low oxygen satura-
tion). This is contrary to a Korean study [84] where serum procalcitonin levels were found to be significantly elevated
in patients with mixed infection of pneumonia than those with 2009 swine flu infection alone. Thus, this clinical feature
can be used as a biomarker to detect swine flu infection and to bring early preventive measures.
The global pattern of the 2009 swine flu pandemic displayed a varying behavior of wave timing across the globe.
For example, in North America a two-wave profile was observed with a spring-summer and fall wave. In Mexico, a
three-wave profile has been identified, with a spring, summer, and fall wave. Whereas in Europe, despite heterogeneity
in pandemic timing, an initial mild wave in the spring and early summer of 2009, which diminished summer, but ree-
merged to produce a more severe second wave when schools were reopened. In India three wave peaks were seen dur-
ing 2009 and 2010.
As of August 2010, the WHO declared officially that the 2009 swine flu pandemic was over and within a span of 15
months it affected .10% of the world population in more than 214 countries including overseas communities, with
incidences 43
89 million and caused over 18,449 deaths until August 2010. However, CDC recently released the first
global mortality by swine flu pandemic in the first year, ranging somewhere between 151,700 and 575,400 deaths,
which is B10
30 times the number of lab-confirmed deaths [85]. The estimation of fatality rates of swine flu relative
to 20th-century pandemics was not based on lab-confirmed cases but rather on statistical attribution of excess deaths
due to all causes [86]. However, the former method does not provide information on unreported cases, either due to
misattribution of death causes or the absence of lab-confirmed infection. Hence, the 2009 swine flu pandemic, relative
to previous pandemics, could have been underestimated in terms of burden and mortality. The same strain which caused
the 2009 pandemic has been active and again caused the 2015 pandemic worldwide infecting a large number of people
in India. The reported number of swine flu cases in India by the National Centre for Disease Control (NCDC) is 29,930
with 1236 deaths as of February 23, 2020.
64 Pandemic Outbreaks in the 21st Century

5.4 Clinical features

5.4.1 Transmission
Transmission of any infectious diseases among humans or animals is the result of the transmission of a pathogen either
directly between hosts or indirectly through the environment or intermediate hosts. Similarly, the infectiousness of
swine flu in the infected host (or hosts) and the susceptibility of uninfected individuals (who are exposed to infection)
decides the efficiency of transmission. Transmission of an infectious disease from one host to another depends upon
several factors that influence and contribute to this process. It is important to understand the pathophysiology of infec-
tious disease. Influenza viruses replicate in the mucous epithelial cells of the respiratory tract (upper and lower). Virus
gains entry to a new host via inhalation or direct or indirect contact including hand contamination followed by self-
inoculation to access the target cells of the exposed respiratory mucosa such as nasal passages, nasopharynx, pharynx,
larynx, trachea, bronchi, bronchioles, and alveoli. Human viruses preferentially attach to epithelial cell surface receptors
(sialyl-oligosaccharides) terminated by an N-acetylsialic acid linked to galactose sugar by an α(2,6)-linkage [87]
through which they make entry inside. The predominance of these receptors reflects the tropism in different tissues. For
example, α(2,6) linkages are mainly found in the human respiratory tract [10]. Hence, entry and shedding of virus in
humans occur via the respiratory tract, that is, predominantly mouth and nose. But recent studies confirmed the pres-
ence of α(2,3) receptors in the LRT [10,11] and also in the eye thus virus can reach the nasopharynx via an ocular route
[88].
Mode of transmission of influenza viruses with typical respiratory illnesses including those caused by severe acute
respiratory syndrome coronavirus (SARS), MERS, is similar among humans, that is, by inhalation of infectious droplets
( . 10 μm) or aerosols (,5 μm) in which the virus resides as well as by direct contact with secretions on the surfaces
(fomites) [89]. Respiratory secretions are released into the environment during events such as coughing, sneezing, and
talking, forming different sized particles such as droplets or aerosols which are governed by basic rules of gravity and
physics. In addition, physicochemical parameters like relative humidity (Rh) and temperature, evaporation, and so on
modulate the viral transmission [90,91]. Influenza viruses prefer cool, dry air with majority of transmission confined
during the onset of winter across temperate latitudes. Adults older than 50 years of age were less vulnerable to A
(H1N1)p09 infection than younger adults or children compared to previous pandemics [92]. A(H1N1)p09 pandemic
profile had three seasonal waves in spring, summer, and fall. The severity was more after summer vacations while open-
ing the schools. Thus, low humidity and poor sunlight during winters are the key factors favorable for the survival of
this virus.
The proportion of cases was higher in temperate regions than in tropical
subtropical regions while the duration of
activity was longer in tropical
subtropical than in temperate countries. In most temperate countries (98%), pandemics
erupted during the fall-winter period [93]. There was a positive correlation between country central latitude and propor-
tion of A(H1N1)p09 than all other IAVs. These findings suggest accounting these potential factors while handling the
future outbreaks [93]. Another recent study reported a statistically significant relationship between the seasonal signa-
tures and latitude which were more clustered along the coast. Thus, understanding the seasonal signatures of influenza
among the geographical regions is helpful to design more effective strategies toward prevention and control [94].
However, a significant spread of infections can also occur during other times. When novel viruses emerge and when
there is a lack of immunity, transmission might occur during unusual circumstances [95].
For transmission to happen, vulnerable people lacking immunity are needed in the exposed population. Factors such
as limiting the contact with a susceptible people or an acquired herd immunity decrease the transmission in a commu-
nity. Novel H1N1 influenza viruses are uninhibited by host immunity factors due to novel mutations and may achieve
high infectivity potential. Thus, A(H1N1) seasonal influenza is regularly emerging at an unusual time. In some tropical
countries H1N1 influenza occurs throughout the whole year but worsens during the return of fall-winter.
For viruses to successfully cause infection in new hosts, three steps have to be passed; they must
1. survive in the environment;
2. reach target cells in a new host; and
3. sufficient inoculum of virus must reach target cells so that infection is initiated.

5.4.2 Infectious versus incubation period

Once exposed to the virus, there is a fairly predictable incubation period for influenza viruses. The average incubation
period for 2009 swine flu is around 2 days in most individuals with a range of 1
4 days following infection. This
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 65

reflects the replication and transcription of the virus primarily occurring in the passages of the upper and LRT from the
time of inoculation and peaks around 48 h in most patients. The infectious period for adults begins about 1 day before
developing the symptoms and lasts around 5
7 days after the onset of symptoms. This hints about the recommended
time of isolation of the infected patient is around 5 days [96]. The infectious period may be longer, 10
14 days in chil-
dren, patients with LRT infections, elderly and immunocompromised patients [96]. Cytotoxic T-lymphocyte activity,
responsible for viral clearance leading to recovery, declines in the elderly as well as in immunocompromised indivi-
duals so that viral shedding could persist longer. Hospitalization may be required for those patients with more severe
disease. On the contrary, lack of clinical illness following exposure indicates no infection, adequate immunity, or sub-
clinical infection [97]. Generally, after 5
6 days of illness, a patient is unlikely to transmit the infection. Knowing the
critical period of viral shedding helps physicians and patients understand the value of self-isolation during the illness.
Patients with chronic diseases of lung, heart, and pregnant women are more prone to severeness such as viral pneumo-
nia, superimposed bacterial pneumonia, and hemorrhagic bronchitis. These complications may develop within 2
3
days from the onset of symptoms. The precise incubation period for A(H1N1)p09 (swine-origin influenza A) infection
is comparable to that of seasonal human influenza A (H1N1) (1
4 days) but longer in the avian origin influenza A
(H5N1) which is 1
8 days.
The serial interval of A/H1N1 (2009) flu was typically short, with a mean value similar to the seasonal flu [98].
Household studies on serial interval of the disease recommended isolation time of the patients and effect of treatment
on transmission [99]. The reproduction number R0 (Reproduction time) for 2009 Pandemic Influenza A (H1N1) has
been estimated to be between 1.4 and 1.6 [100]. The median reproduction number compared with past influenza pan-
demics was similar (1968 pandemic) or slightly smaller (1889, 1918, and 1957 pandemics) [98]. Secondary attack rates
showed significant variation in different regions with estimates of 33% in children and 22% in adults which was higher
in the second season than in the first pandemic season [79,101].

5.4.3 Symptoms
The clinical features of A(H1N1)p09 range from the symptoms of mild flu to severe respiratory illness, possibly lead-
ing to death, depending on the patient’s age, preexisting conditions, vaccination status, and immunity potential.
According to the CDC, the signs and symptoms in humans infected with the 2009 swine flu virus were similar to those
of seasonal influenza. A(H1N1)p09, a subtype of IAV, causes upper and potentially LRT infections resulting in symp-
toms such as nasal secretions/coryza, coughing, sore throat, chills, fever, sneezing, headache, decreased appetite, short-
ness of breath, congested eyes, myalgia, weight loss, dizziness, abdominal pain, and fatigue similar to seasonal flu.
Headache, body aches, fatigue, diarrhea, and vomiting also have been observed [102]. The most frequent symptoms of
swine flu are fever above 104 F for more than 3 days (94%), coughing (92%), sore throat (66%), headache (80%) and
chills (60%), coryza, body aches, and sore throat which are also common in seasonal flu; 25% of patients report diar-
rhea, and a further 25% report vomiting ([102,103]; WHO, 2009). According to CDC, bacterial pneumonia, that is,
accumulation of neutrophils in the alveoli, have been reported in B29% of cases [61
67]. Comparative studies
between A(H1N1)p09 strain and seasonal A(H1N1) strain in ferrets revealed that A(H1N1)p09 strain produced larger
lung consolidation than seasonal flu strain [104]. In addition, A(H1N1)p09 infected ferrets showed multifocal necrotiz-
ing rhinitis, tracheitis, bronchitis and bronchiolitis, and viral antigens were observed from the nasal turbinates to the
bronchioles [105].
Seasonal flu, unlike “2009 swine flu” is usually not associated with gastrointestinal symptoms like diarrhea and
vomiting, with fever rarely above 101  F [106]. In addition, swine flu brings sudden onset of symptoms with much
greater intensity causing weakness and fatigue for up to 2
3 weeks, including muscular aches accompanied by
chills and sweats due to intermittent fevers. Emergency cases among adults include breathing difficulty, pain or
pressure in the chest or abdomen, sudden dizziness, confusion, severe or persistent vomiting, and dehydration [107].
Although the infection rate is highest in young people between 12 and 17 years of age, the risk of hospitalization
and mortality rate is higher in pregnant women, elderly patients with diabetes, asthma, and heart disease. In contrast
to seasonal influenza, a substantial proportion of the swine flu cases of severe illness and death have occurred
among young and previously healthy adults [108]. Hence, swine flu patients while traveling unknowingly transmit
the infection.
As majority of these symptoms are overlapping with seasonal flu, a detailed history needs to be taken for the differ-
ential diagnosis of “2009 swine flu” and henceforth contract tracing of someone already confirmed with swine flu need
to be done. In addition to the common symptoms, the early signs and symptoms in human patients suffering from 2009
swine flu are “asymptomatic” making early diagnosis and treatment significantly delayed, leading to public hysteria
66 Pandemic Outbreaks in the 21st Century

which can lead to intense workloads at laboratories and in hospitals, indirectly affecting the economy of the nation
[109]. Most of the patients, hospitalized, have underlying conditions, such as asthma, lung, heart, diabetes and neuro-
logic diseases and pregnancy [110]. In addition to this, obesity is also reported as a risk factor [111] and hypoxia is the
most common cause of admission to an ICU [61
67].

5.4.4 Complications
A(H1N1)p09, similar to other influenzas is a significant respiratory pathogen. Most happening clinical features were
described above. Respiratory failure was the most common cause of death in severe cases. However, nonrespiratory
symptoms included high fever causing neurological problems, pneumonia-causing sepsis, dehydration and severe hypo-
tension resulting from vomiting and diarrhea, electrolyte imbalance
associated complications, kidney failure, and so
on. More severe cases and fatalities were more likely observed in children younger than 5 years of age and elderly
patients older than 60 years. Since the symptoms of 2009 swine flu are same as seasonal influenzas, similar complica-
tions are expected including those in extra-respiratory tissues: pneumonia, sinusitis, otitis media, bronchiolitis, croup,
status asthmaticus, myocarditis, pericarditis, myositis, rhabdomyolysis, encephalitis, seizures, toxic shock syndrome,
and secondary bacterial pneumonia with or without sepsis [102]. Individuals at extremes of age and with preexisting
medical conditions are at higher risk for complications and exacerbation of the underlying conditions [112]. However,
the complications of swine flu are more severe for some specific people listed in CDC archives (https://www.cdc.gov/
h1n1flu/risks.htm). In some autopsy cases, bacterial coinfections were also identified, such as Streptococcus pneumo-
niae, Streptococcus pyogenes, Staphylococcus aureus, community-acquired methicillin-resistant Staphylococcus aureus,
and Haemophilus influenzae.
Factors leading to higher risk of severity of swine flu illness if infected include:
1. Chronic diseases such as asthma, heart disease, diabetes mellitus, or neuromuscular disease
2. Compromised immune systems due to diseases such as AIDS
3. Gestating females
4. Children younger than 5 years old
5. Older adults more than 65 years old
6. Younger adults and children under age 19 who are on long-term aspirin therapy
In addition, and contrary to seasonal influenza, most cases of swine flu have occurred in young individuals with
approximately 60% of the reported cases in the United States having occurred in persons 18 years of age or younger
[71]. The main reason for the increased infection rate amongst young is not entirely clear, but it has been shown that
adults, especially those born before 1957, have low levels of cross-reacting neutralizing antibodies, likely due to
repeated exposures to seasonal H1N1 viruses [61
67].

5.5 Diagnostics
5.5.1 Diagnostics of H1N1 swine flu (pdm09)
Differential diagnosis of 2009 swine flu from other respiratory pathogens is complex due to the similarity in symptoms.
Influenza swine flu viruses can be collected within 2
7 days from nasal and throat swabs, suspended in transport media
before the test is done. Once the infection commences, the immune system of the host is stimulated to produce specific
antibodies as a function of time-specific for each pathogen. Routine investigations such as serological, biochemical,
virological tests, and so on are required as necessary for evaluation and management of the swine flu infected patient,
including the rapid test kits, which do not always detect zoonotic viruses.
One indication that a novel, probably zoonotic influenza might be present, could be the detection of IAV but not the
hemagglutinins in seasonal human influenza viruses [113]. Zoonotic influenza virus infections are occasionally diag-
nosed retrospectively by serology [114], but serological diagnosis can be complicated by cross-reactivity with human
influenza viruses. A further difficulty is that the HA and NA of some swine influenza viruses (the main targets of anti-
body responses) originally came from human influenza viruses, to which people may have already been exposed.
Serological tests have their own known limitations. In addition to the serological and surveillance tests, biosensors
tests based on antigen
antibody, DNA, RNA, PNA-based binding affinity are being developed. Biosensors, the modern
diagnostic techniques could be better options for filling the gaps for rapid and early detection with high sensitivity and
specificity for pathogens. Ravina et al. [115] have updated the diagnostics for influenza A H1N1 virus with emphasis
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 67

on biosensors. DNA detection tests like RT-PCR and RT-LAMP help in the early detection and treatment of infections.
The available procedures for swine flu diagnosis include:
G virus isolation in cell culture,
G immunofluorescence (IF),
G single radial-immunodiffusion,
G double-immunodiffusion (DI),
G hemagglutinin inhibition (HI) test,
G enzyme-linked immunosorbent assay (ELISA),
G rapid influenza detection test (RIDT),
G reverse transcription loop-mediated isothermal amplification (RT-LAMP), and
G reverse transcription-PCR and real-time PCR.
All the known methods have their own limitations. Some are less specific, time-consuming, less sensitive, nonspe-
cific, laborious, or require expensive instruments or expertise and cannot diagnose rapidly during an outbreak of the dis-
ease. Antigen-based detection tests are not very sensitive. RT-PCR is highly sensitive but limit of detection of test is
high and thus cannot detect small viral load. As the severity of illness with time, it is essential to invent an early detec-
tion for H1N1 infections. Thus, a simple and quick test, which makes detection easy to perform, has become essential.
Virus culture in mammalian cells shows morphological changes, behavior of cells. The established cell types are
Madin Darby canine kidney (MDCK), monkey kidney cells, and A549 cells. Upon infection with virus samples, differ-
ent cell lines show cytopathic effects at different levels and times [116]. To compensate for these, new commercial
mixed cell lines such as R-Mix cells and R-mix Too have been developed, which are hybrid of A549 with MDCK cells
and Mink Lung epithelial cells, respectively (microlab). R-mix Too cells were used for the detection of A(H1N1)09
using kit D3 Ultra 2009 H1N1 ID kit [117]. However, it takes many days to detect pathogen type, hence is not an
option for a quick detection.
Rapid antigen tests are done for quick evaluation of suspected patients in the form of various kits as available in the
market. The sensitivity of rapid antigen testing for detecting S-OIV is probably similar to or lower than the sensitivity
for detecting seasonal influenza [118]. A negative rapid influenza test does not exclude infection but a positive result
should be confirmed by real-time reverse transcriptase (RT)-PCR or culture [119].
IF generally takes 2
4 h in detecting influenza virus under a microscope. Cells are fixed, stained, and bio-
conjugated with specific antibodies (poly or monoclonal; raised against the virus in an animal) with a fluorescent dye
for the detection of the virus with a reasonable specificity and sensitivity [120]. Similar method in the case of tissue
samples infected with viruses takes several hours [121] and also there could be a chance of infection to the researchers.
Though these methods are relatively fast and also less expensive, the sensitivity is not good as that of RT-PCR.
Double immunodiffusion test (DI) for typing of influenza viruses can be performed using 1% agarose [122] with sep-
arate wells for typing influenza A and B in single time using NP and matrix protein. Reference antigens and antiserums
were diffused in selected wells. DI has good sensitivity but virus has to be cultured in a laboratory with a good titer. It
is also time-consuming and laborious method.
Single radial-immunodiffusion test can be employed to assay the HA antigenicity and its content [123]. Specific
antibodies are separated from serum against HA and antigen is poured in wells of agarose gel. Diffusion of antigens in
agarose gel is responsible for the formation of an annulus of precipitation of antigen
antibody. Determination of
unknown antigen concentrations or virus titer is done with the help of antigens with already known concentrations. This
method can detect differences in the HA antigenicity which cannot be identified by DI. It is simple, reproducible, and
sensitive for specific antigenic composition, and used for antigenically active antigen detection [124].
Hemagglutination inhibition assay can be used to detect the virus antigenic type, subtype classification specificity of
antibodies for HA subtypes and to confirm the infection of influenza [125]. HA antigen present on virus envelop can
bind with SA of RBCs forming a complex, known as hemagglutination reaction [126]. This complex formation can be
inhibited by adding antibodies specific to HA. Different dilutions of antibody were poured into the well which already
contains antigens. A negative result indicates an agglutination in well. Reproducibility of HI assay can be increased by
standardization [127].
ELISA is specific and sensitive for influenza detection. Based on the sandwich ELISA method developed in 1993
[128], a new sandwich ELISA diagnostic was developed during A(H1N1)p09 influenza pandemic. In this newly adapted
ELISA method, specific monoclonal antibodies (mAbs) and horseradish-peroxidase coupled rabbit anti-HA polyclonal
antibodies against HA protein were used. ELISA sensitivity decreases after a virus titer of 105 (log 10 mL21) but
QuickVue Influenza A 1 B test sensitivity decreases after a titer of 107 (log 10 mL21) [129]. In a comparative study for
68 Pandemic Outbreaks in the 21st Century

different reference strains (H1N1, H3N2, H1N2, H1N1pdm) from several swineherds, it was shown that sensitivity and
specificity of ELISA to be higher than HI [130]. However, ELISA cannot detect early-stage infection. For improved
sensitivity, a novel ELISA-based immunosensor was developed using gold nanoparticles (AuNP) where EDC-NHS was
used for the binding of anti-HA with AuNP that was already treated with formic acid (HCOOH) [131].

5.5.2 Surveillance methods: advance and quick methods for influenza detection
5.5.2.1 Polymerase chain reaction-based detection methods
Reverse transcriptase
based and/or real-time PCR (polymerase chain reaction) is most preferable at present for detec-
tion of swine flu as well as several other pathogens. In a two-step RT-PCR, purified RNA was first converted into
cDNA using reverse transcriptase enzymes and later in the second step random hexamers were employed for amplifica-
tion of target segments. In the one-step RT-PCR, gene-specific primers along with reverse transcriptase enzyme were
mixed together. Type-specific differentiation of virus is carried out using matrix gene amplification as matrix gene
shows conservation in virus which is useful for the typing of influenza. Subtyping of HA gene is further carried out
using gene-specific primers. These methods are highly sensitive but time-consuming which makes them less favorable
when the patients are in critical conditions. New RT-PCR assays were developed for detection of novel swine flu influ-
enza A(H1N1) pdm09, which can potentially discriminate seasonal H1N1 viruses from other subtypes and also between
other swine viruses and human H1 types [132]. In addition to this, numerous studies have also reported multiplex real-
time PCR assay based on matrix gene for detection of reverse zoonotic influenza infection A(H1N1)p09 from endemic
swine influenza viruses [133]. These modern PCR assays provide better results but they are laborious and expensive.

5.5.3 Rapid influenza detection tests

RIDT detects viral antigens mostly NP and gives colored signal. This can be tested in three forms—dipstick, cassettes,
and cards. The detection is fast but it can give false positives with poor sensitivity which further depends on specimen
type, technician skills, infection time and age of patient, and so on. RIDT can distinguish influenza A and influenza B
but it cannot differentiate between subtypes of influenza A [134]. In a study the data from testing of over thousand sam-
ples by NanoSign Influenza A/B kit were compared with RT-PCR, it was shown that the sensitivity and specificity of
both the tests were found to be 79.4% and 97.2%, respectively, with 94% concordance [135]. In another bulk study
with the results of QuickVue Influenza A 1 B(Quidel) rapid influenza antigen test, the sensitivity and specificity were
found 20% and 99%, respectively [136]. A study performed during the 2009 influenza A H1N1 outbreak, a comparative
study was carried out between rapid influenza tests (TruFlu rapid influenza A and B, rapid assay Directigen EZ detec-
tion) and Direct Immuno Fluorescence assay (DFA) test with real-time PCR for detection of S-OIV. Sensitivities of
both rapid influenza test and DFA test were 9.7%, 20.6%, 32.35% respectively, and the specificities were 98.2%, 99%,
99%, respectively, in comparison with RT-PCR. Hence, based on these studies, these bed-side tests are less sensitive
and cannot be used for 100% accuracy [137].

5.5.4 Non-polymerase chain reaction-based RNA detection methods

One-step amplification method like loop-mediated isothermal amplification is specific and sensitive for detection of
pathogens. This does not require any high-profile machine and can amplify in simple hot water bath. Four primers could
recognize six different sites on target DNA [138]. RT-LAMP method has been developed for specific detection and sub-
typing of influenza viruses. This includes two sets of forward primers and two sets of inner primers in which one set
recognizes outer region and the other set recognizes inner region and gives a result with 93.8% specificity [139]. Some
researchers also add two extra primers for loop regions that enhance their sensitivity and specificity. All primers, DNA
polymerase (isolated from Bacillus stearothermophilus), reverse transcriptase (avian myeloblastosis virus), template
RNA with all buffers can be included in a single PCR tube. Furthermore, strand displacement is considered to be a criti-
cal step as it determines the time for completion of the process.

5.5.5 Nucleotide sequencing and phylogenetic analysis

Amplicons for gene sequencing were generated by reverse transcription, followed by PCR amplification to generate
overlapping double-stranded DNA fragments covering each of eight segments of the H1N1 IAV genome. Phylogenetic
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 69

analysis of sequences contained six gene segments (PB2, PB1, PA, HA, NP, and NS) that were found in triple reassor-
tant swine influenza viruses circulating in pigs. The genes encoding NA and M protein were most closely related to
those in IAVs circulating in swine populations. For confirmation of diagnosis, clinical specimens such as nasopharyn-
geal swab, throat swab, nasal swab, wash or aspirate, and tracheal aspirate (for intubated patients) are to be obtained.
The sample should be collected by a trained physician or microbiologist preferably before administration of the antiviral
drug. Specimens should be kept at 4 C in viral transport media until transported for testing. The samples should be
transported to designated laboratories within 24 h. If they cannot be transported then they must be stored at 270 C.
Paired blood samples at an interval of 14 days for serological testing should also be collected for further analyses
[140,141].
Preparation of cDNA from the patient sample by RT-PCR: Real-time PCR is a standard test for H1N1 detection
recommended by the WHO. A total of 12 suspected samples can be tested using real-time PCR. The proposed
method calculates viral load in real-time because of the presence of fluorescent dye or labeled probes. Every sample
has a different Ct valve that explains the level of viral load in patients. If a sample covers the threshold line in fewer
reaction cycles, it means the patient has a higher viral load. Samples were also tested with conventional PCR using
primers designed specifically for the A(H1N1)p09 HA gene and the primers listed in the WHO updates that are spe-
cific for the H1N1 HA gene and matrix gene. Real-time PCR was performed to diagnose the H1N1 virus in patient
samples and conventional RT-PCR was conducted to check the working efficiency of the designed primers. These
primers helped in selecting a specific probe for the HA gene. Thus, we designed many primers for the selection of a
specific probe that can bind only with A(H1N1)p09 genome. Hence, conventional RT-PCR is useful in the evalua-
tion of the positive and negative samples and helps in the selection of the probe. First, RNA was converted into
cDNA using random primers of the kit with reverse transcriptase. In the next step, PCR was performed using the
designed primers for the HA gene. This method is also specific in detection method. The 1.5% agarose gel was used
for the gel electrophoresis of PCR products and was checked in the presence of UV light. DNA bands appeared in
positive cases and were absent in negative samples. Both methods are specific but time-consuming, laborious, and
costly.

5.6 Biosensors
It is essential to detect the dreadful disease at an early stage. Available diagnostic methods are not quick and suffi-
ciently differentiating, therefore, a new method that can yield specific and fast results is essential. The biosensors
could sense any biological analytes such as antigen and antibody, DNA, RNA, PNA, protein, enzyme/ligand, and so
on, with an affinity toward binding to their complementary segments. Based on these principles several biosensors
have been developed [142]. Any basic unit of biosensor involves four components, a biological analyte and its inter-
acting partner, a transducer of signal, an amplifier, and a processor [115]. Commercial transducers like nanosized
transducers provide high sensitivity and binding affinities, better conductivity and electrical circuits. Biosensors can
be grouped into different types based on their physicochemical parameters as well as interpretation of experimental
results.
Optical biosensors detect pathogens through the change in color or production of color during reaction between bio-
analyte and its target by the visual methods such as optical biosensors such as fluorescent biosensor, luminescent, color-
imetric, and interferometric biosensors. A plastic-based microfluidic immunosensor coated with gold in the detection
channels of the sensors was developed for the detection of antibody or antigen in serum against A (H1N1) pdm09
[143]. A gold-binding polypeptide recombinant influenza HA antigen fusion protein was used as a receptor and an anti-
body with specific fluorescence label (Cy3-labeled anti-H1 Ab) was used as a signal for detection through microchan-
nels [143].
An improved SPR biosensor with an electro-optic modulator sensor detects S-OIV with the help of an antibody
against hemagglutinin (H1) [144]. It is more sensitive and takes less than 20 min. Another SPR-based biosensor
employs a chip on which nine respiratory virus-specific biotinylated primers were immobilized. This biosensor identi-
fies influenza A and influenza B, adenovirus, H1N1, parainfluenza virus 1
3 (PIV1, 2, 3), respiratory syncytial virus,
and SARS from throat swabs [145]. A gold immunosensor with new (M1) matrix protein polyclonal antibody was
developed using cyclic voltammetry and impedance spectroscopy which can detect A (H1N1)p09 virus. This also serves
as a broad-spectrum method for all influenza A serotypes with a sensitivity of 80
100 virions μL21 and in 30 min
[146].
Impedimetric biosensor measures resistance produced due to binding of biological element on the surface of elec-
trode with target molecule in sample with respect to potential applied through potentiostat. Impedimetric detection of
70 Pandemic Outbreaks in the 21st Century

influenza A (H1N1) DNA sequence by way of carbon nanotubes system followed by streptavidin
gold nanoparticle
amplifies the impedance signal and sensitivity [147]. Reduced graphene oxide
based electrochemical immunosensor
was developed, where a stable amide bond formed between COOH group of graphene and NH2 of antibody specific for
H1 of H1N1 influenza A, by way of using EDC-NHS coupling chemistry [148]. A boron-doped diamond sensor of three
electrode system is surface functionalized with polyclonal anti-M1 antibodies upon which M1 protein can be absorbed
via electrochemical impedance spectra [149].
Amperometric sensors generate response in the form of current when specific potential was applied using potentio-
stat. Screen-printed electrodes are the mainly used electrodes for biosensor development and these can be of two elec-
trode sensor or three electrode sensor (working electrode, reference electrode, and counter/auxillary electrode, printed
on the surface of metal, glass, paper, etc.). Single-wall carbon nanotube immunoassay biosensor was designed for 2009
swine flu detection on surface of immunochips coated with poly-L-lysine, antibodies H1N1, where resistance will
increase with concentration of virus [150]. Recently, an H1N1 virus
specific impedimetric aptamer was also developed
for multivalent binding with inactivated H1N1viruses [151]. An antibody-based DPM-Cu(II) redox (a thiol derivative
dipyrromethene) modified with immobilized His6-H1 HA on gold surface can identify anti-H1 antibodies was devel-
oped where Osteryoung square wave voltammetry was used to track the changes that occurred on the surface of elec-
trode with different dilution of antibodies. This voltammetry can quantify the difference between reduction and
oxidation peak current [152]. PNA-based probe involves a synthetic peptide nucleic acid which can bind with RNA and
DNA through complementary binding. PNA-based biosensors can be a good area for A(H1N1)p09 biosensor develop-
ment. It provides specificity of complimentary binding capability of nucleotides and has greater shelf life than DNA
[153]. A DNA biosensor was also developed using phenyl carboxylic acid-modified glassy carbon electrode by immobi-
lization of DNA probe and hybridization with single-stranded complementary DNA of H1N1 by measuring the change
in current [154].
Potentiometric biosensors measure potential difference when a specific current value was applied to the electro-
chemical cell using potentiostat. The underlying concept that HA-specific binding for human SA is adapted here. A
(H1N1)p09 binds to the galactose residue of SA through 2
6 linkage but avian influenza virus binds by 2
3 linkage
(2,3-sialyllactose) of SA. Using this differentiation, a new detection method was designed where a conducting polymer,
that is, poly(3,4-ethylenedioxythiophene) containing SA-terminated trisaccharides, Sia-α2, 60 -Gal-Glu (2,6-sialyllactose)
as a recognition site on a surface. A combination of Quartz crystal microbalance (QCM) and potentiometry makes it
doubly sensitive when compared to commercial kits [155].
Piezoelectric biosensors exhibit a voltage change when mechanical stress or oscillation is applied to the surface of
an electrode bearing a piezoelectric material, which has the ability to show fixed mechanical oscillation upon voltage
introductions. These biosensors are based on the physical property of materials and lack central symmetry in the molec-
ular structures. When a bio-analyte specifically binds (antigen
antibody, DNA
DNA, RNA
DNA, ligand
receptor)
with its target that produces a change in oscillation frequency in oscillation circuit this can be measured as a signal to
find out the type of pathogen [156]. Two significant studies include (1) the detection of influenza A and B samples on
67 nasal samples, using QCM immunosensor in which anti-MA antibodies were immobilized onto gold electrode whose
surface is precoated with protein A [157] with a specificity of 100% and sensitivity of 81%, (2) detection of influenza
using piezoelectric lead zirconate titanate discs using a synthetic sialylglycopolymer sensor coating which has a long
lifetime, a high sensitivity and possibility of getting reproducible results [158].
Magnetic biosensors are formed of paramagnetic or super-paramagnetic particles which detect biological interac-
tions by calculating the changes in magnetic properties or magnetically induced effects such as changes in coil induc-
tance, resistance, or magneto-optical properties. A combination of nitrocellulose membrane with magnetic beads is used
to detect two different influenza viruses. This combination reduces analytical time and makes this assay stable and
reproducible for point-of-care applications [159]. Recently, bacterial-derived quench bodies were used in the detection
of influenza A-HA [160].
Similar to seasonal flu, it takes 2
3 days to manifest its full symptomatic conditions. Classical methods are
mostly based on antigen
antibody titer, time-consuming, less sensitive, and also have a problem of false-negative
results. Real-time PCR is a gold standard recommended by WHO but it is time-consuming, expensive, and reported
L.O.D. is five copies per reaction [161]. RIDT is fast but not sensitive compared to others. It can distinguish influ-
enza A and B but cannot differentiate different subtypes of influenza A. Reported L.O.D. is 103
104 PFU mL21 and
accuracy is ,70% which is still not recommended for regular detection of influenzas ([162,163]). ELISA is better
than HI assay but cannot detect early-stage infections. HI assay and complement fixation methods show sensitivity
and specificity of 91.2%, 38.7% and 25.7%, 85%, respectively. Conventional RT-PCR is good but it is time-
consuming [132,164]. Molecular combination with nanotechnology provides new diagnostic methods effective for
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 71

diagnosis. While many biosensors are based upon immunological properties (antigen
antibody), DNA biosensors
can be a better choice [165]. A user-friendly kit that is easy, fast, sensitive, specific, time-saving, and cost-effective
is the need of the hour.

5.7 Prevention and control

Prevention of swine influenza includes three phases: (1) prevention in swine, (2) prevention of transmission to humans,
and (3) prevention of spread among humans. Preventing the spread of swine flu among the swine is the foremost step
to break the chain of interspecies transmission. Facility management and herd management and vaccination are the
keys. Prevention of swine flu from swine to human (zoonotic transfer) mostly occur when people in close proximity
with swineherds. Since pigs can be infected with both avian and human strains of the H1N1 influenza virus, they are
the prime hosts where antigenic shifts occur leading to the emergence of new strains of swine flu. Individuals dealing
with pigs are highly encouraged to wear face masks. Individuals who smoke and do not wear gloves or masks are more
susceptible to contracting swine flu infection. Mode of transmission from human
humans and preventive measures are
described in above sections. Lifestyle interventions like maintenance of good health, balanced diet, and hygiene habits
minimize the influenza virus transmission. Furthermore, covering coughs and sneezes along with avoiding close contact
with sick people and spitting and the use of face masks play important roles in preventing the transmission. Prevention
and control of influenza depends on the effective communication between public health sectors and clinical interven-
tions. Social distancing, masks mandate, and frequent hand washing are key elements in reducing the transmission.
Surface sanitization can be done by alcohol but the sanitizing effect can long last with the addition of quaternary ammo-
nium compounds. Infection spread can be greatly reduced by closing the heavily crowded premises like schools, restau-
rants, and so on. And finally, the most important factor is to avoid traveling and gatherings. The most important step of
prevention is vaccination of swineherds or humans.

5.7.1 Vaccination
Vaccines are very powerful public health tools to prevent the spread of infectious diseases having pandemic potential.
Seasonal influenza vaccines do not provide protection against swine flu influenza as the HA from the novel swine flu
strain is different from seasonal influenza A (H1N1) [166]. Despite the occurrence of various possible subtype combina-
tions in nature, only three subtypes have consistently persisted in the human population, causing the following pan-
demics in the process as defined by sustained, widespread person-to-person transmission: 1918 and 2009 (H1N1), 1957
(H2N2), and 1968 (H3N2) [167]. At present, only the H1N1 and H3N2 subtypes, as well as the two antigenically dis-
tinct IBV lineages (Victoria and Yamagata), are currently endemic in the human population [168], hence several IAV
vaccines contains two representative IAV and IBV strains [169].
The development of antiviral drugs is much slower than the development of vaccines [59]. Genetic drifting of the
predominant circulating strains of IAVs necessitates reformulation of vaccine every year. Vaccine effectiveness is an
estimate from an observational study, whereas vaccine efficacy is an estimate deduced from a clinical trial. Vaccine
efficacy is the percentage reduction of cases amongst vaccinated individuals from the routinely collected data
[170,171]. In the case of swine flu, the high-risk groups such as children and young adults and adults below 49 years or
people having asthma, heart disease, diabetes, and who are immune-compromised are prioritized first for vaccination.
The most common method to produce influenza vaccine is to grow the virus in fertilized hen’s eggs where the virus
usually gets inactivated after purification and helps to produce an inactivated virus vaccine. Alternatively, the virus can
be grown in eggs until it loses its virulence and a live vaccine is produced from that avirulent strain. As the rate of
mutation of the virus is high, a specific influenza vaccine usually confers protection for no more than a few years and
results in the variability of the effectiveness of these influenza vaccines [60].
Prediction of the new virus strains which are likely possible to be circulated in the succeeding year is periodically
done by the WHO each year. This also permits pharma companies to develop the efficient vaccines against these novel
strains (World Health Organization (WHO) report 2006). There are vaccines available to protect poultry against avian
flu viruses. These vaccines display effectiveness against multiple virus strains, hence can be used as a prevention tool
[172]. Reformulation of vaccines is done every year, but it may not be possible to cover all the active strains currently
infecting population. Approximately a period of 6 months is required to formulate and for the production of millions of
vaccine doses necessary to be dealt with the seasonal or novel epidemics [173].
Young and healthy adults were given priority over elderly, who displayed certain degree of protection during the
2009 pandemic [174], and also it was confirmed that the average age for lab-confirmed deaths was 37 years [86].
72 Pandemic Outbreaks in the 21st Century

Preliminary estimates of mortality and years of life lost associated with the 2009 A/H1N1 pandemic in the United
States and comparison with past influenza seasons. Various studies by the National Institute of Health (NIH) revealed
that a single dose was sufficient to produce enough antibodies to protect within 10 days. Vaccination is prohibited for
those who had a previous allergic reaction to the influenza vaccination. Those who are moderate to critically ill, with or
without fever or asymptomatic should be vaccinated after they recover. The vaccination takes about 2 weeks to develop
protection against the disease. Post-vaccination, the immune system responds in a similar way the body is actually
infected and can cause usual symptoms which are not as long-lasting as a virulent influenza virus. Very rarely, severe
allergic reactions are seen as a lethal side effect (Centers for Disease Control and Prevention (CDC) 2006).
Pregnant women were given top priority to get the A(H1N1)p09 vaccine as they are more likely to get pneumonia
leading to complications due to body’s hormonal, physical, and immunological changes to accommodate the growing
fetus [175]. The typical flu season runs from October to early next year and pregnant women in these months are highly
recommended to get the regular seasonal flu vaccine as well as 2009 swine flu if it is available. Both vaccines can be
given at the same time safely at any time during pregnancy.
Due to limited treatment options, high risk for secondary infection, and frequent need for hospitalization as well as
intensive care of patients with H1N1 pneumonia, preventive measures like environmental control, including vaccination
of high-risk populations and public education are critical to control swine influenza outbreaks. Recently, progress in
influenza surveillances both domestic and international, antiviral therapies, and robustness in the methodology for vac-
cine production [176] is highlighted. On the other hand, information is available on the advancements in the develop-
ment of universal flu vaccines, pertaining to conserved parts, such as matrix protein, NP, HA, and possible cocktail
combinations [177]. These new-age vaccines might offer broad-spectrum protection against new influenza antigenic
variants during novel pandemic situations.

5.8 Clinical management

The clinical management of 2009 swine flu infected patients depends on the extent of severity of symptoms. Most
often, milder to moderate cases can be treated at their residences with adequate rest (and also to prevent spreading), fre-
quent oral hydration and symptomatic treatment, with administration of antipyretics, antihistamines, oral hydration, and
NSAIDS (nonsteroidal antiinflammatory drugs). Avoiding alcohol and tobacco is recommended for those suffering
from swine flu. Paracetamol is recommended to ameliorate swine flu
associated fever and muscle aches.
Aspirin is prohibited for small children and teenagers as it can lead to Reye’s syndrome, a potentially fatal disease
of the liver [178]. Although antibiotics do not work on swine flu infection, they can be used to treat or prevent in the
case of secondary infections, such as bacterial pneumonia.
The patients with progressive or severe symptoms must be admitted to hospitals and preferably in ICU, especially
when the signs are indicative of upcoming respiratory failure or sepsis or multiorgan dysfunction. Extensive supporting
measures such as intravenous hydration, normalizing the electrolyte imbalances, antibiotics for secondary bacterial
infections are to be administered. Patients who are developing ARDS should be treated with mechanical ventilation.
Highly severe cases of swine flu
induced ARDS cases require the utilization of extracorporeal membrane oxygenation.
Prognosis of swine flu indicates that some patients hospitalized are at the risk for sepsis, ARDS, and death which are
predicted by chronic pulmonary disease, obesity, neurological diseases, delayed admission, and other underlying
conditions.
The antiviral medications have been a central component of swine influenza treatment which could help reduce the
severity or possibly prevent the effects of swine flu enabling the patient to feel better and sooner [179]. However, anti-
virals are not a substitute for vaccine but a supplement, while the vaccination still remains the major means of prevent-
ing morbidity and mortality. Antivirals are most effective when administered within the first 48 h of the onset of
symptoms, although they may also be used in severe or high-risk cases. Two types of inhibitors are used as antiviral
drugs for the treatment of swine flu, that is, NAIs (oseltamivir, zanamivir) and M2 inhibitors (MI) or adamantanes
(amantadine, rimantadine). Both groups of inhibitors are effective against swine flu, although they do have few side
effects. Antiviral is only effective until the influenza strains are not resistant to drugs [180].
NAIs: The inhibition of NA is a convenient step in the prevention of H1N1/09 and could serve as a potential drug
target. It can also act as a transition-state analog inhibitor of influenza NA which can prevent the progeny virions from
detaching from the infected cells. Oseltamivir (Tamiflu, Genentech) and Zanamivir (Relenza, GlaxoSmithKline) are
members of a new class of drugs called NAIs and active against both IAVs and IBVs.
Oseltamivir is the first commercially developed, orally active NAI [179] and could act also as a transition-state ana-
log inhibitor of influenza NA preventing the virus progeny from detaching from the infected cells. Oseltamivir is a
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 73

competitive inhibitor of SA, present on the host’s cellular surface proteins. It blocks the viral NA activity and prevents
the release of progeny virions from the infected cells [181]. It is hydrolyzed in the hepatic cells to form the active free
carboxylate of oseltamivir. Its recommended dosage for adults is usually, 75 mg twice daily over a period of 5 days,
whereas in case of children, 30
75 mg twice daily, based upon the bodyweight [15]. Side effects of oseltamivir include
the following. Oseltamivir is generally well tolerated with minimum side effects like nausea, vomiting abdominal pain.
Few sporadic events of transient neuropsychiatric side effects are reported in young adult female patients [182], in the
older patients [183] and adult patients with H3N2 influenza A [184] and in FDA Adverse Event Reporting System
[185]. Very rarely, anaphylaxis and skin rashes are reported.
Zanamivir is another NAI used in the treatment and prophylaxis of influenza virus A and B viruses. At present, it is
marketed with a trade name of Relenza by GlaxoSmithKline. The recommended dosage is 10 mg twice a day for 5
days [186]. This is not recommended for the patients with breathing difficulties [187]. Zanamivir binds to active site of
NA thereby influenza virus cannot escape from the host cells and prevent infecting other cells [187]. Due to an allergy
to eggs, Zanamivir is contraindicated. Other side effects may cause shortness of breath due to constriction of airways,
allergic reactions, seizures, hallucinations, and delirium [186].
Oseltamivir is supplied as an oral capsule. Zanamivir on the other hand is supplied as dry powder which can be
administered by inhalation. Both can be used for treatment of uncomplicated and symptomatic swine flu cases, pref-
erably within 48 h. However, Oseltamivir and Zanamivir are recommended for patients aged .1 year and .5 years,
respectively [188]. Antiviral resistance in many strains of H1N1 may develop quickly and emerge during the treat-
ment and FDA-approved NAI drugs such as Oseltamivir and Zanamivir [189
191]. A small proportion of swine flu
viruses that contained the N2 NA (H1N2, H3N2, and H9N2) developed resistance toward NAIs [188]. Due to side
effects like nausea, vomiting, abdominal pain and headache, and sporadic rash, allergic reactions including anaphy-
laxis, and so on, there is a call for new generation of inhibitors against the H1N1 IAV with less or no side effects.
Oseltamivir and zanamivir can be used to treat swine influenza and are most effective when taken within 48 h of
becoming sick [175].
CDC tested 2008 (Oct) seasonal influenza A (H1N1) and “2009 influenza A (H1N1)” viruses collected in their
respective outbreaks and demonstrated that they behaved differently in terms of ability to develop resistance against
these drugs. Out of 1146 seasonal influenza A (H1N1) viruses, 99.6% of the samples displayed resistance to oseltami-
vir, whereas none showed resistance to zanamivir. Similarly, out of the 853 samples from the 2009 influenza A
(H1N1), only 4% showed resistance to oseltamivir, while none showed resistance to zanamivir.

5.8.1 M2 inhibitors
Antiviral drugs, namely, amantadine and rimantadine block the viral ion channel (M2 protein) and prevent the IAVs
from infecting cells [192]. These drugs are ineffective against IBVs due to the absence of M2 proteins [193].
Amantadine is an organic compound where one of the four methylene positions of the backbone is substituted by an
amino group which is known as 1-amino-adamantane and supplied under the trade name of Symmetrel. In addition to
an antiviral drug, it can also be used as an antiparkinson drug [194]. However, a high-level resistance against these
drugs (adamantine and rimantadine) has been developed in the virus strains, because of global usage to prevent out-
breaks of influenza in farmed poultry [195]. Recommended dosage is 100 mg once daily to IAV-infected people [196].
Viral M2 protein is necessary for uncoating the virus particle inside a cell through endocytosis. This uncoating process
inside the cell is inhibited by amantadine. Furthermore, it blocks the ion channel formation by M2 protein [197]. Side
effects include general neurological problems like nervousness, agitation, anxiety, insomnia and trouble with concen-
trating, and so on.

5.8.2 Rimantadine
Rimantadine, an antiviral drug to prevent IVA infection is commercially traded as Flumadine. It is able to minimize the
duration and reduce the severity of IVA if taken within 1
2 days of becoming sick [198]. The recommended dosage is
100 mg twice a day for 7 days after the onset of symptoms. Children below 16 years of age are not recommended to
take this drug. Patients with kidney failure or liver ailments and older people are recommended to take a lower dose,
that is, 100 mg once daily [199]. Rimantadine inhibits the M2 ion channel and, subsequently, inhibits the replication of
virus [196]. Side effects including nausea, stomach upset, nervousness, tiredness, lightheadedness, trouble sleeping, and
difficulty in concentrating are common [179].
74 Pandemic Outbreaks in the 21st Century

5.8.3 Finding an ultimate cure for the disease

Researchers suggest that shikimic acid is the basic compound for the production of the swine flu drug Tamiflu and is
found in the seeds of an evergreen Chinese plant Star anise (Illicium verum) [200]. Nowadays, shikimic acid plays an
important role against swine flu by reducing the severity of the symptoms. It is obtained from the seeds of Star anise
and, further, is converted to epoxide. In the process of the formation of Tamiflu, the most dangerous part involved is
the azide chemistry, as it is very explosive [201].

5.8.4 Shikimic acid

Shikimic acid occurs in a variety of natural compounds. It is an intermediate compound in the formation of several aro-
matic compounds. It is a white crystalline compound having two types of functional groups in the same molecule, that
is, three hydroxyl groups and a carboxylic group. It is widely used as a chiral building block for the synthesis of phar-
maceuticals [202].

5.8.5 Importance and uses of shikimic acid

Shikimic acid acts as an important compound against swine flu as it serves as the starting material for the production of
Tamiflu (oseltamivir) [203] which is widely used as a chiral building block for the synthesis of pharmaceutical drugs
such as oseltamivir (a NAI), which is used in the treatment and prophylaxis of both IA and IB viruses [204].

5.8.6 Limitations of shikimic acid production

The bulk production of Tamiflu drug is a tedious task, as it not only takes several months to finish but is hazardous
too. Careful handling and milder reaction conditions are necessary for the synthesis, as potentially explosive azide
chemistry is involved. The bulk production requires a sufficient amount of starting material (shikimic acid). The major
harvesting period for Star anise is starting from March to May, which is not enough to stock the bulk of starting mate-
rial to meet the global drug requirements. In addition to Star anise, Ginkgo biloba can also be a potential source of
shikimic acid [205]. As both raw materials are in short supply, the pharma industry needs to find an alternative
sustainable supply.

5.8.7 Alternative approach

Recently, it has been reported that shikimic acid can also be extracted from a few microbes, as it exists as an
intermediate metabolite in the amino acid synthesizing pathway. During the production of shikimic acid, avoiding
the formation of shikimate pathway byproduct is the main problem, as quality and yield are compromised [203].
While carbon-limited growth conditions increase byproduct formation, carbon-rich conditions favor shikimic acid
production when compared to that of byproducts [206,207]. The use of a recombinant strain of Escherichia coli
under fermenter-controlled conditions has resulted in the synthesis of shikimic acid from glucose. However, in
this experiment, along with shikimic acid, quinic acid and dehydro-shikimic acids were also synthesized as bypro-
ducts [208].
Iomantas et al. [209] carried out an alternative approach for shikimic acid production by EPSP synthase-deficient
E. coli strains by blocking the aromatic amino acid pathway after the production of shikimate-3-phosphate (S3P).
Bacterial phosphatases convert S3P to shikimic acid. According to Draths et al. [208], the microbial production of shiki-
mic acid by metabolic engineering is most advanced. Through metabolic engineering, the aromatic amino acid pathway
is blocked soon after the synthesis of shikimic acid. The whole process of blocking is done by the transduction of dis-
rupted aro K and aro L genes encoding shikimate kinase I and II.
Scientists in India have not yet attempted to research on the production of shikimic acid and its applications. A sur-
vey of the literature shows that there are no research publications or patents directly addressing the production and
application of shikimic acid in India. In this context, only Cipla, the Indian generics company, claims to produce a ver-
sion of Tamiflu within months, since they have experience of making the HIV drug AZT, which relies on a similar
chemistry. Currently, the market price of shikimic acid is $1000 per kilogram, increased from the usual price of $40 per
kilogram due to a huge demand in the world market for Tamiflu.
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 75

5.9 Threats and challenges

IAVs pose a serious threat to public health and influenza community. IAVs cause recurrent epidemics and occasional
catastrophic pandemics in humans. The natural reservoir for IAVs is the wild aquatic birds (ducks and geese) which is
the primordial source of all influenza viruses in other species including humans and lower animals. They have been iso-
lated even from penguins in Antarctica [210]. The gene pool from avian reservoir provides all the genetic diversity nec-
essary for emergence of pandemics in humans and pigs. However, for a pandemic to occur, animal IAVs, by acquiring
new viral gene variants, need to cross human species barrier (zoonotic) and further attain an efficient human
human
transmissibility. In 2009 a triple reassortment pandemic H1N1 strain (made up of avian, swine, and human influenza
genes) crossed from pigs to humans, resulting in the co-circulation of three influenza strains [167].
Swine influenza pandemic (2009) was very similar to previous pandemics such as Spanish flu (1918), Asian flu
(H2N2, 1957), Hong Kong flu (H3N2, 1968), and the recent SARS-CoV2 (ongoing), in terms of spreading to different
geographical regions. However, the epidemiological dynamics and wave behavior of each pandemic varied amongst
countries. The common features of pandemics are: (1) contagiousness, fatal spreading to all over the world via human
mobilization such as military troops, national or international travel, and so on, and (ii) the severity, proportional to
chronic diseases. Among all the influenza pandemics, the H1N1 pandemic of 1918 (Spanish flu) was the most disastrous.
Since then, the same strain was in circulation which disappeared in 1957 (during pandemic H2N2), reemerged in 1977,
and again as a variant H1N1 swine flu in the 2009 pandemic [8]. Each pandemic may acquire gene segments from the
bird and/or animal reservoir by genetic reassortment which could result in novel expression of HA and NA surface gly-
coproteins to which the majority of human population has little or no immunity. Hence, the major threat is the constant
evolution of surface antigens (HA and NA) in response to pressure from the host’s immune system [211]. This change
happens by a process called genetic drift or shift. A unique feature of IAVs is that they can circulate not only in humans
but also in domestic animals, wild aquatic birds, poultry, horses, pigs, and so on. The potential ability to change and
adapt to multiple animal species is the main reason why IAVs are more diverse and challenging than IBVs.
76 Pandemic Outbreaks in the 21st Century

Influenza viruses have two different ways to change: a slow change and a fast change. The slow change is called
“genetic drift”—virus progressively accumulates point mutations until its surface glycoproteins HA and NA are no lon-
ger recognized by the host’s immune system. This antigenic drift is driven by low fidelity of virus’ RNA-dependent
RNA polymerase. The individual mutations are positively selected before being fixed resulting in novel strains, which
are antigenically different and may make one of the virus’s progeny unrecognizable to the antibodies generated by the
previous infection or vaccination. Since these point mutations are random, drift is both inevitable and
unpredictable making impossible to predict how exactly the virus will look like to host’s immune system, which is why
influenza epidemics occur each year. The fast change is called “genetic shift”—antigenic shift is even more dramatic.
Instead of accumulating point mutations that progressively change the appearance of its surface glycoproteins, it can
possibly acquire entirely new surface proteins. This is called antigenic shift which is made possible due to a process
called reassortment. Genetic reassortment happens, when different strains simultaneously infect the same cell, the gen-
omes can be shuffled and packaged into new genetic combinations creating a brand-new (hybrid) virus progeny with
novel surface proteins (antigenicity) that are totally different from the previous epidemic causing strain [17].
Based on antigenicity, IAV subtypes are differentiated into two surface glycoproteins: HA and NA, which facilitate
the host cell entry and exit, respectively. A total of 18 HA subtypes and 11 NA subtypes are found in nature, for a theo-
retical total combination with a large genomic diversity of 198 strain variations thus making it more complex to control
than other viruses prevalent across the globe (CDC 2019: http://www.cdc.gov/flu/about/viruses/types.htm).
HA subtypes are further subdivided into two groups [212]: group 1: H1, H2, H5, H6, H8, H9, H11, H12, H13, H16,
H17, and H18; and group 2: H3, H4, H7, H10, H14, and H15. To date, the 16 HA (H1
16) and 9 NA (NA1
9) sub-
types are found to circulate in the different aquatic birds globally [17,213], where they replicate primarily in the gut at
the elevated body temperature of birds (40 C), have a target-cell receptor specificity for α2,3 SA, and are primarily
spread by fecal
oral transmission via water. The remaining 2 HA (H17 and H18) and 2 NA (N10, N11) subtypes of
IAVs are identified in bats but these do not seem to recognize SA [214], they have not been cultured but can be
detected by PCR, cause inapparent disease [215]. However, for over a century, only viruses expressing subtypes,
namely, H1, H2, and H3 are known so far to have successfully caused substantial person-to-person transmission [25].
Avian H5N1 and H7N9 viruses have circulated for a while but without leaking into humans and causing a pandemic
[216].
The pandemic lessons have provided valuable insights into dealing with the continued challenges of influenza, in
terms of its unpredictability and severity. It is difficult to predict a specific IAV pandemic with a specific subtype and
of specific host origin. If we focus on swine flu, we may be hit by avian flu viruses, for example, thus one-health
approach is the need of the hour and global preparedness of any pandemic is recommended. Below are few examples
we witnessed, where a specific anticipation of one pandemic is not appropriate.
First, the A(H1N1)p09 was completely unexpected when the influenza community was facing highly pathogenic
H5N1 influenza in humans (2008) with a possibility of becoming a pandemic. Pandemic plans and single class of anti-
viral drug stockpiles (NA inhibitors) of H5N1 could not provide much help and also the H1N1 seed stocks did not
match antigenically. It took some time to realize that this strain was not of avian origin but of a swine origin, and it
started in the American continent not in Asia. This suggests that a potential pandemic virus can be emerged anywhere,
signifying the importance of swine surveillance in other parts of the globe. The over-enthusiasm of news media in nam-
ing the disease as “swine flu” led to a drastic fall in the global demand for pork, resulting in serious negative economic
consequences.
Second, the emergence of pandemic potential A(H1N1)p09 while the descendants of the 1918 H1N1 Spanish pan-
demic were still causing seasonal flu, was also totally unpredicted. This indicates active recycling of the H1, H2, and
H3 subtypes in humans and the need for continuous risk assessment of H2N2 influenza viruses. The severity of the
infections was another unexpected feature of the 2009 H1N1 pandemic. Although severe in young healthy population,
pregnant women, and obese people, the A(H1N1)p09 was relatively milder than the 1918 Spanish flu. Therefore, it is
often challenging to declare a pandemic and its severity, without causing the global panic [24].
Another unexpected challenge surfaced with an emergence of the H7N9 influenza virus in China in 2013, which
caused atypical disease in poultry and wildlife, but severe respiratory illness in humans through zoonotic transfer.
Therefore, the current challenge is to be prepared if either the H5N1 or H7N9 avian viruses gain sustained human-to-
human transmissibility or the H2N2 influenza virus which is missing from humans for 50 years can reemerge from the
aquatic bird reservoir. At the same time, the swine flu pandemic is not the first and not the last.
The remarkable feature of A/(H1N1)p09, compared with seasonal strains, was its high CFR as well as higher inci-
dence in the younger population. According to WHO, the swine flu 2009 death numbers only include laboratory-
confirmed cases or confirmation in the laboratory was not obtained for all cases thus the magnitude of the event would
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 77

have been underestimated. Hence, it is very important to note that A(H1N1)p09 strain is still circulating among the
populations. Therefore, it is essential to continue the epidemiological and virological surveillance [217]. Very recently
in 2015, the same variant swine flu strain, that is, A(H1N1)p09 which caused the 2009 global pandemic, also infected
the Indian population with over 10,000 cases reported and 774 deaths [7].
The fatality rate of swine flu is low by now and the impact is similar to an average flu season especially when cer-
tain population is immune through infection or immunization. However, antigenic drift/shifts in the A(H1N1)p09 virus
are expected to occur in the future but the timing is unpredictable. The major pathogenicity marker PB1-F2 gene is
implicated in the low mortality of novel swine flu virus. If this strain mutates and changes two nonsense codons cur-
rently present in the PB1-F2 gene through genetic drift, a more threatening phenotype might evolve with possible high-
er influx of neutrophils into lungs and elevated protein expression of cytokines and chemokines in the lung tissues
[218]. However, other factors like the NP or the polymerase also contribute to potential pathogenicity of influenza
viruses. At the same time, through genetic shift, reassortment of the current A(H1N1)p09 with a PB1-F2 expressing
strain might also result in a highly pathogenic strain. To overcome challenges of antigenic drift and shifts, more
advances in vaccine research needs to be carried out especially on increasing the antigenicity via potential role of gly-
coengineering [219].
IAVs are enzootic and can move from swine, avian, equine, feline, and canine species to infect humans; however;
the risk is not the same for all the viruses. Among all the animal IAVs, when exposed equally, swine IAVs can infect
humans most efficiently due to lower barrier followed by avian IAVs [11] and there is a significant barrier for feline,
equine, and canine IAVs in increasing order to transit into humans. Besides, cats, horses, and dogs do not dwell in
dense populations sufficient enough to spread the disease.
Commercial poultry/swine farms, wet markets, backyard poultry farms, slaughtering facilities, global trade of exotic
animals have all been implicated in the spread of IAVs and pose a significant threat to public health through zoonotic
transfer. Multiple highly diverse swine influenza viruses exist and circulate in swine populations [220,221] due to grow-
ing international trades, which can accelerate the virus evolution thus complicating the swine influenza situations world-
wide. Novel genotypes and subtypes of viruses could emerge at any time in pigs due to infections with avian and/or
human influenza viruses. This can result in the emergence of novel swine influenza viruses into naı̈ve swineherds. For
example, the unique H2N3 virus was detected in swine in the United States in 2006, which was infectious and highly
transmissible in swine and ferrets and had undergone some adaptation to the mammalian host [222]. This is a huge
challenge to produce effective vaccines and control swine influenza for the swine industry.
The significance of pig
human interface is not only true for IAVs but also it is the only interface that can be
infected by all other viruses IBVs, ICVs, and IDVs [223]. This nexus, together with rapid expansion of swine industry
can excessively increase the transmission risk and the emergence of novel influenza variants. Hence, S-OIVs are to be
given the top priority for global surveillance. Constant global surveillance of swineherds is essential to provide evidence
on mixing of new genetic elements in swine leading to the emergence of novel viruses so as to prevent the zoonotic
transfer into humans and subsequent human
human transmissibility with pandemic potential.
On the contrary, reverse zoonotic transmissions, that is, transmission of A(H1N1)p09 virus from humans to ferrets
and mice [105], Vietnamese swine [224], and Korean swine [225] were reported. By way of whole-genome sequencing
data and large-scale phylogenetic analyses, at least 49 human-to-swine transmission events that occurred globally dur-
ing 2009
11 have been identified. Therefore, this highlights the ability of the A(H1N1) p09 virus to transmit repeatedly
from humans to swine, even following adaptive evolution in humans. Similarly, it was identified at least 23 separate
introductions of human seasonal (nonpandemic) H1 and H3 influenza viruses globally into swine since 1990 [226].
Centers of Excellence for Influenza Research and Surveillance program discovered that the H1N1pdm09 virus
though was not detected in swine in Asia, but the precursors of it had been circulating in swine for as long as close to 2
decades without detection before its 2009 outbreak [227]. Hence, there is a growing importance of unifying the veteri-
nary and human surveillance, research, and medicine as a one world
one health concept and protect swine/poultry
farms under constant surveillance to prevent a pandemic potential.
Industrial farms in developed countries like the United States and Europe may have been practicing reasonable bio-
security and biosafety measures; however, this is not true in the rest of the world. Intensive training programs with
international quality standards to improve reliable biosecurity and biosafety measures are essential. This includes both
for the farm management as well as for animal workers including the provision of personal protective equipment (ppe
kit) [228]. In addition, similar precautionary measures should also reach wholesale markets and slaughterhouses.
Animal workers would benefit from annual receipt of seasonal human influenza vaccines.
Although genome information of human H1 and H3 influenza viruses are available, similar genome sequences of
wild avian H1, H3, H5, H7, and H9 viruses are absent. It is important to seal the knowledge gaps to explore and
78 Pandemic Outbreaks in the 21st Century

establish those influenza viruses having pandemic threats. Fruitful partnership between animal production farms, veteri-
nary sector, and public health will benefit from resources and latest technologies. It is essential to design a simple,
robust virus sampling preferably via noninvasive surveillance techniques. Bioaerosol samplings coupled with rapid
diagnostics serve as potential screening tools [229]. At present, commercial companies are focusing on developing pen-
side and rapid tests for animal viruses. For example, the FluChip-8G kit, developed by InDevR, is specifically designed
to identify and characterize IAVs and IBVs. High-throughput sequencing platforms such as Oxford’s MinION nanopore
sequencer and Illumina Iseq 100 sequencing system coupled with cloud-based data storage, remote access to bioinfor-
matics software pipelines (Chan Zuckerberg Biohub), and similar such platforms will become more portable and power-
ful to detect the infectious pathogens.
Although various swine flu vaccines are widely administered in swineherds similar to yearly seasonal influenza vac-
cines used for humans, swine flu cannot be efficiently controlled and is still one of the major challenges, causing con-
siderable economic losses in the swine industry. A novel virus could emerge in pigs, with a capacity to infect humans
and a potential to adapt human transmission in the future to instigate an epidemic or pandemic similar to A(H1N1)p09
virus. Global Influenza Surveillance and Response System comprising of .120 laboratories monitors influenza glob-
ally. Antigenic analysis, sequence analysis, and population susceptibility based on antibody levels in human sera are
used to make recommendations on vaccine content reformulations. For successful prediction of the dominant circulating
strain, the proposed recommendations have to be suggested 6
8 months before vaccine development.
Development of a universal vaccine for influenza, effective against all subtypes of influenza, including seasonal
influenza is safe, induces long-lasting immunity in animals and humans would be the ultimate success of influenza vac-
cinology which could save millions of lives. For this, basic knowledge of the structure and immunogenicity of the HA
of influenza virus is essential. A common antigenic domain in the region of the fusion peptide can be used as a determi-
nant. In addition to universal vaccine, development of universal mAbs and the possible development of potential small-
molecule antivirals or nanoparticles are exciting possibilities that could revolutionize the control of influenza. Improved
sanitation and biosecurity in the animal farms and vaccinations would largely prevent the zoonotic transfer to humans.
If intermediate hosts (i.e., pigs, poultry, and horses) were engineered to be resistant to influenza, then transfer between
the avian reservoir and humans would be reduced. Genes that confer resistance to influenza could be inserted into the
susceptible hosts.
However, the 2009 swine flu pandemic, though milder than earlier pandemics in terms of gross fatality rate, had led
to a significant damage to healthcare systems and also economies [230]. Hence there is a growing importance on pan-
demic preparedness beyond health care management but also on global economic consequences downstream. Ever
since, 1918 pandemic, we have made a little but significant contribution to public health as well as scientific advances
in terms of antivirals, vaccines, ICUs, blood thinners, ventilators, and so on. However, in terms of understanding the
molecular biology of influenza viruses and their ability to cause infection in humans we are still in the preliminary
stage, thus it signifies the essential need for continued basic research. The identification of vital molecular determinants
mediating the pathophysiology of swine flu infections will lead to effective drug discovery and development strategies,
including insights on ideal timings for administration of the antivirals and/or antibiotics. The development of universal
influenza vaccines with broad-spectrum efficacy will be the ultimate development.
Influenza pandemics with typical respiratory illnesses are caused by the similar mode of transmission. Hence, focus
should also be given to control the influenza pandemic through nonmedical interventions. This includes regulating and
improving certain parameters in schools, office premises, hospitals, and so on, such as proper ventilation/air filtration,
temperature, humidity (Rh), hygiene, quarantine facilities, household crowding, population density, and so on [92,99].
Current indications and scientific evidence support a potential role for all routes of transmission whose relative signifi-
cances rely on the set of circumstances playing at a specific condition and time. The major factors are related to virus
itself, the host, and the environment. Transmission is a dynamic and opportunistic process that can possibly occur via
multiple routes during the same event. In addition to this, the efficiency of transmission also depends on the R0 of the
influenza virus. Hence, multiple interventions involving both personal responsibilities (masks, hand hygiene, social dis-
tancing, avoiding touching face, and limited time in the crowded places) as well as shared responsibilities (fast and sen-
sitive testing and tracing, ventilation, air filtration, isolation, vaccination, proper communication, etc.) are necessary to
control. And at the same time, no single intervention is safe and secure to prevent the infection.
Effective communication among the members of interprofessional teams helps reduce the morbidity and mortality
of swine flu [99]. It is necessary to bring awareness among the clinicians and communities about the distinction
between clinical manifestations of swine flu and seasonal flu and an early detection and effective therapies. A specific
diagnostic kit for swine flu should be developed so that early interventions can be carried out. Furthermore, strengthen-
ing the collaboration among human, animal, and environmental health sectors is essential to efficiently prevent or detect
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 79

and respond to influenza pandemics and thus achieve one health perspective to improve human, animal, and environ-
mental health and well-being. A hundred years from now, we can imagine how the researchers would look back at the
huge data that we store on a daily basis. Development and maintenance of online genealogy databases, that are crowd
sourced, are important for further investigations in the future research [231] that could also throw light on family link-
age and host genetic risk factors, which could be tested among their descendants. As paper-based archives rapidly decay
or can be lost due to damage or fire, and so on, digitization needs to be done as early as possible.

5.10 Conclusion
In spite of the scientific and public health advancements, including access to influenza vaccines, influenza viruses
remain a global public health threat. There exist still many challenges and unanswered queries on the evolution of IAVs
leading to potential pandemic strains. Therefore, more efficient global surveillance of IAVs in swine is required to bet-
ter comprehend the ecology of IAVs, and more effective swine vaccinations need to be produced to protect animal and
public health. Finally, it is essential to support the research-driven focus to gain more evidence-based infectious biology
from multiple hosts to decipher the pandemic potential of all the IAVs.

References
[1] King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Virus taxonomy. San Diego, CA, USA: Elsevier; 2012. p. 749
61. Family—
Orthomyxoviridae.
[2] Hause BM, Ducatez M, Collin EA, Ran Z, Liu R, Sheng Z, et al. Isolation of a novel swine influenza virus from Oklahoma in 2011 which is
distantly related to human influenza C viruses. PLoS Pathog 2013;9(2):e1003176.
[3] Hause BM, Collin EA, Liu R, Huang B, Sheng Z, Lu W, et al. Characterization of a novel influenza virus in cattle and Swine: proposal for a
new genus in the Orthomyxoviridae family. MBio 2014;5 e00031-00014.
[4] Shen CF, Ho TS, Wang SM, Liao YT, Hu YS, Tsai HP, et al. The cellular immunophenotype expression of influenza A virus and influenza B
virus infection in children. Clin Immunol 2020;219:108548.
[5] Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE, Fanning TG. Initial genetic characterization of the 1918 “Spanish” influenza virus.
Science 1997;275:1793
6.
[6] Taubenberger JK, Morens DM. 1918 Influenza: the mother of all pandemics. Rev Biomed 2006;17(1):69
79.
[7] Kshatriya RM, Khara NV, Ganjiwale J, Lote SD, Patel SN, Paliwal RP. Lessons learnt from the Indian H1N1 (swine flu) epidemic: predictors
of outcome based on epidemiological and clinical profile. J Family Med Prim Care 2018;7(6):1506
9.
[8] Nickol ME, Kindrachuk J. A year of terror and a century of reflection: perspectives on the great influenza pandemic of 1918
1919. BMC
Infect Dis 2019;19(1):117.
[9] Klenk HD, Matrosovich M, Stech J. Avian influenza: molecular mechanisms of pathogenesis and host range. In: Mettenleiter TC, Sobrino F,
editors. Molecular biology of animal viruses. Wymondham: Caister Academic Press; 2008. p. 253
303.
[10] Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza virus receptors in the human airway. Nature 2006;440
(7083):435
6.
[11] Borkenhagen LK, Salman MD, Ma MJ, Gray GC. Animal influenza virus infections in humans: a commentary. Int J Infect Dis
2019;88:113
19.
[12] Lagacé-Wiens PRS, Rubinstein E, Gumel A. Influenza epidemiology—past, present, and future. Crit Care Med 2010;38(4):e1
9.
[13] Garten RJ, Davis CT, Klimov AI, Cox NJ, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulat-
ing in humans. Science 2009;325(5937):197
201.
[14] Keenliside J. Pandemic influenza A H1N1 in swine and other animals. Curr Top Microbiol Immunol 2013;370:259
71.
[15] Rewar S, Mirdha D, Rewar P. Treatment and prevention of pandemic H1N1 influenza. Ann Glob Health 2015;81(5):645
53.
[16] Peiris JS, Poon LL, Guan Y. Emergence of a novel swine-origin influenza A virus (S-OIV) H1N1 virus in humans. J Clin Virol 2009;45
(3):169
73.
[17] Dou D, Revol R, Östbye H, Wang H, Daniels R. Influenza A virus cell entry, replication, virion assembly and movement. Front Immunol
2018;9:1581.
[18] Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat
Med 2001;7(12):1306
12.
[19] Lamb RA, Lai CJ, Choppin PW. Sequences of mRNAs derived from genome RNA segment 7 of influenza virus: colinear and interrupted
mRNAs code for overlapping proteins. Proc Natl Acad Sci U S A 1981;78(7):4170
4.
[20] Kochs G, Garcia-Sastre A, Martinez-Sobrido L. Multiple anti-interferon actions of the influenza A virus NS1 protein. J Virol 2007;81
(13):7011
21.
[21] Briedis DJ, Lamb RA. Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1
and NS2 proteins. J Virol 1982;42(1):186
93.
[22] Hatta M, Kawaoka Y. The NB protein of influenza B virus is not necessary for virus replication in vitro. J Virol 2003;77(10):6050
4.
80 Pandemic Outbreaks in the 21st Century

[23] Horvath CM, Williams MA, Lamb RA. Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypep-
tide. EMBO J 1990;9(8):2639
47.
[24] Webster RG, Govorkova EA. Continuing challenges in influenza. Ann N Y Acad Sci 2014;1323(1):115
39.
[25] Krammer F, Smith GJD, Fouchier RAM, Peiris M, Kedzierska K, Doherty PC, et al. Influenza. Nat Rev Dis Primers 2018;4:3.
[26] Fauquet CM, Fargette D. International committee on taxonomy of viruses and the 3,142 unassigned species. Virol J 2005;2:64. Available from:
https://doi.org/10.1186/1743-422X-2-64.
[27] Palese P, Shaw ML. Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields virology. Philadelphia:
Lippincott Williams & Wilkins; 2007.
[28] Bouvier NM, Palese P. The biology of influenza viruses. Vaccine 2008;26(Suppl. S4):D49
53. Available from: https://doi.org/10.1016/j.
vaccine.2008.07.039.
[29] Baudin F, Bach C, Cusack S, Ruigrok RW. Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in pan-
handle RNA and exposes the bases to the solvent. EMBO J 1994;13(13):3158
65.
[30] Badham MD, Rossman JS. Filamentous influenza viruses. Curr Clin Microbiol Rep 2016;3:155
61. Available from: https://doi.org/10.1007/
s40588-016-0041-7.
[31] Seladi-Schulman J, Steel J, Lowen AC. Spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing
strains are selected in vivo. J Virol 2013;87:13343
53.
[32] Calder LJ, Wasilewski S, Berriman JA, Rosenthal PB. Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci U S A
2010;107:10685
90. Available from: https://doi.org/10.1073/pnas.1002123107.
[33] Zebedee SL, Lamb RA. Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol
1988;62(8):2762
72.
[34] Hutchinson EC, Charles PD, Hester SS, Thomas B, Trudgian D, Martinez-Alonso M, et al. Conserved and host-specific features of influenza
virion architecture. Nat Commun 2014;5:4816. Available from: https://doi.org/10.1038/ncomms5816.
[35] Suzuki Y. Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biol Pharm Bull 2005;28:399
408.
[36] Hamilton BS, Whittaker GR, Daniel S. Influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and exper-
imental approaches for assessing virus fusion. Viruses 2012;4(7):1144
68.
[37] Sakai T, Nishimura SI, Naito T, Saito M. Influenza A virus hemagglutinin and neuraminidase act as novel motile machinery. Sci Rep
2017;7:45043.
[38] Kalthoff D, Globig A, Beer M. (Highly pathogenic) avian influenza as a zoonotic agent. Vet Microbiol 2010;140:237
45.
[39] Beare AS, Webster RG. Replication of avian influenza viruses in humans. Arch Virol 1991;119(1
2):37
42.
[40] Dou D, Hernández-Neuta I, Wang H, Östbye H, Qian X, Thiele S, et al. Analysis of IAV replication and co-infection dynamics by a versatile
RNA viral genome labeling method. Cell Rep 2017;20(1):251
63.
[41] Pinto LH, Holsinger LJ, Lamb RA. Influenza virus M2 protein has ion channel activity. Cell 1992;69(3):517
28.
[42] Bullough PA, Hughson FM, Skehel JJ, Wiley DC. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature
1994;371:37
43. Available from: https://doi.org/10.1038/371037a0.
[43] Martin K, Helenius A. Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol 1991;65(1):232
44.
[44] Martin K, Helenius A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits
import. Cell 1991;67:117
30. Available from: https://doi.org/10.1016/0092-8674(91)90576-K.
[45] Bui M, Whittaker G, Helenius A. Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol
1996;70:8391
401.
[46] Wharton SA, Belshe RB, Skehel JJ, Hay AJ. Role of virion M2 protein in influenza virus uncoating: specific reduction in the rate of membrane
fusion between virus and liposomes by amantadine. J Gen Virol 1994;75(Pt 4):945
8.
[47] Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. A universal influenza A vaccine based on the extracellular domain of the
M2 protein. Nat Med 1999;5(10):1157
63.
[48] Cros JF, Palese P. Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res
2003;95(1
2):3
12.
[49] Eisfeld AJ, Neumann G, Kawaoka Y. At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol 2015;13(1):28
41.
[50] Gabriel G, Klingel K, Otte A, Thiele S, Hudjetz B, Arman-Kalcek G, et al. Differential use of importin-α isoforms governs cell tropism and
host adaptation of influenza virus. Nat Commun 2011;2:156.
[51] Pflug A, Lukarska M, Resa-Infante P, Reich S, Cusack S. Structural insights into RNA synthesis by the influenza virus transcription-replication
machine. Virus Res 2017;234:103
17. Available from: https://doi.org/10.1016/j.virusres.2017.01.013.
[52] Fodor E. The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication. Acta Virol 2013;57:113
22. Available
from: https://doi.org/10.4149/av_2013_02_113.
[53] Reich S, Guilligay D, Cusack S. An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase. Nucleic Acids
Res 2017;45(6):3353
68.
[54] Poon LL, Pritlove DC, Fodor E, Brownlee GG. Direct evidence that the poly(A) tail of influenza A virus mRNA is synthesized by reiterative
copying of a U track in the virion RNA template. J Virol 1999;73(4):3473
6.
[55] Reich S, Guilligay D, Pflug A, Malet H, Berger I, Crépin T, et al. Structural insight into cap-snatching and RNA synthesis by influenza poly-
merase. Nature 2014;516(7531):361
6.
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 81

[56] Colman PM, Varghese JN, Laver WG. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature 1983;303(5912):41
4.
[57] Palese P, Compans RW. Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid
(FANA): mechanism of action. J Gen Virol 1976;33(1):159
63.
[58] Ghosh A, Nandy A, Nandy P. Computational analysis and determination of a highly conserved surface exposed segment in H5N1 avian flu and
H1N1 swine flu neuraminidase. BMC Struct Biol 2010;10:6.
[59] Lynch 3rd JP, Walsh EE. Influenza: evolving strategies in treatment and prevention. Semin Respir Crit Care Med 2007;28(2):144
58.
[60] Hilleman MR. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 2002;20:3068
87.
[61] Centers for Disease Control and Prevention (CDC). Intensive-care patients with severe novel influenza A (H1N1) virus infection—Michigan,
June 2009. MMWR Morb Mortal Wkly Rep 2009;58(27):749
52.
[62] Centers for Disease Control and Prevention (CDC). Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza
A (H1N1)—United States, May-August 2009. MMWR Morb Mortal Wkly Rep 2009;58(38):1071
4.
[63] Centers for Disease Control and Prevention (CDC). Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccina-
tion with seasonal influenza vaccine. MMWR Morb Mortal Wkly Rep 2009;58(19):521
4.
[64] Centers for Disease Control and Prevention (CDC). Bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza
A (H1N1)—United States, May-August 2009. MMWR Morb Mortal Wkly Rep 2009;58(38):1071
4.
[65] Centers for Disease Control and Prevention (CDC). Swine influenza A (H1N1) infection in two children
Southern California, March-April
2009. MMWR Morb Mortal Wkly Rep 2009;58(15):400
2.
[66] Centers for Disease Control and Prevention (CDC). Update: drug susceptibility of swine-origin influenza A (H1N1) viruses, April 2009.
MMWR Dispatch 2009;58:1
3.
[67] Centers for Disease Control and Prevention (CDC). Update: drug susceptibility of swine origin influenza A (H1N1) viruses, April 2009.
MMWR Morb Mortal Wkly Rep 2009;58:433e5.
[68] Henderson DA, Courtney B, Inglesby TV, Toner E, Nuzzo JB. Public health and medical responses to the 1957
58 influenza pandemic.
Biosecur Bioterror 2009;7(3):265
73.
[69] Baudon E, Chu DKW, Tung DD, Thi Nga P, Vu Mai Phuong H, Le Khanh Hang N, et al. Swine influenza viruses in Northern Vietnam in
2013
2014. Emerg Microbes Infect 2018;7(1):123.
[70] Nelson MI, Souza CK, Trovão NS, Diaz A, Mena I, Rovira A, et al. Human-origin influenza A(H3N2) reassortant viruses in swine, Southeast
Mexico. Emerg Infect Dis 2019;25(4):691
700.
[71] Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, Garten RJ, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in
humans. N Engl J Med 2009;360(25):2605
15.
[72] McNeil J, Donald G. In new theory, swine flu started in Asia, not Mexico. The New York Times 2009;6:23.
[73] Trifonov V, Khiabanian H, Rabadan R. Geographic dependence, surveillance, and origins of the 2009 influenza A (H1N1) virus. N Engl J Med
2009;361(2):115
19.
[74] Fraser C, Donnelly CA, Cauchemez S, et al. Pandemic potential of a strain of influenza A (H1N1): early findings. Science 2009;324
(5934):1557
61.
[75] Chowell G, Bertozzi SM, Colchero MA, Lopez-Gatell H, Alpuche-Aranda C, Hernandez M, et al. Severe respiratory disease concurrent with
the circulation of H1N1 influenza. N Engl J Med 2009;361(7):674
9.
[76] Siston AM, Rasmussen SA, Honein MA, Fry AM, Seib K, et al. Pandemic 2009 influenza A(H1N1) virus illness among pregnant women in the
United States. JAMA 2010;303:1517
25.
[77] Nguyen-Van-Tam JS, Openshaw PJ, Hashim A, Gadd EM, Lim WS, Semple MG, et al. Risk factors for hospitalisation and poor outcome with
pandemic A/H1N1 influenza: United Kingdom first wave (May-September 2009). Thorax 2010;65(7):645
51.
[78] Trovato M, Sartorius R, D’Apice L, Manco R, De Berardinis P. Viral emerging diseases: challenges in developing vaccination strategies. Front
Immunol 2020;11:2130.
[79] Viboud C, Simonsen L. Global mortality of 2009 pandemic influenza A H1N1. Lancet Infect Dis 2012;12:651
3.
[80] Wong JY, Kelly H, Cheung C-MM, Shiu EY, Wu P, et al. Hospitalization fatality risk of influenza A(H1N1) pdm09: a systematic review and
meta-analysis. Am J Epidemiol 2015;182:294
301.
[81] Fraser C, Donnelly C, Cauchemez S, Hanage WP, Van Kerkhove MD, Hollingsworth TD, et al. Pandemic potential of a strain of influenza A
(H1N1): early findings. Science 2009;324:1557
61.
[82] Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 2009;459(7249):931
9.
[83] Chien YW, Klugman KP, Morens DM. Bacterial pathogens and death during the 1918 influenza pandemic. N Engl J Med 2009;361
(26):2582
3.
[84] Ahn S, Kim WY, Kim SH, Hong S, Lim CM, Koh Y, et al. Role of procalcitonin and C-reactive protein in differentiation of mixed bacterial
infection from 2009 H1N1 viral pneumonia. Influenza Other Respir Viruses 2011;5(6):398
403.
[85] Rockman S, Laurie K, Barr I. Pandemic influenza vaccines: what did we learn from the 2009 pandemic and are we better prepared now?
Vaccines (Basel) 2020;8(2):211.
[86] Viboud C, Miller M, Olson D, Osterholm M, Simonsen L. Preliminary estimates of mortality and years of life lost associated with the 2009
A/H1N1 pandemic in the United States and comparison with past influenza seasons. PLoS Curr 2010;2:RRN1153.
[87] Rogers GN, Paulson JC. Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hem-
agglutinin based on species of origin. Virology 1983;127(2):361
73.
82 Pandemic Outbreaks in the 21st Century

[88] Bischoff WE, Reid T, Russell GB, Peters TR. Transocular entry of seasonal influenza-attenuated virus aerosols and the efficacy of n95 respira-
tors, surgical masks, and eye protection in humans. J Infect Dis 2011;204:193
9.
[89] Blachere FM, Lindsley WG, Pearce TA, Anderson SE, Fisher M, Khakoo R, et al. Measurement of airborne influenza virus in a hospital emer-
gency department. Clin Infect Dis 2009;48(4):438
40.
[90] Marr LC, Tang JW, Van Mullekom J, Lakdawala SS. Mechanistic insights into the effect of humidity on airborne influenza virus survival,
transmission and incidence. J R Soc Interface 2019;16(150):20180298. Available from: https://doi.org/10.1098/rsif.2018.0298.
[91] Božič A, Kanduč M. Relative humidity in droplet and airborne transmission of disease. J Biol Phys 2021;1
29.
[92] Akin L, Gözel MG. Understanding dynamics of pandemics. Turk J Med Sci 2020;50(SI-1):515
19. Available from: https://doi.org/10.3906/
sag-2004-133.
[93] Storms AD, Van Kerkhove MD, Azziz-Baumgartner E, Lee WK, Widdowson MA, Ferguson NM, et al. Worldwide transmission and seasonal
variation of pandemic influenza A(H1N1)2009 virus activity during the 2009
2010 pandemic. Influenza Other Respir Viruses 2013;7
(6):1328
35.
[94] Almeida A, Codeço C, Luz PM. Seasonal dynamics of influenza in Brazil: the latitude effect. BMC Infect Dis 2018;18(1):695. Available
from: https://doi.org/10.1186/s12879-018-3484-z.
[95] Lowen AC, Mubareka S, Steel J, Palese P. Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog
2007;3(10):1470
6.
[96] Calore EE, Uip DE, Perez NM. Pathology of the swine-origin influenza A (H1N1) flu. Pathol Res Pract 2011;207(2):86
90.
[97] Cox NJ, Subbarao K. Influenza. Lancet 1999;354(9186):1277
82.
[98] Boëlle PY, Ansart S, Cori A, Valleron AJ. Transmission parameters of the A/H1N1 (2009) influenza virus pandemic: a review. Influenza
Other Respir Viruses 2011;5(5):306
16.
[99] Cauchemez S, Ferguson NM, Wachtel C, Tegnell A, Saour G, Duncan B, et al. Closure of schools during an influenza pandemic. Lancet
Infect Dis 2009;9(8):473
81.
[100] Coburn BJ, Wagner BG, Blower S. Modeling influenza epidemics and pandemics: insights into the future of swine flu (H1N1). BMC
Medicine 2009;7:30.
[101] Casado I, Martı́nez-Baz I, Burgui R, Irisarri F, Arriazu M, Elı́a F, et al. Household transmission of influenza A(H1N1)pdm09 in the pandemic
and post-pandemic seasons. PLoS One 2014;9(9):e108485. Available from: https://doi.org/10.1371/journal.pone.0108485.
[102] Lim BH, Mahmood TA. Influenza A H1N1 2009 (Swine Flu) and pregnancy. J Obstet Gynaecol India 2011;61:386e93.
[103] Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans.
N Engl J Med 2009;361:1
10.
[104] Munster VJ, de Wit E, van den Brand JM, Herfst S, Schrauwen EJ, Bestebroer TM, et al. Pathogenesis and transmission of swine-origin 2009
A (H1N1) influenza virus in ferrets. Science 2009;325(5939):481
3.
[105] Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, et al. Transmission and pathogenesis of swine-origin 2009 A (H1N1)
influenza viruses in ferrets and mice. Science 2009;325:484
7.
[106] Sabharwal AD, Sandhu BS, Singh B. Investigations of effect of target thickness and detector collimation on 662 keV multiply backscattered
gamma photons. Radiat Meas 2009;44:411
14.
[107] Cheng PKC, Leung TWC, Ho ECM, Leung PKC, Ng AYY, Lai MYY, et al. Oseltamivir- and amantadine-resistant influenza viruses A
(H1N1). Emerg Infect Dis 2009;15:966
8. Available from: https://doi.org/10.3201/eid1506.081357.
[108] Lee CW, Koh CW, Chan YS, Aw PP, Loh KH, Han BL, et al. Largescale evolutionary surveillance of the 2009 H1N1 influenza A virus using
resequencing arrays. Nucleic Acids Res 2010;38(9):e111.
[109] Tandon VR, Mahajan A, Sharma S, Gupta SK, Kumar D, Sharma Y. Swine flu (H1N1)—pandemic or bioterrorism. JK Sci 2009;11(4):
161
2.
[110] Jain S, Kamimoto L, Bramley AM, Schmitz AM, Benoit SR, Louie J, et al. Hospitalized patients with 2009 H1N1 influenza in the United
States, April-June 2009. N Engl J Med 2009;361(20):1935
44.
[111] Domı́nguez-Cherit G, Lapinsky SE, Macias AE, Pinto R, Espinosa-Perez L, de la Torre A, et al. Critically ill patients with 2009 influenza A
(H1N1) in Mexico. JAMA 2009;302(17):1880
7.
[112] Saha A, Jha N, Dubey NK, Gupta VK, Kalaivani M. Swine-origin influenza A (H1N1) in Indian children. Ann Trop Paediatr 2010;30:51e5.
[113] Kumar S, Henrickson KJ. Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic. Clin Microbiol Rev
2012;25:344e61.
[114] Shi J, Xie J, He Z, et al. A detailed epidemiological and clinical description of 6 human cases of avian-origin influenza A (H7N9) virus infec-
tion in Shanghai. PLoS One 2013;8:e77651.
[115] Ravina R, Dalal A, Mohan H, Prasad M, Pundir CS. Detection methods for influenza A H1N1 virus with special reference to biosensors: a
review. Biosci Rep 2020;40(2) BSR20193852.
[116] Roa PL, Catalán P, Giannella M, de Viedma DG, Sandonis V, Bouza E. Comparison of real-time RT-PCR, shell vial culture, and conventional
cell culture for the detection of the pandemic influenza A (H1N1) in hospitalized patients. Diagn Microbiol Infect Dis 2011;69:428
31.
[117] Higgins AD, Shaw CJ, Johnson JG, Navarro A, Chapman NA, Ewers SD, et al. Monoclonal antibody kit for identification of the novel 2009
H1N1 influenza A virus. J Clin Microbiol 2010;48:2677
82.
[118] Ginocchio CC, Zhang F, Manji R, Arora S, Bornfreund M, Falk L, et al. Evaluation of multiple test methods for the detection of the novel
2009 influenza A (H1N1) during the New York City outbreak. J Clin Virol 2009;45:191.
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 83

[119] Hurt AC, Baas C, Deng YM, Roberts S, Kelso A, Barr IG. Performance of influenza rapid point-of-care tests in the detection of swine lineage
A(H1N1) influenza viruses. Influenza Other Respir Viruses 2009;3:171
6.
[120] Miller E, Hoschler K, Hardelid P, Stanford E, Andrews N, Zambon M. Incidence of 2009 pandemic influenza A H1N1 infection in England: a
cross-sectional serological study. Lancet North Am (Ed.) 2010;375:1100
8.
[121] Mahony JB. Detection of respiratory viruses by molecular methods. Clin Microbiol Rev 2008;21:716
47. Available from: https://doi.org/
10.1128/CMR.00037-07.
[122] Tulloch WJ. Observations on the virus of influenza, with a view to elaborating a simple diagnostic test whereby its presence in the respiratory
tract of man may be revealed—part I. Edinburgh Med J 1939;46:117
43.
[123] Schild GC, Wood JM, Newman RW. A single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: proposals
for an assay method for the haemagglutinin content of influenza vaccines. Bull World Health Organ 1975;52:223
31.
[124] Choi Y, Lee S, Kwon SY, Lee Y, Park YK, Ban SJ. Analysis of the proficiency of single radial immunodiffusion assays for quality control of
influenza vaccines in Korea. Biologicals 2017;50:137
40. Available from: https://doi.org/10.1016/j.biologicals.2017.08.001.
[125] Pedersen JC. Hemagglutination-inhibition assay for influenza virus subtype identification and the detection and quantitation of serum antibo-
dies to influenza virus. Animal influenza virus. New York, NY: Humana Press; 2014. p. 11
25.
[126] Reber A, Katz J. Immunological assessment of influenza vaccines and immune correlates of protection. Expert Rev Vaccines
2013;12:519
36.
[127] Zacour M, Ward BJ, Brewer A, Tang P, Boivin G, Li Y, et al. Standardization of hemagglutination inhibition assay for influenza serology
allows for high reproducibility between laboratories. Clin Vaccine Immunol 2016;23:236
42.
[128] Rodda SJ, Gallichio HA, Hampson AW. The single radial immunodiffusion assay highlights small antigenic differences among influenza virus
hemagglutinins. J Clin Microbiol 1981;14:479
82.
[129] Lee BW, Bey RF, Baarsch MJ, Simonson RR. ELISA method for detection of influenza A infection in swine. J Vet Diagn Invest
1993;5:510
15.
[130] Rizzo F, Lovecchio C, Ingravalle F, Ceccarelli L, Sona B, Mandola ML. Swine influenza A serology: ELISA versus HI test. Int J Infect Dis
2016;53:105.
[131] Ahmed SR, Kim J, Suzuki T, Neethirajan S, Lee J, Park EY. In situ self-assembly of gold nanoparticles on hydrophilic and hydrophobic sub-
strates for influenza virus-sensing platform. Sci Rep 2017;7:44495. Available from: https://doi.org/10.1038/srep44495.
[132] Poon LL, Chan KH, Smith GJ, et al. Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-
time quantitative RT-PCR assays. Clin Chem 2009;55:1555e8.
[133] Harmon K, Bower L, Kim WI, Pentella M, Yoon KJ. A matrix gene
based multiplex real-time RT-PCR for detection and differentiation of
2009 pandemic H1N1 and other influenza A viruses in North America. Influenza Other Respir Viruses 2010;4:405
10.
[134] Baas C, Barr IG, Fouchier RA, Kelso A, Hurt AC. A comparison of rapid point-of-care tests for the detection of avian influenza A (H7N9)
virus, 2013. Eurosurveillance 2013;18:20487.
[135] Lee GC, Jeon ES, Kim WS, Le DT, Yoo JH, Chong CK. Evaluation of a rapid diagnostic test, NanoSigns Influenza A/B Antigen, for detec-
tion of the 2009 pandemic influenza A/H1N1 viruses. Virol J 2010;7:244.
[136] Lucas PM, Morgan OW, Gibbons TF, Guerrero AC, Maupin GM, Butler JL, et al. Diagnosis of 2009 pandemic influenza A (pH1N1) and sea-
sonal influenza using rapid influenza antigen tests, San Antonio, Texas, April
June 2009. Clin Infect Dis 2011;52:S116
22.
[137] Al Johani SM, Al Balwi M, Al Alwan B, Al Hefdhi R, Hajeer A. Validity of two rapid point of care influenza tests and direct fluorescence
assay in comparison of real time PCR for swine of origin influenza virus. J Infect Public Health 2011;4:7
11. Available from: https://doi.org/
10.1016/j.jiph.2010.10.004.
[138] Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic
Acids Res 2000;28:e63. Available from: https://doi.org/10.1093/nar/28.12.e63.
[139] Sharma V, Chaudhry D, Kaushik S. Evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay
for detection and subtyping of Influenza A viruses. J Virol Methods 2018;253:18
25. Available from: https://doi.org/10.1016/j.
jviromet.2017.12.005.
[140] Lam WY, Leung TF, Lee N, et al. Development and comparison of molecular assays for the rapid detection of the pandemic influenza A
(H1N1) 2009 virus. J Med Virol 2010;82:675e83.
[141] Whiley DM, Bialasiewicz S, Bletchly C, et al. Detection of novel influenza A(H1N1) virus by real-time RT-PCR. J Clin Virol 2009;45:203e4.
[142] Mehrotra P. Biosensors and their applications—a review. J Oral Biol Craniofacial Res 2016;6:153
9.
[143] Lee KG, Lee TJ, Jeong SW, Choi HW, Heo NS, Park JY, et al. Development of a plastic-based microfluidic immunosensor chip for detection
of H1N1 influenza. Sensors 2012;12:10810
19.
[144] Su LC, Chang CM, Tseng YL, Chang YF, Li YC, Chang YS, et al. Rapid and highly sensitive method for influenza A (H1N1) virus detection.
Anal Chem 2012;84:3914
20.
[145] Shi L, Sun Q, He JA, Xu H, Liu C, Zhao C, et al. Development of SPR biosensor for simultaneous detection of multiplex respiratory viruses.
Bio-med Mater Eng 2015;26(s1):S2207
16.
[146] Nidzworski D, Pranszke P, Grudniewska M, Król E, Gromadzka B. Universal biosensor for detection of influenza virus. Biosens Bioelectron
2014;59:239
42.
[147] Bonanni A, Pividori MI, Del Valle M. Impedimetric detection of influenza A (H1N1) DNA sequence using carbon nanotubes platform and
gold nanoparticles amplification. Analyst 2010;135:1765
72.
84 Pandemic Outbreaks in the 21st Century

[148] Singh R, Hong S, Jang J. Label-free detection of influenza viruses using a reduced graphene oxide-based electrochemical immunosensor inte-
grated with a microfluidic platform. Sci Rep 2017;7:42771. Available from: https://doi.org/10.1038/srep42771.
[149] Nidzworski D, Siuzdak K, Niedziałkowski P, Bogdanowicz R, Sobaszek M, Ryl J, et al. A rapid-response ultrasensitive biosensor for influenza
virus detection using antibody modified boron-doped diamond. Sci Rep 2017;7:15707.
[150] Lee D, Chander Y, Goyal SM, Cui T. Carbon nanotube electric immunoassay for the detection of swine influenza virus H1N1. Biosens
Bioelectron 2011;26:3482
7.
[151] Bai C, Lu Z, Jiang H, Yang Z, Liu X, Ding H, et al. Aptamer selection and application in multivalent binding-based electrical impedance
detection of inactivated H1N1 virus. Biosens Bioelectron 2018;110:162
7. Available from: https://doi.org/10.1016/j.bios.2018.03.047.
[152] Mikuła E, Silva CE, Kopera E, Zdanowski K, Radecki J, Radecka H. Highly sensitive electrochemical biosensor based on redox-active mono-
layer for detection of anti-hemagglutinin antibodies against swine-origin influenza virus H1N1 in sera of vaccinated mice. BMC Vet Res
2018;14:328.
[153] Metaferia B, Wei JS, Song YK, Evangelista J, Aschenbach K, Johansson P, et al. Development of peptide nucleic acid probes for detection of
the HER2 oncogene. PLoS One 2013;8:e58870.
[154] Hyo EL, Yun OK, Seong HC. Electrochemical- DNA biosensor development based on a modified carbon electrode with gold nanoparticles for
influenza A (H1N1) detection: effect of spacer. Int J Electrochem Sci 2014;9:6793
808.
[155] Hai W, Goda T, Takeuchi H, Yamaoka S, Horiguchi Y, Matsumoto A, et al. Specific recognition of human influenza virus with PEDOT bear-
ing sialic acid-terminated trisaccharides. ACS Appl Mater Interfaces 2017;9:14162
70.
[156] Pohanka M. Overview of piezoelectric biosensors, immunosensors and DNA sensors and their applications. Materials 2018;11:448. Available
from: https://doi.org/10.3390/ma11030448.
[157] Hewa TM, Tannock GA, Mainwaring DE, Harrison S, Fecondo JV. The detection of influenza A and B viruses in clinical specimens using a
quartz crystal microbalance. J Virol Methods 2009;162:14
21.
[158] Erofeev AS, Gorelkin PV, Kolesov DV, Kiselev GA, Dubrovin EV, Yaminsky IV. Label-free sensitive detection of influenza virus using PZT
discs with a synthetic sialylglycopolymer receptor layer. R Soc Open Sci 2019;6(9):190255.
[159] Hong HB, Krause HJ, Song KB, Choi CJ, Chung MA, Son SW, et al. Detection of two different influenza A viruses using a nitrocellulose
membrane and a magnetic biosensor. J Immunol Methods 2011;365:95
100.
[160] Jeong HJ, Dong J, Ueda H. Single-step detection of the influenza virus hemagglutinin using bacterially-produced quenchbodies. Sensors
2019;19:52. Available from: https://doi.org/10.3390/s19010052.
[161] Shu B, Wu KH, Emery S, Villanueva J, Johnson R, Guthrie E, et al. Design and performance of the CDC real-time reverse transcriptase PCR
swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus. J Clin Microbiol 2011;49:2614
19.
[162] Kwon D, Shin K, Kwon M, Oh HB, Kang C, Lee JY. Development and evaluation of a rapid influenza diagnostic test for the pandemic
(H1N1) 2009 influenza virus. J Clin Microbiol 2011;49:437
8.
[163] Kuo CY, Huang YC, Huang CG, Tsao KC, Lin TY. Symptomatic predictors for 2009 influenza A virus (H1N1) infection with an emphasis
for patients with a negative rapid diagnostic test. PLoS One 2011;6:e28102.
[164] Veguilla V, Hancock K, Schiffer J, Gargiullo P, Lu X, Aranio D, et al. Sensitivity and specificity of serologic assays for detection of human
infection with 2009 pandemic H1N1 virus in United States populations. J Clin Microbiol 2011;49:2210
15.
[165] Hideshima S, Hinou H, Ebihara D, Sato R, Kuroiwa S, Nakanishi T, et al. Attomolar detection of influenza A virus hemagglutinin human H1
and avian H5 using glycan-blotted field effect transistor biosensor. Anal Chem 2013;85:5641
4.
[166] Molinari NA, OrtegaSanchez IR, Messonnier ML, Thompson WW, Wortley PM, Weintraub E, et al. The annual impact of seasonal influenza
in the United States: measuring disease burden and costs. Vaccine 2007;25:5086
96.
[167] Morens DM, Taubenberger JK, Fauci AS. The persistent legacy of the 1918 influenza virus. N Engl J Med 2009;361(3):225
9.
[168] Rota PA, Wallis TR, Harmon MW, Rota JS, Kendal AP, Nerome K. Cocirculation of two distinct evolutionary lineages of influenza type B
virus since 1983. Virology 1990;175(1):59
68.
[169] Houser K, Subbarao K. Influenza vaccines: challenges and solutions. Cell Host Microbe 2015;17(3):295
300.
[170] Legrand N, Weijer K, Spits H. Experimental models to study development and function of the human immune system in vivo. J Immunol
2006;176:2053
8.
[171] Skowronski DM, Masaro C, Kwindt TL, Mak A, Petric M, Li Y, et al. Estimating vaccine effectiveness against laboratory-confirmed influenza
using a sentinel physician network: results from the 2005
2006 season of dual A and B vaccine mismatch in Canada. Vaccine
2007;25:2842
51.
[172] Capua I, Alexander DJ. The challenge of avian influenza to the veterinary community. Avian Pathol 2006;35(3):189
205.
[173] Holmes EC, Ghedin E, Miller N, Taylor J, Bao Y, St George K, et al. Whole-genome analysis of human influenza A virus reveals multiple
persistent lineages and reassortment among recent H3N2 viruses. PLoS Biol 2005;3(9):e300.
[174] Taubenberger JK, Baltimore D, Doherty PC, Markel H, Morens DM, Webster RG, et al. Reconstruction of the 1918 influenza virus: unex-
pected rewards from the past. MBio 2012;3(5).
[175] Littauer EQ, Esser ES, Antao OQ, Vassilieva EV, Compans RW, Skountzou I. H1N1 influenza virus infection results in adverse pregnancy
outcomes by disrupting tissue-specific hormonal regulation. PLoS Pathog 2017;13(11):e1006757.
[176] Jester B, Uyeki T, Jernigan D. Readiness for responding to a severe pandemic 100 years after 1918. Am J Epidemiol 2018;187(12):2596
602.
[177] Epstein SL. Universal influenza vaccines: progress in achieving broad cross-protection in vivo. Am J Epidemiol 2018;187(12):2603
14.
[178] Glasgow JF, Middleton B. Reye syndrome—insights on causation and prognosis. Arch Dis Child 2001;85(5):351
3.
 Swine-origin influenza A (H1N1) virus: current status, threats, and challenges Chapter | 5 85

[179] Kumar DS, Banji D, Rajashekar K, Rahul CH, Kranthi A, Verma MA, et al. Drug therapy for swine flu—an overview. Int J Pharm Bio Sci
2010;1(1):1
9.
[180] Hurt AC, Ho HT, Barr I. Resistance to anti-influenza drugs: adamantanes and neuraminidase inhibitors. Expert Rev Anti Infect Ther 2006;4
(5):795
805.
[181] Lew W, Chen X, Kim CU. Discovery and development of GS 4104 (oseltamivir): an orally active influenza neuraminidase inhibitor. Curr
Med Chem 2000;7(6):663
72.
[182] Shaik AB, Prabhu M, Shenoy S, Thomson SR. Oseltamivir-induced neuropsychiatric symptoms. J Pharmacol Pharmacotherap 2018;9(1):43.
[183] Clifford KM, Feighner L, McCarrell JL, Hong P. Oseltamivir-induced neuropsychiatric event in an older person: a case report. Sr Care Pharm
2020;35(9):394
7. Available from: https://doi.org/10.4140/TCP.n.2020.394.
[184] Chen R, Fang Z, Huang Y. Neuropsychiatric events in an adult patient with influenza a (H3N2) treated with oseltamivir (Tamiflu): a case
report. BMC Infect Dis 2019;19(1):224. Available from: https://doi.org/10.1186/s12879-019-3827-4.
[185] Hoffman KB, Demakas A, Erdman CB, Dimbil M, Murali Doraiswamy P. Neuropsychiatric adverse effects of oseltamivir in the FDA
Adverse Event Reporting System, 1999
2012. BMJ 2013;347.
[186] Hayden FG. Perspectives on antiviral use during pandemic influenza. Philos Trans R Soc Lond B Biol Sci 2001;356(1416):1877
84.
Available from: https://doi.org/10.1098/rstb.2001.1007.
[187] Cyranoski D. Avian flu special: masking our ignorance. Nature 2005;435(7041):408.
[188] Orozovic G, Orozovic K, Lennerstrand J, Olsen B. Detection of resistance mutations to antivirals oseltamivir and zanamivir in avian influenza
A viruses isolated from wild birds. PLoS One 2011;6:e16028.
[189] Moscona A. Global transmission of oseltamivir-resistant influenza. N Engl J Med 2009;360:953
6.
[190] Collins PJ, Haire LF, Lin YP, Liu J, Russell RJ, Walker PA, et al. Crystal structures of oseltamivir-resistant influenza virus neuraminidase
mutants. Nature 2008;453:1258
61.
[191] Heneghan CJ, Onakpoya I, Thompson M, Spencer EA, Jones M, Jefferson T. Zanamivir for influenza in adults and children: systematic review
of clinical study reports and summary of regulatory comments. BMJ 2014;348:g2547.
[192] Pinto LH, Lamb RA. The M2 proton channels of influenza A and B viruses. J Biol Chem 2006;281(14):8997
9000.
[193] Stephenson I, Nicholson KG. Chemotherapeutic control of influenza. J Antimicrob Chemother 1999;44(1):6
10.
[194] Maugh TH. Amantadine: an alternative for prevention of influenza. Science 1976;192(4235):130
1.
[195] Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane resistance among influenza A viruses isolated early during the 2005
2006
influenza season in the United States. JAMA 2006;295(8):891
4.
[196] Jing X, Ma C, Ohigashi Y, Oliveira FA, Jardetzky TS, Pinto LH, et al. Functional studies indicate amantadine binds to the pore of the influ-
enza Avirus M2 proton-selective ion channel. Proc Natl Acad Sci USA 2008;105(31):10967
72.
[197] Wang C, Takeuchi K, Pinto LH, Lamb RA. Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. J
Virol 1993;67(9):5585
94.
[198] Govorkova EA, Fang HB, Tan M, Webster RG. Neuraminidase inhibitor-rimantadine combinations exert additive and synergistic anti-
influenza virus effects in MDCK cells. Antimicrob Agents Chemother 2004;48(12):4855
63.
[199] Beigel J, Bray M. Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res 2008;78(1):91
102.
[200] Gibson MI, Gibson F, Doy CH, Morgan P. The branchpoint in the synthesis of the aromatic amino acids. Nature 1962;195:1173
5.
[201] Tiedtke J. Information requirements for botanical cosmetic ingredients. Cosmetic Science Technology; 2006. pp. 15
21.
[202] Evans IA, Osman MA. Carcinogenicity of bracken and shikimic acid. Nature 1974;250:348
9.
[203] Krämer M, Bongaerts J, Bovenberg R, Kremer S, Müller U, Orf S, et al. Metabolic engineering for microbial production of shikimic acid.
Metab Eng 2003;5:277
83.
[204] Rohloff JC, Kent KM, Postich MJ, Becker MW, Chapman HH, Kelly DE, et al. Practical total synthesis of the anti-influenza drug GS-4104. J
Org Chem 1998;63(13):4545
50.
[205] Bradley D. Star role for bacteria in controlling flu pandemic? Nat Rev Drug Discov 2005;4(12):945
6.
[206] Johansson L, Lindskog A, Silfversparre G, Cimander C, Nielsen KF, Lidén G. Shikimic acid production by a modified strain of E. coli
(W3110.shik1) under phosphate-limited and carbon-limited conditions. Biotechnol Bioeng 2005;92:541
52.
[207] Knop M, Miller KJ, Mazza M, Feng D, Weber M, Keränen S, et al. Molecular interactions position Mso1p, a novel PTB domain homologue,
in the interface of the exocyst complex and the exocytic SNARE machinery in yeast. Mol Biol Cell 2005;16:4543
56.
[208] Draths KM, Knop DR, Frost JW. Shikimic acid and quinic acid: replacing isolation from plant sources with recombinant microbial biocataly-
sis. J Am Chem Soc 1999;121:1603
4.
[209] Iomantas YAV, Abalakina EG, Polanuer BM, Yampolskaya TA, Bachina TA, Kozlov YI. Method for producing shikimic acid. United States
Patent No. 6436664; 2002.
[210] Hurt AC, Su YC, Aban M, Peck H, Lau H, Baas C, et al. Evidence for the introduction, reassortment, and persistence of diverse influenza A
viruses in Antarctica. J Virol 2016;90:9674
82. Available from: https://doi.org/10.1128/JVI.01404-16.
[211] Bedford T, Suchard MA, Lemey P, Dudas G, Gregory V, Hay AJ, et al. Integrating influenza antigenic dynamics with molecular evolution.
Elife 2014;3 e01914.
[212] Russell RJ, Kerry PS, Stevens DJ, Steinhauer DA, Martin SR, Gamblin SJ, et al. Structure of influenza hemagglutinin in complex with an
inhibitor of membrane fusion. Proc Natl Acad Sci U S A 2008;105(46):17736
41.
[213] Yoon SW, Webby RJ, Webster RG. Evolution and ecology of influenza A viruses. Curr Top Microbiol Immunol 2014;385:359
75.
86 Pandemic Outbreaks in the 21st Century

[214] Maruyama J, Nao N, Miyamoto H, Maeda K, Ogawa H, Yoshida R, et al. Characterization of the glycoproteins of bat-derived influenza
viruses. Virology 2016;488:43
50.
[215] Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, et al. New world bats harbor diverse influenza A viruses. PLoS Pathog 2013;9(10):
e1003657.
[216] Palese P, Wang TT. H5N1 influenza viruses: facts, not fear. Proc Natl Acad Sci USA 2012;109:2211
13.
[217] Baldo V, Bertoncello C, Cocchio S, Fonzo M, Pillon P, Buja A, et al. The new pandemic influenza A/(H1N1) pdm09 virus: is it really “new”?
Prev Med Hyg 2016;57(1):E19
22.
[218] Tumpey TM, Garcı́a-Sastre A, Taubenberger JK, Palese P, Swayne DE, Pantin-Jackwood MJ, et al. Pathogenicity of influenza viruses with
genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in
mice. J Virol 2005;79(23):14933
44.
[219] Chen JR, Liu YM, Tseng YC, Ma C. Better influenza vaccines: an industry perspective. J Biomed Sci 2020;27(1):33.
[220] Lewis NS, Russell CA, Langat P, Anderson TK, Berger K, Bielejec F, et al. The global antigenic diversity of swine influenza A viruses. Elife
2016;5:e12217.
[221] Anderson TK, Chang J, Arendsee ZW, Venkatesh D, Souza CK, Kimble JB, et al. Swine influenza a viruses and the tangled relationship with
humans. Cold Spring Harb Perspect Med 2020;11(3):a038737.
[222] Ma W, Vincent AL, Gramer MR, Brockwell CB, Lager KM, Janke BH, et al. Identification of H2N3 influenza A viruses from swine in the
United States. Proc Natl Acad Sci U S A 2007;104(52):20949
54.
[223] Bailey ES, Choi JY, Fieldhouse JK, Borkenhagen LK, Zemke J, Zhang D, et al. The continual threat of influenza virus infections at the
human
animal interfaceWhat is new from a one health perspective? Evol Med Public Health 2018;2018(1):192
8.
[224] Trevennec K, Leger L, Lyazrhi F, Baudon E, Cheung CY, Roger F, et al. Transmission of pandemic influenza H1N1 (2009) in Vietnamese
swine in 2009
2010. Influenza Other Respir Viruses 2012;6(5):348
57.
[225] Song MS, Lee JH, Pascua PN, Baek YH, Kwon HI, Park KJ, et al. Evidence of human-to-swine transmission of the pandemic (H1N1) 2009
influenza virus in South Korea. J Clin Microbiol 2010;48(9):3204
11.
[226] Nelson MI, Gramer MR, Vincent AL, Holmes EC. Global transmission of influenza viruses from humans to swine. J Gen Virol 2012;93(Pt
10):2195
203.
[227] Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, et al. Origins and evolutionary genomics of the 2009 swine-origin
H1N1 influenza A epidemic. Nature 2009;459:1122
5.
[228] Ramirez A, Capuano AW, Wellman DA, Lesher KA, Setterquist SF, Gray GC. Preventing zoonotic influenza virus infection. Emerg Infect
Dis 2006;12(6):996
1000.
[229] Bui VN, Nguyen TT, Nguyen-Viet H, Bui AN, McCallion KA, Lee HS, et al. Bioaerosol sampling to detect avian influenza virus in Hanoi’s
largest live poultry market. Clin Infect Dis 2018;68(6):972
5.
[230] Saunders-Hastings PR, Krewski D. Reviewing the history of pandemic influenza: understanding patterns of emergence and transmission.
Pathogens 2016;5(4):66.
[231] Dahal S, Mizumoto K, Bolin B, Viboud C, Chowell G. Natality decline and spatial variation in excess death rates during the 1918
1920 influ-
enza pandemic in Arizona, United States. Am J Epidemiol 2018;187(12):2577
84.
Chapter 6

Molecular mechanisms of Zika fever in

inducing birth defects: an update
Hema Masarapu1 and Naga Charan Konakalla2
1
Department of Virology, Sri Venkateswara University, Tirupati, India, 2Department of Plant Protection Biology, Swedish University of Agricultural
Sciences, Alnarp, Sweden

6.1 Introduction
Zika virus (ZIKV) is an emergent mosquito-borne member of the genus Flavivirus of the family Flaviviridae and it
poses a significant global threat to public health due to its link to the serious complications such as neuropathies in the
pregnant women and teratogenic damage to the developing fetus. ZIKV was first isolated in 1947 from a rhesus monkey
in the Zika forest of Kampala, Uganda, Africa [1] and later it was reported in humans in Uganda and Tanzania in 1952,
while the virus was isolated for the first time from Nigeria in 1954 [2]. It had remained sporadic with fewer than 20
human infections for decades until the major outbreaks in Pacific islands and French Polynesia in 2007 and 2013,
respectively [3
5]. Then the virus was introduced into Brazil, subsequently spreading rapidly throughout Brazil and the
Americas, caused a large epidemic in 2015 [6
8]. Currently, there are 86 countries, territories, or subnational areas
with evidence of ZIKV transmission (http://www.who.int/emergencies/zika-virus/classificationtables/en/; http://www.
cdc.gov/zika/geo/index.html; http://www.who.int/mediacentre/factsheets/zika/en/). As ZIKV advances across the globe,
more imported cases of human infection are being reported worldwide in places with no previous record [8]. World
Health Organization (WHO) declared ZIKV as major international public health concern in 2016 with the growing evi-
dences that the ZIKV is causally associated with Guillain
Barre syndrome (GBS) in the adults and neonatal complica-
tions such as microcephaly and other severe neurological disorders [8
10]. To date, there are no approved vaccines or
specific antiviral treatments available for ZIKV control (covered in [11,12]).
ZIKV is closely linked to dengue virus (Flavivirus) both genetically and serologically, with approximately 43%
amino acid sequence homology [13]. As per the literature, there is one ZIKV serotype, two sublineages (African and
Asian), and three genotypes (West African, East African, and Asian). During the last decade, the clinical manifestations,
mode of transmission of the virus have changed due to the increased infectivity of ZIKV, resulting in larger outbreaks
with more severe symptoms and increased sexual transmission cases [6,14,15]. It has been postulated that genetic
changes of the ZIKV may have contributed to its increased transmissibility and infectivity, enhanced neurotropic poten-
tial, and greater replicative capacity with its ability to breach the placental protection and to trigger permanent fetal
brain injuries. The epidemiological associations between ZIKV and congenital neuropathologies have been confirmed
during the last few years by using in vivo animal models and in vitro cellular systems (reviewed in [8,9,16
24]).
The placenta and the fetal brain appear to be the primary targets of ZIKV, as evident due to the presence of ZIKV
particles, RNA and proteins in human placental and fetal brain remnants. Experimental studies showed potential trans-
placental passage of the ZIKV and subsequent selective targeting of neural progenitor cells (NPCs), the founder cells
that generate all neuronal and glial cells, leading to changes in the expression of genes and proteins related to the cell
cycle, stress response, cell death, intracellular structural organization, cell differentiation, innate and adaptive immune
responses, cell proliferation, cell migration, cell adhesion, and synaptic organization (reviewed in [23,25
29]).
Depletion of NPCs by ZIKV is associated with restricted brain growth in mice with suspected transplacental ZIKV pas-
sage recapitulates the features of congenital ZIKV-associated microcephaly in humans. Other neonatal and pediatric
neural disorders associated with ZIKV infection include chorioretinopathy, sensory neural hearing loss, and epilepsy
and other motor deficits [16,17,23]. Evidence indicated that ZIKV infection cripples the host cells’ innate immune

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00005-7

© 2021 Elsevier Inc. All rights reserved. 87
88 Pandemic Outbreaks in the 21st Century

responses, allowing productive replication and potential dissemination of the virus. Microcephaly can result from ZIKV
infection at any gestational stage, but the risk is greater during the first trimester. Although a number of recent studies
revealed some specific molecular and cellular roles of ZIKV proteins, the mechanisms by which it produces the sus-
pected pathophysiological effects are not completely understood (reviewed in [8,22,23]).
In this review, we focus on the ZIKV-associated neurological abnormalities, ZIKV
host cell interactions that ulti-
mately result in virus multiplication, ZIKV-mediated cell death and immune evasion mechanisms and discuss the poten-
tial molecular mechanisms in inducing microcephaly and other birth defects in newborns. This understanding is
essential and critical for the development of vaccines and therapeutic interventions that can limit ZIKV infections.

6.2 Molecular biology of ZIKV

6.2.1 Genome organization of ZIKV
ZIKV particles are spherical in shape, 50 nm in size with an icosahedral outer envelope and a dense inner core of
B30 nm in diameter [10]. ZIKV consists of positive sense, linear, monopartite single-stranded RNA [ssRNA(1)]
genome of 10,794 nucleotides (nt) (Fig. 6.1). The genomic RNA consists of single open reading frame (ORF) which is
flanked by two terminal noncoding regions (NCRs), that is, the 50 NCR (107 nt) and the 30 NCR (428 nt). The 50 end of
the genome contains a type 1 cap structure [32], to ensure efficient translation of viral genome, and also to evade host
immune response. The 30 untranslated region ends with CUOH and lacks a poly-A tail [21,30]. The ZIKV genome is
directly translated into a polyprotein of 3149 amino acids, which is processed cotranslationally and posttranslationally
by viral and host proteases (PRs) to produce three structural proteins (capsid-C, envelope-E, and precursor membrane-
prM/membrane-M) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins as shown in
Fig. 6.1. While the virus is comprised of structural proteins, the nonstructural proteins are involved in viral genome rep-
lication and infection as well as inhibiting cellular innate immune responses. Among the structural proteins, the mature
capsid protein interacts with the viral RNA to form the nucleocapsid. Membrane protein inhibits premature fusion of

5'NCR 3'NCR
5'm7G Structural Genes Nonstructural Genes 3'OH
ssRNA (10,794 nts)

Polyprotein
3149 amino acids
Polyprotein processing by host and viral proteases
pr M
NS1 NS2A NS2B NS3 NS4A 2K NS4B NS5
C prM E P H M R

Protease
Capsid Envelope Assembly Helicase
binding & Replication RdRp
RNA
fusion Methyl
triphosphatase
transferase

Membrane Replication Protease Assembly

associated Immune cofactor Replication
protein evasion

FIGURE 6.1 Schematic representation of Zika virus (ZIKV) genome organization (not to scale) and polyprotein processing. At the top is the virion
ssRNA of about 10.7 kb with the structural and nonstructural protein-coding regions and the 50 - and 30 -noncoding regions (NCRs). The single open
reading frame encodes a polyprotein precursor that is posttranslationally cleaved into three structural (capsid, C; membrane, M; envelope, E) proteins
and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). P, H, M, and R symbols indicate the localization of the NS3 pro-
tease, NS3 RNA helicase, NS5 methyltransferase, and NS5 RNA-dependent RNA polymerase (RdRp) domains, respectively. prM represents precursor
membrane protein; 2K represents signal peptide 2K. Modified from Simmonds P, Becher P, Bukh J, Gould EA, Meyersm G, Monath T, et al. ICTV
virus taxonomy profile: Flaviviridae. J Gen Virol 2017;98(1):2
3 and Lee JK, Shin OS. Advances in Zika virus hostcell interaction: current knowl-
edge and future perspectives. Int J Mol Sci 2019;20(5), 1101.
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 89

the virus with host cell membranes and it forms a complex to protect the envelope protein from degradation during
virion assembly. The envelope protein, which makes up the majority of the virion surface and the primary target for
neutralizing antibodies by the host, facilitates several crucial steps of the viral life cycle, including membrane attach-
ment, membrane fusion, release of viral genome into the host cell, and viral assembly (reviewed in [10,22,23]).
In flaviviruses, NS1, NS3, and NS5 are large, highly conserved proteins, whereas NS2A, NS2B, NS4A, and NS4B
proteins are small, hydrophobic proteins. Flaviviral NS1 is involved in viral replication and infection and interacts with
the host immune factors when secreted extracellularly for immune evasion and pathogenesis [22]. The NS2B and NS3
proteins are important for protein synthesis and replication of viral genome. NS3 consists of an N-terminal protease
domain, which is linked to a membrane-associated NS2B cofactor to form a protease complex, and NS3pro is responsi-
ble for proteolytic processing of the viral polyprotein, whereas the NS2B cofactor is required for enhancing enzymatic
activity and substrate specificity. The C-terminal domain of NS3 protein produces ZIKV helicase, which plays a critical
role in NTP-dependent RNA unwinding and translocation during viral replication [10,18,23]. ZIKV NS5 is a multitask-
ing protein, contains a C-terminal RNA-dependent RNA polymerase (RdRp) domain, which is essential for viral repli-
cation, and an N-terminal methyltransferase (MTase) domain, which is involved in RNA capping, translation, and
evasion of host immune response (reviewed in [10,18,22,23]). The 30 NCR forms a loop structure that may play a role
in cyclization, translation, RNA packaging, recognition by cellular factors, and genome stabilization. The 50 NCR
allows translation via a methylated nucleotide cap or a genome-linked protein. In addition, ZIKV produces abundant
noncoding subgenomic flavivirus RNAs (sfRNAs) from the 30 UTR in infected cells, which may play a role in the viral
life cycle, subversion of innate immunity, and RNA interference [8,21]. The crystal structure of ZIKV envelope is
similar to other flaviviruses and possesses three distinct domains: a central β-barrel-shaped domain I, an elongated
finger-like domain II, and a C-terminal immunoglobulin-like domain III [33,34]. ZIKV genome organization, proteo-
lytic processing, and the translation products were shown in Fig. 6.1.

6.2.2 Replication of ZIKV

The life cycle of ZIKV follows four basic steps: RNA translation into viral proteins, replication of viral RNA, assembly
of viral particles in the endoplasmic reticulum (ER), and virion release from the host cell. The virion seeks the entry
into the host cell by binding to one or more cellular receptors through surface envelope protein spikes followed by
clathrin-mediated endocytosis. After internalization, virus particles fuse with the endosomal membrane in a pH-
dependent manner and the envelope is removed. This will lead to disruption of nucleocapsid and release of genomic
RNA into the cytoplasm of the host cell (reviewed by [35]). The replication of the virus takes place in intracellular
membrane-associated compartments located on the surface of the ER. The positive-sense ZIKV genomic ssRNA is
translated into a single polyprotein which is subsequently processed to generate viral structural and nonstructural pro-
teins. Then the positive-sense RNA genome of ZIKV is replicated via negative-strand RNA intermediate by the utiliza-
tion of ZIKV RdRP [36]. The viral C protein helps in packaging new RNA genomes into progeny virions which are
secreted from the ER lumen as immature virus particles. After acquiring posttranslational processing of E protein and
prM protein in the trans-Golgi network, matured infectious virus particles are released from the host cell by exocytosis
[12,37,38]. Intriguingly, ZIKV has evolved to contain a nucleotide composition and RNA modifications, such as meth-
ylation and the formation of G-quadruplexes that allow effective replication in mosquitoes and primates [8,39].

6.3 ZIKV transmission

6.3.1 Vector-borne transmission
The transmission of ZIKV shows a high degree of variance. The most common mode of transmission of ZIKV is hori-
zontal transmission through the bite of Aedes mosquito species, where they take a viremic blood meal and then inject
infectious saliva into the host during blood feeding. During a mosquito bite, the virus is delivered into extravascular
spaces in the epidermal and dermal areas and the initial cycle begins after the virus gains access to resident and migra-
tory cells of the skin in a vertebrate host [7,8]. There are two distinct life cycles of ZIKV transmission have been
reported. ZIKV lineage is maintained in nature through enzootic or sylvatic cycle occurring between Aedes mosquitoes
(Ae. africanus, Ae. furcifer, Ae. taylori, and Ae. luteocephalus) and nonhuman primates including apes and monkeys in
Africa [40] (Fig. 6.2). Humans are the incidental host in sylvatic transmission cycle and carry the virus further to epi-
demic or urban cycle in which humans are the main host and serve as amplifier and carrier of infection to uninfected
mosquitoes (Fig. 6.2). Ae. aegypti and Ae. albopictus are the principle species involved in such transmissions and have
90 Pandemic Outbreaks in the 21st Century

Skin
Mosquito ƒ Fibroblasts Embryonic brain tissue
Aedes species Testis ƒ Epidermal
ƒ Neural progenitor stem cells
Human ƒ keratinocytes
Semen ƒ Radial Glial progenitor cells
ƒ ƒ Immature
Epididymis ƒ Microglia
dendritic cells
Sylvatic
transmission Maternal– fetal interface
Cycle
ƒ Decidual stromal cells
Nonhuman Nonhuman ƒ Trophoblasts
primate primate ZIKV ƒ Fibroblasts
ƒ Endothelial cells
ZIKV

Mosquito ƒ Hofbauer macrophages

Aedes species ƒ Amniotic epithelial cells

Human Women Man Maternal–fetal

Urban transmission
transmission Non–vector transmission (vertical)
Cycle (Sexual, Blood
transfusion, Maternal–
Human
fetal routes)
Transplacental route
Intrauterine route
Mosquito ZIKV–infected Transvaginal route?
Aedes species Pregnant women
Newborn with microcephaly
FIGURE 6.2 Transmission cycle of ZIKV. ZIKV adopted the sylvatic (enzootic) transmission cycle in Africa, which involves various species of
Aedes mosquitoes and nonhuman primates (e.g., apes, rhesus monkeys). In the urban cycle, ZIKV has adopted the transmission passages from the
human
mosquito and human
human transmission mode in Asia. Non-vector-borne transmission of ZIKV infection in human population can be
caused through sexual contact (heterosexual or homosexual transmission), from mothers to their fetuses, and infected blood transfusions. Another part
of the illustration shows the tissues and cell types that are targeted by ZIKV in testis, skin, embryonic brain tissue, maternal
fetal interface. ZIKV,
Zika virus. Modified from Gorshkov K, Shiryaev SA, Fertel S, Lin Y-W, Huang C-T, Pinto A, et al. Zika virus: origins, pathological action, and treat-
ment strategies. Front Microbiol 2019;9:3252; Kazmi SS, Ali W, Bibi N, Nouroz F. A review on Zika virus outbreak, epidemiology, transmission and
infection dynamics. J Biol Res (Thessalon) 2020; 27:5; and Rather IA, Lone JB, Bajpai VK, Paek WK, and Lim J. Zika virus: an emerging worldwide
threat. Front Microbiol 2017;8:1417.

been noted in the majority of ZIKV outbreaks in Asia [12,37,41,42]. The transmission cycle of the virus is mainly from
reservoir host to mosquito with an incubation period of 2
5 days in host and 5
7 days in mosquito. Vertical transmis-
sion of the virus has been shown in Ae. aegypti mosquitoes [9,10,43].

6.3.2 Nonvector transmission

Nonvector-borne transmission of ZIKV infection in human population can be caused through sexual contact (heterosex-
ual or homosexual transmission), from mothers to their fetuses, and infected blood transfusions (Fig. 6.2) (reviewed in
[8,10,40,44
47,128]). Sexual transmission of ZIKV was suspected in 2008 when a scientist infected in Senegal trans-
mitted the virus to his wife upon returning to the United States, and in 2013 when infectious viral particles were
detected in the semen of a French Polynesian patient. Sexual transmission of the virus can occur through contact of
male-to-female, female-to-male, or male-to-male partners from both asymptomatic and symptomatic infections, via
unprotected vaginal, oral, or anal intercourse [48,49]. Zika viral shedding has been detected in the genital fluids of both
female and male patients. ZIKV is the first arbovirus to be detected in the semen samples of infected individuals with
high viral loads for prolonged periods [50,51]. The prolonged presence of virus in reproductive tracts of both sexes cre-
ates serious concerns of teratogenicity, which is reflected by WHO and Centre for Disease Control (CDC)’s updated
Zika prevention guidelines, and both the organizations outline the use of contraception for a period of 6 months in
infected men and a period of 8 weeks in infected women from the time of symptom onset [41,52].
Prenatal transmission of ZIKV has been detected in neonates born to mothers with a history of ZIKV infection dur-
ing the French Polynesian outbreak in 2013
14 [53]. The entire mechanism behind the ability of ZIKV to infect and
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 91

damage a developing fetus is not completely understood, but fetal infection implies that the virus can cross and/or
bypass the placental barrier [54
57]. There are adequate reports that ZIKV has the capability to be transmitted from a
mother to her fetus through transplacental route during the first trimester, resulting in infection of fetal brain, placental
damage, and fetal abortion, suggesting different susceptibilities and distinct mechanisms of pathogenesis at specific
times during pregnancy [16,54
56,58]. The transplacental transmission of ZIKV from mother to fetus may occur by
the infection of placental Hofbauer cells (HCs, placental macrophages) and cytotrophoblasts (Fig. 6.2) [59]. ZIKV parti-
cles and its RNA has been recovered from amniotic fluid, placental tissue, fetal brain tissue, and infants with high rates
of microcephaly among children born to mothers with proven acute ZIKV infection during pregnancy provided strong
evidence linking central nervous system (CNS) anomalies to maternal infection [9,12,60
62]. High ZIKV RNA load
has been detected in breast milk, but no cases have been reported via breastfeeding. Other body fluids (sweat, tears, and
saliva) might also be a source of infection and can increase the magnitude of threat caused by nonmosquito transmission
of the virus [37,52]. Blood transfusion is another potential novel route for ZIKV transmission, and the first case of
blood transfusion transmission is reported on December 2015 in Brazil [10,12]. The other reported instances of
nonvector-borne transmission are through bite of infected monkey and accidental exposure in a laboratory environment
[37,63].

6.4 Clinical manifestations associated with ZIKV infection

The clinical presentation of uncomplicated ZIKV infection often termed as “Zika fever” is usually mild, nonspecific,
self-limiting within a week in a large proportion of individuals and can be misdiagnosed as other arbovirus diseases
such as dengue and chikungunya. The symptoms of the virus in approximately 20% of adults usually include a low-
grade fever, maculopapular pruritic rash, arthralgia, or nonpurulent conjunctivitis headaches, myalgias, retro-orbital
pain, gastrointestinal disorders, cervical lymphadenopathy, while 80% of the ZIKV-infected people remain asymptom-
atic [5,12]. Incubation period of ZIKV is usually 3
12 days and it is estimated that about one in five people infected
with ZIKV becomes symptomatic with clinical manifestations in many ZIKV outbreaks [9,64,65].
The association of chronic ZIKV infections with neonatal complications including microcephaly, a serious neurolog-
ical disorder, characterized by abnormally small heads and underdeveloped brains in children/infants and it was recog-
nized as a matter of serious concern during the ZIKV outbreak in Brazil in late 2015 [66]. Additionally, other fetal
complications reported include fetal death, placental insufficiency, fetal intrauterine growth restriction (IUGR) and
CNS injury and birth defects such as craniofacial disproportion, brainstem dysfunction, ocular anomalies, and auditory
dysfunctions, which have been collectively termed “congenital Zika syndrome (CZS)” (reviewed in [9,18,41,43,67,68]).
The development of CZS may occur in any pregnant mother with ZIKV infection, regardless of the presence of symp-
toms [6,11,47,69]. Major neurological anomalies consistently observed with CZS in fetuses and neonates include
decreased brain matter, cerebral atrophy, diffuse cortical and subcortical calcifications, varying degrees of ventriculo-
megaly, signs of abnormal gyration, cortical development, and ocular anomalies that might lead to severe mental retar-
dation and substantial motor disabilities, and visual and auditory impairments [41,47]. Other serious sequelae of ZIKV
include IUGR [70], abnormal umbilical artery flow, placental insufficiency, hydrops fetalis, and fetal demise [71,72]. In
addition to neuronal abnormalities, reactive gliosis, microglial hyperplasia, corpus callosum hypoplasia, and delayed
myelination have also been subsequently reported [19]. Some infected newborns will remain asymptomatic or develop
only minor symptoms later in life. However, blindness, hearing loss, hypotonia, paresy, epilepsy, and severe neurodeve-
lopmental delay are the long-term outlook for children severely infected in utero [9]. Apart from the microcephaly,
infants present with diverse ocular manifestations including the following: focal pigment mottling, cataracts, intraocular
calcifications, chorioretinal macular atrophy, optic nerve abnormalities, conjunctival injections, optic disc cupping,
microphthalmia, lens subluxation in addition to bilateral iris coloboma, foveal reflex loss, macular hypoplasia, and scar-
ring. Retinal lesions, including well-defined chorioretinal atrophy and gross pigmentation, generally affecting the macu-
lar region, are unique to ZIKV infection [68,73,74].
During the ZIKV outbreaks in French Polynesia in 2013 and the Americas in 2015, number of adult patients with
ZIKV infection was found to be associated with GBS and acute disseminated encephalomyelitis. GBS is a serious, life-
threatening autoimmune neuropathy that can result in gradual muscle weakness, sensory abnormalities, reduced nerve
function that can ultimately trigger flaccid paralysis, and respiratory failure as a consequence of the immune-mediated
demyelination of peripheral nervous system neurons [16,41,75,76]. However, scientific evidences for the pathogenesis
of ZIKV-associated GBS are still lacking [10,43].
92 Pandemic Outbreaks in the 21st Century

6.5 Molecular mechanisms underlying ZIKV-induced birth defects

6.5.1 Cellular targets and entry of ZIKV
Epidemiological studies suggested that ZIKV infections in pregnant women may be linked to a rise in the number of
cases of babies born with microcephaly in Brazil during the 2015 outbreak [66]. The first line of evidence came from
the clinical case reports revealing the presence of ZIKV in the brains of fetuses with microcephaly in women who were
infected with ZIKV during pregnancy [22,45,77]. But it was unclear whether developing neurons and/or proliferating
progenitor cells were direct targets of ZIKV and there is very limited knowledge about the possible mechanisms of how
ZIKV infection leads to fetal brain damage. Recent investigations utilizing laboratory testing, in vitro cellular systems
[neural cell precursors (neurospheres), brain organoids, advance embryonic stem cell (ESC), induced pluripotent stem
cell (iPSC), spinal cord neuroepithelial stem cells (NESCs), and neocortical cell models] and in vivo animal models
(mouse, rats, chickens, and nonhuman primates) profoundly helped to understand and explain the cellular and molecular
basis of microcephaly development, level of selective neurotropism, spectrum of changes occurring to the brain, and
pathophysiology of viremia in ZIKV-infected fetuses [78].
ZIKV infection exhibits broad cellular tropism and persistence in body tissues and fluids, a characteristic that distin-
guishes ZIKV from other arthropod-transmitted flaviviruses. Cellular targets of ZIKV range from the brain, placenta,
skin, testis, kidney, and retina (neural stem cells, oligodendrocyte precursor cells, microglia, and astrocytes) (Fig. 6.2)
[8,19,21,62]. Initial ZIKV infection most likely occurs via mosquito bite in human skin by directly affecting permissive
human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells. Viral replication is further supported
by the formation of autophagosomes containing viral particles inside the skin fibroblasts. After a period of replication
in the primary inoculation site, the virus is disseminated to dendritic cells via the lymphatics and bloodstream [78
80].
ZIKV was found to successfully infect several cell types in the human placenta such as HCs, cytotrophoblasts, and pla-
cental endothelial cells (Fig. 6.2), thereby placenta plays a significant role in transmitting the virus to fetal brains via
blood [81]. Other developmentally relevant stem cell populations are also highly susceptible to ZIKV infection, such as
neocortical and spinal cord NESCs, which are the earliest population of resident NPCs present during neurodevelop-
ment [82], and cranial neural crest cells (CNCCs), which give rise to most cranial bones and exert paracrine effects on
the developing brain [83]. Primary human endometrial stromal cells are greatly permissive to ZIKV infection and sup-
port its in vitro replication [22,84].
In addition to NPCs, studies of ZIKV infection in organotypic cultures from primary human brain tissue or necropsy
brain tissues from fetuses and newborns revealed that iPSC-derived astrocytes, microglia, and oligodendrocyte precur-
sor cells located throughout the developing cortex can be productively infected by ZIKV, while the infection rate of
neurons is low [85,86]. Infected Sertoli cells in human testis were found to act as ZIKV reservoirs [87]. Congenital ocu-
lar disease triggered by ZIKV could be due to direct invading of fetal ocular cells. In addition, retinal neurons appeared
to be targeted by ZIKV and this was associated with infections in the lateral geniculate, suprachiasmatic nuclei, and
superior colliculus, indicating a potential for the virus to infect cells along the visual pathway. This selective infection
may be due to axonal transport of viral particles [17].
The mode of cellular entry by ZIKV is a matter of controversy. Studies showed that putative ZIKV entry receptors
include DC-SIGN (dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin) receptor, heat shock
proteins, and tyrosine-kinase receptors TIM (TIM-1, TIM-4) and TAM (Tyro3, AXL, and Mer). TAM receptors belong
to a family of tyrosine kinase receptors which are important in the regulation of immune responses, including mediating
the physiologies of the brain’s immune cells (microglia). TAM receptors maintain neurogenesis in adult brain, and they
are important for survival, proliferation, and differentiation of neural stem cells. Of the TAM receptors, AXL, a member
of the phosphatidylserine phagocytic protein receptors, appears to be a receptor exploited by ZIKV, although the virus
may use a combination of different receptors to gain cellular entry [23,88
90]. AXL protein is overexpressed in devel-
oping human brain cells, including radial glia, astrocytes, endothelial, and microglia. It was found that the TAM ligand
Gas6 (growth arrest
specific protein 6) acts as a cofactor to recruit the virus to the AXL receptor, resulting in the inter-
nalization of ZIKV usually by clathrin-mediated endocytosis. ZIKV-containing vesicles then translocate to Rab5 1
endosomes to establish productive infection and induce the transcription of Toll-like receptor 3 (TLR3, innate immunity
receptor), DDX58, and IFIH1 as well as several interferon (IFN)-stimulated genes (ISGs), leading to suppression of the
innate immune response. Downregulation of AXL by an engineered AXL decoy receptor, MYD1 or the AXL kinase
inhibitor R428; or an antibody specific for the extracellular domain of AXL significantly reduced but did not abolish
the ZIKV infection, suggesting the AXL receptor might be the primary but not the only receptor that is required for
ZIKV infection [21,79,91]. Examination of the receptor repertoire of human radial glia cells in the fetal brain involved
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 93

in ZIKV attachment and entry during neurogenesis by single-cell RNA-sequencing revealed that conserved and high
AXL expression in NPCs could render them selectively susceptible to ZIKV infection [92]. Consistently, many candi-
date ZIKV entry receptors were significantly elicited, most strikingly AXL in animal models of ZIKV-induced micro-
cephaly [22,23,26].
Studies indicated that ZIKV may invade host cells by TAM receptor-independent mechanism(s). Genetic ablation of
AXL failed to protect NPCs and cerebral organoid against ZIKV infection [93]. Chen et al. [94] showed that AXL is
not a viral entry receptor but rather enhances infection by suppressing ZIKV-induced activation of type 1 IFN genes.
Recently, the ZIKV entry into the host cells tracked by chemical proteomics approach showed neural cell adhesion mol-
ecule I as a potential and unique receptor for ZIKV entry [95]. Further investigations on the entry of ZIKV using AXL
and other receptors will provide better insight into the detailed molecular mechanism of ZIKV-induced neurovirulence
[17,22,23].

6.5.2 Induction and suppression of innate immune mechanisms mediated by ZIKV

ZIKV, like other flaviviruses, induces a humoral antibody response that is thought to provide lifelong protection against
reinfection, although this assumption to be further confirmed through multiple human cohort studies. In commonly
occurring endemic ZIKV areas, the secondary infections result in heterotypic, cross-reacting antibody response with
multiple flavivirus antigens, making the identification by serological assays a challenging task [62]. The amount of
background immunity required in the population to prevent the emergence or recirculation of the virus is unknown.
Although in vitro studies have shown that preexisting dengue virus antibodies might drive increased ZIKV replication
(termed as antibody-dependent enhancement), there is no evidence that this phenomenon plays a part in viral pathogen-
esis [96]. Studies in rhesus macaques suggest that immunity against the ZIKV African lineage confers immunity against
the heterologous Asian lineage and vice versa [97]. Very little is known about the innate and cellular immune responses
to ZIKV [9,20,62].
Inflammation is one of the first-line responses of the cellular immune system to viral infection, which is typically
ignited by releasing cytokines including chemokines. ZIKV triggers various host cell pro-inflammatory responses
[98
100]. ZIKV stimulates CD8 1 T cell-mediated polyfunctional immune responses to induce NF-κB-mediated pro-
duction of cytokines such as interleukin-1β (IL-1β), IL-6, MIP1α, as well as chemokines including IP10 and RANTES.
These ZIKV-induced T cell immune responses are antiviral because when CD8 1 cells from previously ZIKV-infected
mice are introduced to naive mice prior to ZIKV infection, viral clearance is enhanced. Conversely, depletion of
CD8 1 T cells from infected animals compromises viral clearance. ZIKV structural proteins (C, prM, and E) are the
major targets of CD8 1 T cell and CD4 1 T cell responses [6,20,80].
ZIKV infection of placenta will lead to morphological changes secondary to inflammation in the fetal brain and tis-
sue. Inflammatory processes in the placenta may lead to altered expression and regulation of neuropeptides and growth
factors, leading to microcephaly [18,101]. Signals induced within the placenta affect the response of the maternal
immune system to antigenic signals. The resultant inflammation may have systemic effects on the morphology of the
fetus. Although induction of innate antiviral responses in glia cells would dampen ZIKV infection, glia-mediated
inflammation is likely to be harmful to neural or glial cell development, as well as neuron survival and function, which
has been detected in different ZIKV-infected mouse models [18]. Considering the central role of inflammation in neuro-
logical disorders, future studies in animals are required to test the hypothesis that ZIKV infection-induced activation of
astrocytes and microglia triggers a strong immune response, which in turn induces neuronal death and dysfunction [19].
Apart from ZIKV-mediated inflammatory and humoral responses, ZIKV also triggers a series of host cell innate
immune responses [20]. ZIKV is recognized by an endosomal TLR3 that can be found in macrophages or Langerhans
cells. Replication of the ZIKV induces the production of type I IFN and pro-inflammatory cytokines, resulting in
increased expression of type1 ISGs via TLR3 activation and the activation of pattern recognition receptors (PRRs)
(Fig. 6.3A) [79,89]. ZIKV replication has been shown to be inhibited by type I IFNs in several human cell types and in
mouse models (reviewed in [7,18,20,23,102]). ZIKV-induced TLR3 activation promotes phosphorylation of interferon
regulatory factor 3 (IRF3) by TANK-binding kinase 1 (TBK1), leading to induction of type 1 IFN signaling pathways
[98]. This initiates a cascade that further activates cytoplasmic RIG-I-like receptors (RLRs) responses, subsequently
inducing transcription of RIG-I, MDA5, and several type I and III ISGs including OAS2, ISG15, and MX1 [79].
Activation of the type I IFN signaling pathway results in the production and secretion of IFN-β. Secreted IFN-β binding
to IFN-β receptor activates JAK1 (Janus kinase I) and Tyk2 kinases that in turn phosphorylate STAT1 (signal trans-
ducer and activator of transcription) and STAT2. Upon ZIKV infection, association of the phosphorylated STAT1/
94 Pandemic Outbreaks in the 21st Century

ZIKV
A B Type I IFNs; Type III IFNs;
IFNs IFN-α,β, ε IFN-λ,1,2,3,4

AXL
IFNAR1 IFNAR2 IFNλR1 IL10Rβ
Viral RNA Viral RNA Cytosol
Cytosol
RIG-1 MDA5 JAK2 JAK1
TYK2 JAK1
NS3

Translocation
Translocation

14-3-3ε 14-3-3π

TLR3 P P
STAT1 STAT2
MAVS Mitochondria MAVS IFNs

P
NS4A

STAT1

STAT2
P
TBK1 ISGF3
RIG-1
NS4A P
MDA5
IRF9
P P
IRF3 IRF7

STAT2
STAT1
P
IRF3 P
IRF7
IFNs
P
>300 ISGs
IRF9 ISRE

Type I IFNs
Nucleus Type III IFNs Nucleus
Antiviral response

FIGURE 6.3 Induction and suppression of cellular innate immune mechanisms mediated by ZIKV. (A) During ZIKV infection, viral RNA sensors
and interferon-mediated signaling through downstream adapter molecules and transcription factors can be targeted for immune evasion strategies.
ZIKV nonstructural (NS) proteins are involved in the inhibition of signal transduction mediated by pattern-recognition-receptors (PRRs) leading to
type I, III interferons (IFNs) (left panel) and IFN-mediated expression of ISGs (right panel). ZIKV NS3 interacts with the scaffold proteins 14
3
3ε
and 14
3
3π and thereby prevents the translocation of the sensors RIG-I and MDA5 from the cytosol, where viral RNA binding occurs, to the mito-
chondria, where RIG-I and MDA5 transmit downstream signaling via the adapter protein mitochondrial antiviral signaling (MAVS). Furthermore,
ZIKV NS4A directly binds to both RIG-I and MDA5, thereby blocking their interaction with MAVS. Downstream of MAVS, ZIKV NS1, and NS4B
have been found to reduce the phosphorylation of TBK1, which suppresses TBK1 activation. ZIKV NS4A and NS5 have also been shown to inhibit
IRF3 phosphorylation, which inhibits the downstream IFN transcription. In addition, several ZIKV proteins inhibit signaling downstream of the IFNα/
β receptor (IFNAR). ZIKV NS2B3 degrades the kinase JAK1 in a proteasome-dependent manner, while ZIKV NS5 induces the proteasomal degrada-
tion of STAT2. Moreover, it has been shown that ZIKV NS5 inhibits the activating phosphorylation of STAT1 and of STAT2. ZIKV NS proteins are
colored in red and can interfere with IFN responses by suppressing the induction of signaling pathways at multiple steps. - indicates a positive inter-
action, whereas denotes inhibitory action. Small yellow colored circles with P are used to indicate phosphorylation. ZIKV, Zika virus. Modified from
Lee I, Bos S, Li G, Wang S, Gadea G, Desprès P, et al. Probing molecular insights into Zika virus hostinteractions. Viruses 2018;10(5):233; Lee JK,
Shin OS. Advances in Zika virus hostcell interaction: current knowledge and future perspectives. Int J Mol Sci 2019;20(5):1101; and Serman TM,
Gack MU. Evasion of innate and intrinsic antiviral pathways by the Zika virus. Viruses 2019;11(10):970.

STAT2 heterodimer with IRF9 promotes ISRE3 (IFN-stimulated response element)-mediated transcription of antiviral
ISGs (Fig. 6.3B) [21,102].
The efficient replication and pathogenesis of ZIKV is associated with the ability to mount an effective antagonistic
response by employing multiple immune evasion mechanisms through the inhibition of induction of IFNs and down-
stream signaling pathways (Fig. 6.3A and B) [21,103]. Monel et al. [104] showed that ISGs, IFITM3 and IFITM1 inhib-
ited the replication of ZIKV. ZIKV infection of human astrocytes in CNS resulted in decreased IFITM3 levels, which is
associated with increased cytoplasmic vacuoles derived from the ER, an indication of para-apoptotic cell death [23].
Apart from that, type III IFN, IFNλ1 that are constitutively secreted by placental trophoblasts, protect human astrocytes
from ZIKV infection. However, no antiviral activity was detected when ZIKV-infected cells were treated with IFNl-λ1
indicating that ZIKV may antagonize IFNλ1 signaling [55]. Frumence et al. [99] demonstrated that early ZIKV infec-
tion causes cells to produce IFN-β resulting in apoptosis of potential ZIKV host cells [23,102]. It is also likely that
ZIKV stimulates cytokines in the infected CNCCs. This cytokine storm contributes to the death and aberrant
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 95

differentiation of neural progenitors, affecting the signaling crosstalk among the developing brain regions and destroy-
ing the normal brain and facial development programs (Fig. 6.4A) [83]. ZIKV-induced inflammation may also drive the
death of NPCs, via paracrine mode. During early development, CNCCs that form the cranial bones exert paracrine

A
IFN signaling
ZIKV Gas6

IFNAR AXL
UPR genes NS4A Abnormal
(IRE1- AKT
centrosome
XBP1..) NS4B

NF-κB STAT1
mTOR Mitotic NS5
signal disruption STAT2
P53

NS2A ZO1
NUMB Proteosome IL-1β
ER stress Autophagy
(BIP, ATF6) (LC3)
Microcephaly Cytokine production and response
(ASPM, CEP152…) (TNF,TLR3…..)

Cell death Cell cycle deregulation Immune response

B
ZIKV Immune
infection response
Deregulated signaling pathways

Abnormal
Disrupted glial cell
neurogenesis
development
development

Microglial
OPC activation
NPC
depletion
depletion
Astrocyte
reactivation

Neuron
loss Myelination defect and
demyelination loss

FIGURE 6.4 Mechanisms underlying ZIKV-induced pathologies during brain development. (A) Schematic diagram showing the activated cellular
pathways regulating cell death, cell cycle, and immune response upon ZIKV infection. (B) Mechanisms associated with impaired brain development
upon ZIKV infection. ZIKV prefers to target NPCs and OPCs during the early and later stages of brain development, respectively, and activates innate
immune response, which could lead to deregulation of gene networks involved in cell cycle, neurogenesis, oligodendrogenesis, and apoptosis, resulting
in increased cell death, disrupted cell cycle progression, reduced proliferation, and premature differentiation of NPCs and OPCs. However, ZIKV
infection of glial cells, including astrocytes and microglia, leads to their activation, as well as immune response (inflammation). Inflammation would
elicit non-cell-autonomous effects on NPCs, OPCs, neurons, and oligodendrocytes, resulting in the impaired neurogenesis, gliogenesis (especially oli-
godendrogenesis), and myelination of neurons, accompanied by massive neuronal death. Together, these pathogeneses impair brain development and
lead to CZS, including microcephaly and other serious congenital neurological complications. AXL, AXL receptor tyrosine kinase; CZS, congenital
Zika syndrome; ER, endoplasmic reticulum; IFN, type I interferon; NPC, neural progenitor cell; OPC, oligodendrocyte progenitor cell; TLR3, Toll-
like receptor 3; UPR, unfolded protein response ZIKV, Zika virus. Modified from Xu D, Li C, Qin CF, Xu Z. Update on the animal models and under-
lying mechanisms for ZIKV-induced microcephaly. Annu Rev Virol 2019;6(1):459
479.
96 Pandemic Outbreaks in the 21st Century

effects on the developing brain. An in vitro study showed that ZIKV-infected CNCCs, although resistant to apoptosis,
secrete significant levels of cytokines that resulted in NPCs death [83].
ZIKV has adopted various strategies to counteract host antiviral responses to establish its successful infection. ZIKV
becomes resistant to IFN treatment after the establishment of infection, suggesting ZIKV might have deployed effective
counteractive measures against host innate immune responses [21,105]. Resultant to this finding, no secreted type I and
type III IFNs were detectable from ZIKV-infected cells [98]. ZIKV impairs the induction of type I IFN by binding to
IRF3 [105
107]. These ZIKV-mediated counteracting/antagonizing effects are achieved through multiple nonstructural
ZIKV proteins (NS1, NS2A, NS2B, NS4A, NS4B, and NS5). All these ZIKV proteins suppress IFN-β production to
various degrees by targeting distinct components of the RIG-I pathway [106]. NS1, NS4A, and NS5 proteins specifi-
cally inhibit IRF3 and NF-kB [105]. N-terminal region of flavivirus NS4B is shown to be responsible for inhibition of
alpha/beta IFN signaling [108]. NS1 and NS4B proteins of ZIKV functions as the main suppressors of type I IFN induc-
tion by targeting TBK1, which is required for phosphorylation of IFN regulatory factor 3 to initiate type I IFN transcrip-
tions and they, in turn, stabilize NS2B-NS3 (Fig. 6.3A) [21,106,109].
ZIKV has also developed mechanisms to block JAK1/Tyk2-mediated STAT1 and STAT2 phosphorylation resulting
in ISGF3 transcription and ISGs translation shutdown [21,98]. ZIKV NS5 selectively binds to STAT2, resulting in pro-
teosomal degradation of this protein in human cells but not in mice, presumably inhibiting type I IFN signaling
[105,110]. NS5 also selectively activates type II IFN, IFN-β, signaling to induce inflammation [107]. ZIKV NS4A sup-
presses type I IFN induction via inhibition of mitochondrial antiviral signaling protein interaction as a way of immune
evasion (Fig. 6.3A and B) [111]. African ZIKV lineage infection in human dermal fibroblasts and human epidermal ker-
atinocytes resulted in distinct expression changes in RLRs, such as RIG-I and MDA5. Inhibition of RIG-I using small
interfering RNA resulted in increased viral gene expression and reduced induction of IFNs and ISGs. Furthermore,
ZIKV NS1 directly interacted with RIG-I or MDA5 and downregulated RLR-mediated antiviral signaling pathways
[80].
Among the multiple key cellular signaling cascades, Akt-mTOR pathway is critical for neurogenesis of NPCs, as
well as for their subsequent migration and maturation, and autophagy regulation in brain development. After invading
NPCs in the fetal brain, ZIKV proteins NS4A and NS4B target and suppress Akt-mTOR signaling pathway, leading to
an impaired neurogenesis and aberrant activation of autophagy in the host cell, ultimately resulting in enhanced viral
replication [8,17,112]. ZIKV NS2B-NS3 impairs JAK/STAT signaling pathways by promoting proteosomal-directed
degradation of JAKI and reduces virus-induced apoptotic cell death (Fig. 6.4A). Furthermore, NS1, NS4B, and NS2B-
NS3 cooperate to enhance ZIKV infection by blocking IFN-induced autophagic degradation of NS2B-NS3. These puta-
tive roles by ZIKV proteins may limit neurogenesis, by interfering with the growth of neural cell precursors
[23,31,109]. Thus the nonstructural proteins of ZIKV work synergistically at multiple levels by interfering important
cellular survival and homeostasis mechanisms to restrict host antiviral responses [31,80].

6.5.3 ZIKV-mediated mechanisms to induce congenital Zika syndrome

6.5.3.1 Study of ZIKV-associated microcephaly using in vitro and in vivo models
NPCs proliferate, differentiate in diverse neuronal cell fates, and migrate to their final position in the cortical plate,
where they mature into adult neurons during the forebrain development. The temporal and spatial precisions with which
these processes occur during telencephalic development are important for higher-order functions including language,
emotion, and cognition. Therefore loss of NPCs during telencephalic development can lead to severe neurodevelopmen-
tal problems including primary congenital microcephaly [23]. Though ZIKV has been in existence for .50 years, only
2013
15 ZIKV epidemics of ZIKV showed the association of virus infection with a marked increase of risk for con-
genital microcephaly and other serious neurologic complications, which triggered widespread efforts to understand the
molecular basis of infection [20,24]. One hypothesis is that the nonsynonymous nucleotide mutations found in ZIKV
strains from the Brazilian outbreak in 2015 may contribute to the increased incidence of microcephaly. The Brazilian
strain contains mutations in three of the nonstructural proteins, including three in NS1 (K940E, T1027A, and M1143V),
which is implicated in immune evasion; one in NS4B (T2509I), which is involved in the inhibition of type I IFN signal-
ing; and one in NS5, which is known to mask the viral RNAs from host recognition (M2634V) [24,77]. In addition to
the adaptation of nonstructural proteins, the current pathology may also be consequent to mutations in the prM protein
and around the Asn-154 glycosylation site in the E protein of the Brazilian ZIKV strains. These modifications have
been speculated to contribute to viral adhesion to host cells and facilitate unique aspects of ZIKV transmission [113].
Furthermore, ZIKV also produces sfRNAs in their 30 untranslated regions that accumulate during infection and resist
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 97

degradation by host exonucleases in infected cells, which are directly linked to pathologic effects [114]. All these
genetic and epigenetic hypotheses remain to be fully tested experimentally, which may provide critical insight into the
viral
host interaction and pathogenesis of ZIKV infection [24,88].
ZIKV-induced microcephaly could be possible through its passage from maternal blood via placenta, a physical and
immunological barrier separating the maternal and fetal compartments. Detection of ZIKV particles and mRNA in the
placental tissue, amniotic fluid, and corresponding fetal brain tissues suggest transplacental passage by the virus
[18,61,85]. Syncytotrophoblast, outer layer of trophoblasts, invades the wall of uterus and establishes nutritional supply
between mother to fetus. ZIKV is shown to have the ability to infect placental macrophages and cytotrophoblasts
in vitro [55]. Other routes of fetal ZIKV infection during pregnancy could include movement of ZIKV into the amniotic
and yolk sacs of the developing fetus or leakage through the trophoblastic plugs [115]. Presence of ZIKV in the semen
indicates its early access to the embryo through sexual transmission [44].
Identification of cell types that are particularly vulnerable to viral infection in the developing brain after ZIKV
breaches the placental barrier is the key step to understand the impact and molecular/cellular mechanisms that implicate
ZIKV induced fetal and pediatric neuropathologies. During the last few years, researchers have successfully conducted
studies to investigate the ZIKV pathogenesis and its affinity for neurons and neural stem cells by using cellular systems
and animal models, which have broadened the understanding of ZIKV-induced teratogenic effect on fetal brain
(reviewed in [6,12,16,17,19,21
23,78]).

6.5.3.1.1 ZIKV-mediated cell cycle disruption, cell death, and altered gene expression
Cell death either by apoptosis or necrosis leads to the catastrophic destruction of placental architecture and vasculature
by ZIKV, which can result in fetal brain injury. Upon infection of the placental cells, ZIKV not only suppresses the
innate and adaptive immune responses but may also proceed to induce the production of inflammatory cytokines and
trigger the upregulation of genes involved in apoptosis and necrosis. Analyses of human placental tissues obtained from
pregnant women showed chronic inflammatory lesions 1 week after ZIKV infection in the first trimester, and examina-
tion of corresponding brain tissues revealed microcalcification and viral proteins and particles in glial cells [19,85].
ZIKV infection of placental chorionic villi tissues resulted in increased expression of IFNα, IFNβ, and IFNλ (but not
IFNγ) and the upregulation of transcripts associated with apoptosis, cell death, and necrosis (Fig. 6.4A and B) [56].
Cell death also contributes to ZIKV-induced microcephaly, which has been detected extensively in human fetal brains
infected with ZIKV during the major outbreaks [45,77]. In a reported clinical case, postmortem analysis revealed dif-
fuse cerebral cortex thinning in a fetal brain infected by ZIKV followed by excessive apoptosis in the neocortex [45].
Following clinical observations that ZIKV could be found in brains of fetuses from infected pregnant women
[45,77,116], a human NPCs (hNPCs)-based cellular model provided the first evidence that ZIKV efficiently infects
human cortical NPCs, causing dysregulation of the progenitor cell cycle, mitotic abnormalities, significant reorganiza-
tion of intermediate filament and microtubule networks and remodeling of the cytoskeleton, differential expression of
genes related to cell cycle dynamics, transcription, and protein localization, caspase-3 activation, increased apoptosis
and autophagy, and decreased cell growth, viability and diminished cortical layers, leading to CNS abnormalities
including microcephaly [16,17,28,78,117]. Notable intracellular changes resulting from ZIKV infection of neurons
include aberrant granulation pattern in the cytoplasm, irregular cell shape, partial nuclear condensation, pyknosis or
“vacuolar nuclei” appearance, swollen mitochondria, disorganized ER architecture, and supernumerary mitotic spindle
[29,79,82]. In summary, cell death, neuronal migration defects, and misdirected and reduced proliferation associated
with ZIKV infection of NPCs can lead to the formation of a thinner cortex of the developing forebrain [23].
A study using fission yeast for genome-wide analysis of ZIKV proteins attributed intracellular changes to specific
ZIKV proteins. All 14 structural and nonstructural ZIKV proteins and peptides were expressed under an inducible pro-
moter in fission yeast and their intracellular localization and cytopathic activities were subsequently determined [118].
The authors found that membrane-anchored capsid (anaC), C, prM, M, E, NS2B, and NS4A caused elongation of cells,
while cellular hypotrophy was a consequence of prM, NS2B, and NS4A [23,118]. Global transcriptome analyses
revealed that many genes involved in cell proliferation, differentiation, and migration and organ development are down-
regulated, with enrichment of downregulated genes in cell cycle
related and DNA replication/repair pathways and
upregulation of genes related to apoptosis-related pathways upon ZIKV infection of human and mouse NPCs and these
multiple significantly downregulated genes are found to be microcephaly associated genes [26,28,117], many of which
encode proteins localized at the centrosome and play an important role in cell cycle progression, suggesting a direct
mechanistic link of ZIKV infection to microcephaly at the molecular level [24]. The apoptosis-induced cell death could
be mediated by activation of tumor suppressor protein p53 (TP53), as the ZIKV infection significantly upregulated
98 Pandemic Outbreaks in the 21st Century

TP53 gene and protein expression levels and Ser15 phosphorylation, which are correlated with genotoxic stress and
apoptosis induction (Fig. 6.4A) [117,119]. NPC apoptosis upon ZIKV infection can also be caused by activation of the
immune response. Infection by the Asian strain of ZIKV, which is more closely related to current epidemic strains,
leads to significant upregulation of viral response genes involved in IFN response and the type II IFN signaling, TLR
signaling, and tumor necrosis factor α (TNFα) signaling pathways in hNPCs [117]. On the other hand, the prototypical
African ZIKV strain does not elicit such a virus response in hNPCs, suggesting that different ZIKV strains have specific
neurotropism and unique molecular signatures [41].
Activation of an immune response leading to upregulation of several genes involved in cytokine production and
response has been observed in mouse fetal brains infected with Asian ZIKV strains [26,120], suggesting that cytokines
may play a key role in the pathogenesis of ZIKV infection. Activation of TLR3 may mediate the ZIKV-induced apopto-
sis and impaired neurogenesis. Inhibition of TLR signaling by a TLR3 competitive inhibitor largely rescued an African
strain of ZIKV Mr766-induced apoptosis and organoid shrinkage, while a TLR3 agonist, poly(I:C), mimicked the phe-
notypes induced by ZIKV infection in brain organoids [121]. ZIKV infection can also activate genes coding for binding
immunoglobulin protein (Bip), activating transcription factor 6 (Atf6), CCAATenhancer-binding protein homologous
protein (Chop), and many other genes involved in ER stress. Unfolded protein response (UPR) genes (e.g., Ire1-Xbp1)
are activated to alleviate stress responses induced by ZIKV in mouse brain cells [122]. Importantly, the dysregulation
of gene networks associated with autophagy, ER stress, and UPR are confirmed in a mouse model of infected brains
(Fig. 6.4A) [19,123]. These evidences strongly suggest neurotropism of ZIKV, and the demonstrated disruption of early
neurological development may provide some explanation for neural anomalies seen with CZS [41].
Subsequently, studies using more recent clinical isolates confirmed similar productive infection and death of NPCs
in 2D cultures as well as premature differentiation, aberrant expression of centrosomal proteins, and disrupted centroso-
mal structure [124]. Disruption of the centrosome and spindle positioning was also observed in HeLa cells after ZIKV
infection [125]. In neurospheres derived from hiPSCs, over 500 genes were differentially expressed following ZIKV
infection, many of which were enriched for previously identified gene ontology categories related to cell cycle, differ-
entiation, and cellular stress pathways (Fig. 6.4A and B). In addition, this study also revealed a significant involvement
of genes related to RNA processing, microRNA biogenesis, and ribosomal proteins [22,29].
The analysis of human NESCs, organotypic fetal brain slices, and ZIKV-infected microencephalic brain samples
revealed that ZIKV affected both neocortical and spinal NESCs as well as their fetal homolog, causing disrupted mito-
sis, supernumerary centrosomes, impaired mitotic spindle positioning, structural disorganization, and cell death.
Inhibition of neural migration in ZIKV-infected radial glial (RG) cells (important for the development of CNS) resulted
in ZIKV-associated microcephaly. RGs are playing important role in projecting radial fibers to the surface of the cortex
and these fibers guide migrating newborn neurons to their correct positions in the cortical plate. RGs also act as neural
stem cell pools, with the potential to differentiate into diverse neuronal and glial cell types, including neurons, oligoden-
drocytes, and astrocytes. ZIKV-induced translocation of the centrosomal protein phosphor-TBK1 to the mitochondria of
human NESCs and RG cells leads to disrupted mitosis, accumulation of chromosomal abnormalities and cell division
defects, resulting in impaired NPC proliferation and cell death (Fig. 6.4B) [82]. Examination of postmortem forebrain
of ZIKV-associated microcephaly revealed massive death of cells that correspond to NPCs [24,82,126]. ZIKV infection
of RGs might be facilitated by the expression of the putative ZIKV candidate receptor AXL in the cortex, bordering the
lateral ventricle and the outer subventricular zones (SVZs), a region where RGs are known to reside in humans [92].
Yoon et al. [127] demonstrated the involvement of ZIKV NS2A protein in reducing the proliferation of RGs and also
degrading the adherent junction (AJ) proteins in mouse cerebral cortex and human forebrain organoids. ZIKV-
associated loss of AJ can result in aberrant glial scaffold and misdirected neurons [23].
Perturbation of cell cycle progression may result from impaired dynamic gene expression during neurogenesis and
disrupted mitosis. Infection of human ESC-derived cerebral organoid cultures with ZIKV caused impaired development
and severe inhibition of growth of organoids by affecting the expression of around 41 genes responsible for neurogen-
esis, the differentiation of NPCs, and apoptosis via disrupting the activation of TLR3-mediated innate immune
responses. Identification of these putative genes will improve the understanding of ZIKV-induced microcephaly
[16,89,121,126]. There appears to be a relationship with ZIKV-related birth defects and gestational age, where babies
infected in earlier gestational ages were born with severe neurological abnormalities while the pregnant women infected
with ZIKV during the third trimester did not have babies with birth defects, and further studies are needed to elucidate
the underlying mechanism [18,62,128]. The development of neurospheres and brain organoids mark the early phases of
neurogenesis during the first trimester of fetal brain development. The ZIKV-infected hNPCs have been shown to gen-
erate very few numbers of neurospheres with reduced growth of brain organoids in comparison to their healthy counter-
parts, which resulted in microcephaly like symptoms, such as increased cell death, decline in cellular proliferation, and
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 99

neuronal cell volume, suggesting significant damage to the developing fetal brain during this time period. For the mech-
anism of human brain injury, ZIKV is thought to reduce the formation of brain matter by affecting autophagy pathway,
chromosomal stability, and centrosome segregation (Fig. 6.4A). Strong colocalization of ZIKV E protein in NPCs in 3D
brain organoids again indicates relatively specific tropism of ZIKV toward NPCs. These studies suggest that ZIKV
might impair neurogenesis during human brain development and induce severe brain damage when infection occurs
during the first trimester [22,24,25,37,78,118,129].
In addition to cell-autonomous effects on NPCs in the brain, non-cell-autonomous effects might also contribute to
ZIKV pathology. Apoptosis was observed in both infected and noninfected cortical cells taken from a 20-week-old fetus
[130]. Cell culture studies were the first to demonstrate that ZIKV induces apoptosis in a non-cell-autonomous way
through the release of cytotoxic factors, such as TNFα, IL-1β, and glutamate [131]. Other sources of non-cell-
autonomous effects and viral reservoirs that could exert long-term consequences are glial cells and microglia.
Conditioned media alone, taken from infected primary microglia isolated from newborn mice, can inhibit the prolifera-
tion of NPCs [132], and microglia infection led to the subsequent infection of NPCs when cocultured, resulting in
increased NPC death [22,133].

6.5.3.1.2 ZIKV-mediated autophagy and cell stress responses

Apoptosis and autophagy are two independent mechanisms of cell death and ZIKV infection can affect the expression
of genes involved in both processes [23]. Autophagy appears to play both proviral and antiviral roles in complex
host
virus interactions in a cell type-specific manner. For example, ZIKV nonstructural proteins NS4A and NS4B have
been shown to work together to suppress protein kinase B
mammalian target of rapamycin (AKT-mTOR) and autop-
hagy pathways to influence human fetal NPC function through autophagy and inhibiting neurogenesis (Fig. 6.4A)
[112]. Inhibition of AKT-mTOR also has a proviral role in ZIKV replication and vertical transmission [134]. However,
in human umbilical vein endothelial cells, ZIKV induces autophagy that serves to limit viral replication [135].
Consistent with this finding, KO of Atg16/1, an essential autophagy gene, improved placental and fetal outcomes in a
mouse model for ZIKV vertical transmission [22,62].
Like the context-dependent role of autophagy, ZIKV has a differential effect on stress granule formation depending
on the cellular state [136]. Stress granules are large aggregates of stalled translation preinitiation complexes. Under con-
ditions of oxidative stress, phosphorylation of eIF2a inhibits protein synthesis, inducing the formation of stress granules,
which restrict the bioavailability of intracellular machinery for translation. In response to virus entry, the formation of
stress granules reflects a defensive strategy within the host to reduce viral replication, and many viruses actively sup-
press the stress response and stress granule assembly. Some research groups found that ZIKV blocks stress granule for-
mation in a phospho-eIF2a-dependent manner [136], while others showed that ZIKV blocks both phosphorylation of
eIF2a and stress granule assembly, independent of suppressing host cell protein translation [137]. Another group
reported that ZIKV infection induced the phosphorylation of eIF2a and inhibition of translation in addition to the inhibi-
tion of stress granule formation [138]. There are several factors that may contribute to these apparently conflicting data,
including differences in the host cell type, ZIKV strain, and states of oxidative stress induced by different stressors
[22].
Being a membrane-associated virus, ZIKV utilizes host ER for its multiplication along the cellular secretory path-
way. ZIKV can trigger autophagy through those cellular membrane interactions in a cell-dependent manner. This cellu-
lar process is normally involved in the removal of aggregated or erroneously folded proteins through lysosomal
degradation. Activation of cellular autophagy is a hallmark of flavivirus infection, which was thought to be part of the
host innate immune response to eliminate invading intracellular pathogens [21]. Because autophagy activation could
halt cellular growth and trigger apoptosis, ZIKV-induced autophagy was implicated in the ZIKV-mediated microceph-
aly (covered in [21]). Activation of autophagy elicits antiviral activities by removing viral proteins through reticulo-
phagy, a selective form of autophagy that leads to ER degradation, or inclusion of viral proteins in autophagosomes
destined for lysosomal degradation [134]. The ER-localized reticulophagy receptor FAM134B serves as a host cell
restriction factor to ZIKV and other flaviviruses [139]. However, ZIKV-induced autophagy could be a double-edged
sword, which shows activities of both pro- and anti-ZIKV infection [134]. Activation of cellular autophagy counteracts
ZIKV infection by actively removing viral proteins. As part of the host cell’s antiviral responses, type I IFN signaling
also limits ZIKV replication by promoting autophagic destruction of the viral NS2B/NS3pro protease in a STAT1-
dependent manner [109]. Conversely, ZIKV takes advantage of autophagosome formation, whose presence was associ-
ated with enhanced viral replication [79]. ZIKV activates autophagy through the cellular mTOR stress pathway that
connects oxidative stress and reactive oxygen species production. This virus
host interaction appears to be highly
100 Pandemic Outbreaks in the 21st Century

conserved, as in human fetal neural stem cells, ZIKV triggers autophagy through inhibition of the mammalian mTOR
pathway via AKT (Fig. 6.4A) [112]. Altogether, ZIKV infection elicits RIG-1/MDA5- and TLR3-mediated innate
immune responses leading to releases of type I and type III IFNs to protect cells from viral invasion (Fig. 6.3A). ZIKV
concurrently triggers cellular activation of the stress TOR signaling pathway that induces autophagy. The balance
between pro- and anti-ZIKV activities of autophagy, at least in some cells, determines whether infected cells are pro-
tected through viral elimination, or destined to apoptosis as the result of viral propagation in host cells (Figs. 6.3A,B
and 6.4A) [21].
In addition to evading antiviral responses, ZIKV infection induces multiple cellular death pathways by mediating
changes in cell survival, death, and metabolism. Immunohistochemistry analysis of brain tissues showed significantly
higher expression of NLRP1, NLRP3, and AIM2, cytokines IL-1, IL-18, IL-33, and caspase 1 in cases of ZIKV-
induced microcephaly, highlighting IL-33 as one of the cytokines that exerts multiple actions in relation to necroptosis,
pyroptosis, and activation of inflammasome [140]. ZIKV-induced large cytoplasmic vacuoles from the ER and
paraptosis-like cell death have been demonstrated in various cell types including HeLa cells, primary human astrocytes,
and skin fibroblasts. Some of these changes are consistent with viral infection of cells and subcellular changes associ-
ated with congenital brain malformation (e.g., microcephaly) [31,104].
In recent years, several animal models such as chicken embryo, mouse, monkey, and hamsters were developed with
established in utero ZIKV infection to study the plausibility of casual relationships between ZIKV and neurological dis-
eases and to assess the mechanisms of congenital abnormalities, including microcephaly (reviewed in
[7,16,17,19,78,141]). ZIKV-induced intracellular structural disorganization may be related to the deregulation of genes
involved in microcephaly. Molecular genetic analyses identified mutations in at least 12 genes mapped to the micro-
cephaly (MCPH) loci. Most of these genes are expressed by NPCs where they play important roles in mitotic orienta-
tion and positioning and centrosomal integrity, and their mutant variants are associated with severe brain malformation,
including microcephaly (reviewed in [142]).
Gene expression studies of embryonic and fetal brain samples of ZIKV-infected mice and ZIKV-infected NPCs
revealed the downregulation of several MCPH genes, including those that code for centrosomal proteins, cell-cycle
arrest, apoptosis and inhibited proliferation. In addition, ZIKV infections resulted in increased caspase-3 (CASP3) activ-
ities in in vitro and in vivo models; in proliferative, immature (e.g., NPCs) and mature neurons; and in human NESCs.
The activation of CASP3 may be a result of ZIKV suppression of cellular immune response. Cells of the anterior SVZs
of the forebrain and subgranular zone of the hippocampal dentate gyrus of ZIKV-infected mice, deficient in IFN
response factor, showed elevated CASP3 activities [26] Moreover, ZIKV induced cell death by autophagy in hNPCs
generated from iPSCs, neurospheres raised from NPCs, and in vivo studies using mice [26
28,82,143,144]. ZIKV
invaded fetal brain results in cellular damage accompanied by caspase-3 activation and DNA fragmentation in NPCs,
resulting in a reduction of the cortical NPC pool and smaller brains with significant damage to brain structure.
Computational modeling analyses revealed a number of ZIKV miRNAs (microRNAs) that can target human MCPH
genes [145]. However, experimental evidence is needed to further elucidate the precise role(s) of ZIKV in the deregula-
tion of MCPH genes leading to microcephaly. Such investigations may involve the use of ZIKV-derived miRNAs to
inhibit the expression of specific MCPH gene and study the changes in embryonic, fetal, and neonatal brains in animal
models [17,23,26,27,143].
Inoculation of ZIKV into the uterine wall of immunocompromised pregnant mice revealed that ZIKV infection dur-
ing pregnancy caused several fetal abnormalities, which included microcephaly, IUGR, and enlarged ventricle
[12,120,146]. ZIKV infection was mimicked in mouse models, showing efficient Brazilian ZIKV infection and replica-
tion in NPCs located in the ventricular zones and SVZs of the fetal mouse cortex, leading to cerebral malformations in
the surviving fetuses such as cortical thinning, dysregulated autophagy resulting in apoptosis pathologies linked to fetal
microcephaly in humans [82,120,144]. Intraperitoneal injection of a contemporary ZIKV strain into the maternal mice
resulted in vertical transmission of the ZIKV from the pregnant mice to their fetuses. Pathology studies indicate that
immunocompetent murine models of ZIKV pathogenesis mimic some of the features of human neurological disease
such as damage to the placental barrier, placental insufficiency, IUGR, and fetal demise [54,89]. Furthermore, the effect
of fetal brain development due to vertical transmission of ZIKV was characterized using contemporary ZIKV strains in
mouse models to establish a link between ZIKV infection in neural cells and microcephaly. These studies indicate that
the ZIKV crossed the placenta and caused microcephaly by targeting cortical progenitor cells, causing cell death by
apoptosis and autophagy. Other features observed are impaired fetal neurodevelopment, IUGR, reduced cavity of lateral
ventricles, and a decrease in surface areas of the cerebral cortex (covered in [19,89]).
ZIKV also exhibits tropism for all principal brain cells, including postmitotic neuron and glial cells in mice. Glial
cells (astrocytes and oligodendrocytes) develop during the late stage of brain development and after birth, and they
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 101

represent at least half of the total brain cells (approximately 90% in specific parts of the human brain). Studies on preg-
nant mice inoculated with ZIKV resulted in infection of embryonic RG cells of the dorsal ventricular zone, reduction in
lateral ventricles cavity, placental infection, reduced number of dorsal and ventricular RG cells of brain, and viremia,
restricted intrauterine growth, stunted heads, and ocular anomalies. Transplacental route has been suggested to involve
viral spread to the chorionic villi, amnio chorionic membranes, and from the basal to the parietal decidua [8,19,147].
Kumar et al. [148] have shown dysregulation of retinoic acid-dependent genes that may affect the formation of the neu-
ral tube in developing brain cells, causing neurological malformations. In vivo, ZIKV infection at embryonic day 12.5
in IFN-deficient mice showed reduced numbers of NPCs and neurons as well as fewer blood vessels in the brain, retina,
and placenta at embryonic day 15.5 [149]. A study using both human postmortem tissue and mouse models showed that
ZIKV led to ER stress and an UPR that resulted in a net loss of neurons by affecting neurogenesis (Fig. 6.4B) [150].
Collectively, it appears that there are both direct and indirect mechanisms that contribute to overall restrictions on
neurogenesis.
A global gene expression analysis of ZIKV-infected mouse brains indicated that 63 genes involved in gliogenesis,
cell fate commitment, glial cell differentiation, and glial cell migration were significantly dysregulated [151]. Recent
study on ZIKV-mediated pathogenesis employing large-scale metabolomics revealed that the ZIKV reprograms placen-
tal lipidome by impairing the lipogenesis pathways. Furthermore, lipidome reprogramming by ZIKV is associated with
mitochondrial dysfunction and inflammatory immune imbalance, which contributes to placental damage [152]. This
indicates that ZIKV infection leads to an attenuated expansion of glial progenitors (i.e., OPCs) through virally induced
dysregulation of their proliferation and differentiation (Fig. 6.4B) [19].
To map pathology in the developing brain following in utero exposure to ZIKV, one study characterized CNS
lesions in stillborn or newborn babies who died within 48 h of birth [153]. In several animal models, postnatal ZIKV
infections led to various developmental complications, including sustained structural and functional changes in the brain
[154], delayed brain atrophy [155], and transient seizure activity [156]. One of the most critical outstanding questions
about ZIKV-mediated pathophysiology is how infections in the pregnant mother or developing child, which may go
undiagnosed, could lead to long-lasting effects on nervous system function later in life. More long-term animal studies
are required to answer these questions [22].
Although mouse models were instrumental in generating the early data needed to determine that ZIKV was causal for
microcephaly, primate models are often better choice to represent specific features of human physiology. A recent study
in nonhuman primates revealed that infection during early pregnancy results in microcalcifications and vascular abnor-
malities, in addition to cell death of NPCs [157]. Investigation of ZIKV-infected pregnant female rhesus monkeys using
placenta-specific magnetic resonance imaging revealed severely compromised oxygen delivery and a clear
maternal
placental
fetal inflammatory response, although no microcephaly was observed [158]. Similar to studies in
humans, pregnant rhesus macaques took longer to clear viremia than nonpregnant animals [159]. Supporting evidence for
sexual transmission was provided by a marmoset model showing that ZIKV was present in bodily fluids such as semen,
urine, and saliva, and ZIKV RNA was detectable for up to 2 weeks postinfection, despite viremia lasting only about 1
week and an absence of symptoms [160]. Recently, other models have been developed in piglets [161,162], which reca-
pitulate many features of neurodevelopmental pathology, including microcephaly, when infected in utero, and olive
baboons (Papio anubis) could be more amenable to in vivo monitoring of placental integrity and maternal
fetal immune
responses [163]. A chicken embryo model revealed dose-dependent ZIKV-induced developmental abnormalities that
included reduced brain volume and microcephaly-like pathology; this model has several advantages based on the exten-
sive literature of developmental biology and virology in this system ([164]; covered in [22]).

6.5.3.2 ZIKV-associated congenital ocular anomalies

In both the developing fetus and adults, ZIKV exhibits tropism for components of the visual system [165]. Another
study found retinopathy and the presence of viral RNA in retinal cells following in utero or early postnatal exposure to
ZIKV but minimal effects following adult exposure, which suggests a selective vulnerability before the retina
blood
barrier is fully established [166]. Examination of eye tissue from fetuses with CZS revealed the expression of NS2B in
the retina, choroid, and optic nerve as well as muscle derived from the neuroectoderm [167]. Müller cells, the principal
glial cells in the retina, were also found to be permissive to ZIKV and exhibited a pro-inflammatory response with the
activation of many pathways upon infection [168], which could be partly ameliorated by blocking p38 mitogen-
activated protein kinase activation. As with most neurological sequelae, ophthalmic complications appear to be more
severe during fetal development [22,169]. ZIKV targets retinal endothelial cells, retinal pericytes, and retinal pigmented
epithelial cells of the blood
retinal barrier, where it replicates productively and stimulates the expression of the
102 Pandemic Outbreaks in the 21st Century

inflammatory cytokine, RANTES, potentially disrupting the permeability of the retinal vasculature, and resulting in
chorioretinal atrophy [170,171]. These data, taken together, suggest that ZIKV may produce eye diseases, leading to
blindness in newborns arising from infected pregnancies [23].
Ocular anomalies were reported with congenital ZIKV infection in several clinical studies, specifically in the retina
and choroid of infants (covered in [23]). ZIKV invades optic nerve, retina, iris, and cornea, and trigger panuveitis, con-
junctivitis, and neuroretinitis in mice, and viral RNA was detected in the intraocular fluid [172]. The results from
human studies also reveal that ZIKV analogously infects the human ophthalmic tissues and trigger adverse eye illnesses
including optic neuritis, chorioretinal atrophy, and blindness in neonates, and conjunctivitis and uveitis in adults [172].
In addition, AXL expression has been observed in the outer margin of the neural retina and in cells of the ciliary mar-
ginal zone adjacent to neural retina, suggesting a possible underlying mechanism for macular atrophy (leading to blind-
ness) in babies born to ZIKV-infected mothers [23].

6.5.3.3 Hearing implications related to ZIKV infection

In utero transmission of ZIKV to the developing fetus can result in impaired hearing [173] and adults infected with
ZIKV have reported transient hearing loss [174,175]. Hearing impairment modeled in AG129 mice prenatally infected
with ZIKV shows conceded auditory thresholds without any detectable hair cell loss [176]. Yee et al. [177] examined
the effects of ZIKV infection on inner ear cellular integrity in mice and indicated the presence of ZIKV antigens in
mice blood and alteration in the expression of genes that encodes proteins for signal damage (S100B), transport fluids
(AQP1), gaseous transmitters (eNOs). The study also showed the compromised cochlear structures by the ZIKV infec-
tion and damage that occurs in vestibular end organs [177]. A recent study by Thawani et al. [178] addressed the
mechanisms of sensorineural hearing loss by delivering ZIKV directly into the otic cup/otocyst of chicken embryos,
and the infection of inner ear tissues was evaluated using immunohistochemistry. During the study, the detection of the
virus peaked earlier in ganglion and vestibular compartments and later in cochlea. ZIKV infection to the ganglial cells
increased the apoptosis proving that ZIKV infects the inner ear epithelium and showed unknown inner ear dysmorpho-
genesis phenotypes [178].

6.6 Summary
ZIKV infection and its causal association with neonatal microcephaly have gained importance only after the ZIKV pan-
demic in 2015. Since then, several researchers across the globe have significantly contributed to the understanding of
the ZIKV etiology and its associated human diseases through their unprecedented efforts. Recent in vitro and in vivo
studies firmly demonstrated that ZIKV is a potential teratogenic agent due to its ability to cross the placental
fetal bar-
rier, breach the fetal brain, and invade neural stem cells as the primary target population. Preferential disruption of neu-
ral stem cells by ZIKV results in neural cell death due to alterations in gene expression, autophagy, and apoptosis,
leading to impaired brain development followed by microcephaly and other serious congenital neurological complica-
tions. ZIKV infection could activate TLR3 signaling, which in turn perturbs neurogenesis and apoptotic pathways, thus
they both share common molecular signatures. However, it is not clear whether TLR3 activation is depending on ZIKV
strain, context, and cell type. Apart from that, inflammatory cytokines derived from ZIKV-induced placenta may also
contribute to the death of NPCs in uterus. ZIKV may bypass the strong IFN-mediated defense in placenta through a
variety of mechanisms. ZIKV is capable to access immune-privileged sites, such as the blood
brain barrier or mater-
nal
fetal placental barrier, indicating its tissue tropism. However, definitive proofs are still required to confirm the cau-
sality between the ZIKV infection during epidemics and malformations in fetal brains.
Despite the several studies on pathogenesis of ZIKV, the precise mechanisms by which the virus crosses the pla-
centa and infects the developing fetus remain inconclusive. It remains unclear about the host and viral factors contribut-
ing to ZIKV persistence in the placenta and other immune-privileged sites and the role of noncoding RNAs during
ZIKV infection. The hypothesis that indicates that the genetic changes in the currently circulating strains of ZIKV are
contributing to its altered virulence, pathogenesis, and cell tropism to induce fetal abnormalities needs to be tested.
Much more studies are to be done to determine the factors that impact ZIKV infection, in utero transmission, and neuro-
pathogenic mechanisms. ZIKV infection is shown to alter microRNA-associated genes, gene splicing isoform composi-
tion, DNA methylation, and long noncoding RNAs, and the relationship between these factors and the pathology
associated with ZIKV infection remain to be explored. Employment of combination of transcriptomic, proteomic, and
epigenomic techniques to analyze ZIKV-infected neurotropic cases at different developmental stages along with the elu-
cidation of involvement of long noncoding RNA and siRNA would contribute to determining the actual molecular
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 103

pathways underlying the induction of microcephaly and other neurological abnormalities. Although different mouse
models have reproduced most of the clinical features associated with ZIKV infection in human brains there are struc-
tural, functional, and immunological dissimilarities between human and mouse brains, indicating the requirement of a
nonhuman primate model to confirm the mechanisms of ZIKV-induced pathogenesis. As per the recent literature,
female rhesus macaques were found to be susceptible to ZIKV infection via efficient maternal
fetal ZIKV transmission
and they recapitulate many features of CZS in humans during early pregnancy, thus facilitating more relevant mechanis-
tic studies in the future. Further investigations, insights, and complete understanding of ZIKV
host cell interactions
may shed light on molecular mechanisms underlying the birth defects, including microcephaly and other neurological
anomalies, which in turn facilitate the design and development of diagnostic tools and safer, potent vaccines and thera-
peutic drugs for the treatment and management of ZIKV-associated diseases.

Acknowledgments
We gratefully acknowledge Dr. Zhiheng Xu, State Key Laboratory of Molecular Developmental Biology, CAS Center for Excellence in
Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China,
for providing the permission to use the content and figures of his publication.

References
[1] Dick GWA, Kitchen SF, Haddow AJ. Zika virus (I). Isolations and serological specificity. Trans R Soc Trop Med Hyg 1952;46(5):509
20.
Available from: https://doi.org/10.1016/0035-9203(52)90042-4.
[2] MacNamara FN. Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg
1954;48(2):139
45. Available from: https://doi.org/10.1016/0035-9203(54)90006-1.
[3] Musso D. Zika virus transmission from French Polynesia to Brazil. Emerg Infect Dis 2015;21(10):1887. Available from: https://doi.org/
10.3201/eid2110.151125.
[4] Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, et al. Zika virus, French polynesia, South pacific, 2013. Emerg Infect
Dis 2014;20(6):1085
6. Available from: https://doi.org/10.3201/eid2006.140138.
[5] Duffy MR, Chen TH, Hancock WI, Powers AM, Kool JL, Lanciotti RS, Pretrick M, et al. Zika virus outbreak on Yap island, Federated States
of Micronesia. N Engl J Med 2009;360(24):2536
43. Available from: https://doi.org/10.1056/NEJMoa0805715.
[6] Musso D, Gubler DJ. Zika virus. Clin Microbiol Rev 2016;29:487
524. Available from: https://doi.org/10.1128/CMR.00072-15.
[7] Pawitwar SS, Dhar S, Tiwari S, Ojha CR, Lapierre J, Martins K, et al. Overview on the current status of Zika virus pathogenesis and animal
related research. J Neuroimmune Pharmacol 2017;12(3):371
88. Available from: https://doi.org/10.1007/s11481-017-9743-8.
[8] Gorshkov K, Shiryaev SA, Fertel S, Lin Y-W, Huang C-T, Pinto A, et al. Zika virus: origins, pathological action, and treatment strategies.
Front Microbiol 2019;9:3252. Available from: https://doi.org/10.3389/fmicb.2018.03252.
[9] Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet 2017;390(10107):2099
109. Available from:
https://doi.org/10.1016/S0140-6736(17)31450-2.
[10] Kazmi SS, Ali W, Bibi N, Nouroz F. A review on Zika virus outbreak, epidemiology, transmission and infection dynamics. J Biol Res
(Thessalon) 2020;27:5. Available from: https://doi.org/10.1186/s40709-020-00115-4.
[11] Heymann DL, Hodgson A, Sall AA, Freedman DO, Staples JE, Althabe F, et al. Zika virus and microcephaly: why is this situation a PHEIC?
Lancet 2016;387(10020):719
21. Available from: https://doi.org/10.1016/S0140-6736(16)00320-2.
[12] Rather IA, Lone JB, Bajpai VK, Paek WK, Lim J. Zika virus: an emerging worldwide threat. Front Microbiol 2017;8:1417. Available from:
https://doi.org/10.3389/fmicb.2017.01417.
[13] Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an
epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis 2008;14(8):1232
9. Available from: https://doi.org/10.3201/eid1408.080287.
[14] Faye O, Freire CC, Iamarino A, Faye O, de Oliveira JV, Diallo M, et al. Molecular evolution of Zika virus during its emergence in the 20(th)
century. PLoS Negl Trop Dis 2014;8(1):e2636. Available from: https://doi.org/10.1371/journal.pntd.0002636.
[15] Jun SR, Wassenaar TM, Wanchai V, Patumcharoenpol P, Nookaew I, Ussery DW. Suggested mechanisms for Zika virus causing microcephaly:
what do the genomes tell us. BMC Bioinforma 2017;18(Suppl 14):471. Available from: https://doi.org/10.1186/s12859-017-1894-3.
[16] Faizan MI, Abdullah M, Ali S, Naqvi IH, Ahmed A, Parveen S. Zika virus-induced microcephaly and its possible molecular mechanism.
Intervirology 2016;59(3):152
8. Available from: https://doi.org/10.1159/000452950.
[17] Zhou K, Wang L, Yu D, Huang H, Ji H, Mo X. Molecular and cellular insights into Zika virus-related neuropathies. J Neurovirol 2017;23
(3):341
6. Available from: https://doi.org/10.1007/s13365-017-0514-3.
[18] Mittal R, Nguyen D, Debs LH, et al. Zika virus: an emerging global health threat. Front Cell Infect Microbiol 2017;7:486. Available from:
https://doi.org/10.3389/fcimb.2017.00486.
[19] Xu D, Li C, Qin CF, Xu Z. Update on the animal models and underlying mechanisms for ZIKV-induced microcephaly. Annu Rev Virol 2019;6
(1):459
79. Available from: https://doi.org/10.1146/annurev-virology-092818-015740.
104 Pandemic Outbreaks in the 21st Century

[20] Lee CY, Ng LFP. Zika virus: from an obscurity to a priority. Microbes Infect 2018;20(11
12):635
45. Available from: https://doi.org/
10.1016/j.micinf.2018.02.009.
[21] Lee I, Bos S, Li G, Wang S, Gadea G, Desprès P, et al. Probing molecular insights into Zika virus hostinteractions. Viruses 2018;10(5):233.
Available from: https://doi.org/10.3390/v10050233.
[22] Christian KM, Song H, Ming GL. Pathophysiology and mechanisms of Zika virus infection in the nervous system. Annu Rev Neurosci 2019;8
(42):249
69. Available from: https://doi.org/10.1146/annurev-neuro-080317-062231.
[23] Gharbaran R, Somenarain L. Putative cellular and molecular roles of Zika virus in fetal and pediatricneuropathologies. Pediatr Dev Pathol
2019;22(1):5
21. Available from: https://doi.org/10.1177/1093526618790742.
[24] Wen Z, Song H, Ming GL. How does Zika virus cause microcephaly? Genes Dev 2017;31(9):849
61. Available from: https://doi.org/10.1101/
gad.298216.117.
[25] Garcez PP, Loiola EC, da Costa RM, Higa LM, Trindade P, Delvecchio R, et al. Zika virus impairs growth in human neurospheres and brain
organoids. Science 2016;352(6287):816
18. Available from: https://doi.org/10.1126/science.aaf6116.
[26] Li H, Saucedo-Cuevas L, Regla-Nava JA, Chai G, Sheets N, Tang W, et al. Zika virus infects neural progenitors in the adult mouse brain and
alters proliferation. Cell Stem Cell 2016;19(5):593
8. Available from: https://doi.org/10.1016/j.stem.2016.08.005.
[27] Wu KY, Zuo GL, Li XF, Ye Q, Deng YQ, Huang XY, et al. Vertical transmission of Zika virus targeting the radial glial cells affects cortex
development of offspring mice. Cell Res 2016;26(6):645
54. Available from: https://doi.org/10.1038/cr.2016.58.
[28] Tang H, Hammack C, Ogden SC, Wen Z, Qian X, Li Y, et al. Zika virus infects human cortical neural progenitors and attenuates their growth.
Cell Stem Cell 2016;18(5):587
90. Available from: https://doi.org/10.1016/j.stem.2016.02.016.
[29] Garcez PP, Nascimento JM, de Vasconcelos JM, Madeiro da Costa R, Delvecchio R, et al. Zika virus disrupts molecular fingerprinting of
human neurospheres. Sci Rep 2017;7:40780. Available from: https://doi.org/10.1038/srep40780.
[30] Simmonds P, Becher P, Bukh J, Gould EA, Meyersm G, Monath T, et al. ICTV virus taxonomy profile: Flaviviridae. J Gen Virol 2017;98
(1):2
3. Available from: https://doi.org/10.1099/jgv.0.000672.
[31] Lee JK, Shin OS. Advances in Zika virus hostcell interaction: current knowledge and future perspectives. Int J Mol Sci 2019;20(5):1101.
Available from: https://doi.org/10.3390/ijms20051101.
[32] Dong H, Fink K, Züst R, Lim SP, Qin CF, Shi PY. Flavivirus RNA methylation. J Gen Virol 2014;95(Pt 4):763
78. Available from: https://
doi.org/10.1099/vir.0.062208-0.
[33] Cox BD, Stanton RA, Schinazi RF. Predicting Zika virus structural biology: challenges and opportunities for intervention. Antivir Chem
Chemother 2015;24(3
4):118
26. Available from: https://doi.org/10.1177/2040206616653873.
[34] Dai L, Song J, Lu X, Deng YQ, Musyoki AM, Cheng H, et al. Structures of the Zika virus envelope protein and its complex with a Flavivirus
broadly protective antibody. Cell Host Microbe 2016;19(5):696
704. Available from: https://doi.org/10.1016/j.chom.2016.04.013.
[35] Agrelli A, de Moura RR, Crovella S, Brandão LAC. Zika virus entry mechanisms in human cells. Infect Genet Evol 2019;69:22
9. Available
from: https://doi.org/10.1016/j.meegid.2019.01.018.
[36] Cortese M, Goellner S, Acosta EG, Neufeldt CJ, Oleksiuk O, Lampe M, et al. Ultrastructural characterization of Zika virus replication factories.
Cell Rep 2017;18(9):2113
23. Available from: https://doi.org/10.1016/j.celrep.2017.02.014.
[37] Sharma V, Sharma M, Dhull D, Sharma Y, Kaushik S, Kaushik S. Zika virus: an emerging challenge to public health worldwide. Can J
Microbiol 2020;66:87
98. Available from: https://doi.org/10.1139/cjm-2019-0331.
[38] de Paula Freitas B, de Oliveira Dias JR, Prazeres J, Sacramento GA, Ko AI, Maia M, et al. Ocular findings in infants with microcephaly associ-
ated with presumed Zika virus congenital infection in Salvador, Brazil. JAMA Ophthalmol 2016;134(5):529
35. Available from: https://doi.
org/10.1001/jamaophthalmol.2016.0267.
[39] Goertz GP, Abbo SR, Fros JJ, Pijlman GP. Functional RNA during Zika virus infection. Virus Res 2017;254:41
53. Available from: https://
doi.org/10.1016/j.virusres.2017.08.015.
[40] Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika virus. N Engl J Med 2016;374(16):1552
63. Available from: https://doi.org/
10.1056/NEJMra1602113.
[41] Vasudevan J, Skandhan A, Skandhan AKP, Balakrishnan S, Skandhan KP. Zika virus. Rev Med Microbiol 2018;29(2):43
50. Available from:
https://doi.org/10.1097/MRM.0000000000000126.
[42] Ciota AT, Bialosuknia SM, Zink SD, Brecher M, Ehrbar DJ, Morrissette MN, et al. Effects of Zika virus strain and Aedes mosquito species on
vector competence. Emerg Infect Dis 2017;23(7):1110
17. Available from: https://doi.org/10.3201/eid2307.161633.
[43] Al-Qahtani AA, Nazir N, Al-Anazi MR, Rubino S, Al-Ahdal MN. Zika virus: a new pandemic threat. J Infect Dev Ctries 2016;10(3):201
7.
Available from: https://doi.org/10.3855/jidc.8350.
[44] Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis 2015;21
(2):359
61. Available from: https://doi.org/10.3201/eid2102.141363.
[45] Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jaaskelainen AJ, Smura T, Rosenberg A, et al. Zika virus infection with prolonged maternal
viremia and fetal brain abnormalities. N Engl J Med 2016;374:2142
51. Available from: https://doi.org/10.1056/NEJMoa1601824.
[46] Zanluca C, de Noronha L, Duarte dos Santos CN. Maternal-fetal transmission of the zika virus: an intriguing interplay. Tissue Barriers 2018;6
(1):e14. Available from: https://doi.org/10.1080/21688370.2017.1402143.
[47] Antoniou E, Orovou E, Sarella A, Iliadou M, Rigas N, Palaska E, et al. Zika virus and the risk of developing microcephaly in infants: a system-
atic review. Int J Environ Res Public Health 2020;17(11):3806. Available from: https://doi.org/10.3390/ijerph17113806.
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 105

[48] Russell K, Hills SL, Oster AM, Porse CC, Danyluk G, Cone M, et al. Male-to-female sexual transmission of Zika virus—United States,
January
April 2016. Clin Infect Dis 2017;64(2):211
13. Available from: https://doi.org/10.1093/cid/ciw692.
[49] Hills SL, Russell K, Hennessey M, Williams C, Oster AM, Fischer M, et al. Transmission of Zika Virus through sexual contact with travelers
to areas of ongoing transmission - continental United States, 2016. MMWR Morb Mortal Wkly Rep 2016;65(8):215
16. Available from:
https://doi.org/10.15585/mmwr.mm6508e2.
[50] Medina FA, Torres G, Acevedo J, Fonseca S, Casiano L, Leon-Rodriguez CM, et al. Duration of the presence of infectious Zika virus in semen
and serum. J Infect Dis 2018;219:31
40. Available from: https://doi.org/10.1093/infdis/jiy462.
[51] Atkinson B, Hearn P, Afrough B, Lumley S, Carter D, Aarons EJ, et al. Detection of zika virus in semen. Emerg Infect Dis 2016;22(5):940.
Available from: https://doi.org/10.3201/eid2205.160107.
[52] Baud D, Musso D, Vouga M, Alves MP, Vulliemoz N. Zika virus: a new threat to human reproduction. Am J Reprod Immunol 2016;77(2).
Available from: https://doi.org/10.1111/aji.12614.
[53] Besnard M, Lastere S, Teissier A, Cao-Lormeasu V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December
2013 and February 2014. Euro Surveill 2014;19:20751.
[54] Miner JJ, Cao B, Govero J, Smith AM, Fernandez E, Cabrera OH, et al. Zika virus infection during pregnancy in mice causes placental damage
and fetal demise. Cell 2016;165(5):1081
91. Available from: https://doi.org/10.1016/j.cell.2016.05.008.
[55] Bayer A, Lennemann NJ, Ouyang Y, Bramley JC, Morosky S, et al. Type III interferons produced by human placental trophoblasts confer pro-
tection against Zika virus infection. Cell Host Microbe 2016;19(5):705
12. Available from: https://doi.org/10.1016/j.chom.2016.03.008.
[56] Weisblum Y, Oiknine-Djian E, Vorontsov OM, Haimov-Kochman R, et al. Zika virus infects early- and midgestationhuman maternal decidual
tissues, inducing distinct innate tissue responses in the maternal-fetalinterface. J Virol 2017;91(4):e01905
16. Available from: https://doi.org/
10.1128/JVI.01905-16.
[57] Chiu CF, Chu LW, Liao I, Simanjuntak Y, Lin YL, Juan CC, et al. The mechanism of the Zika virus crossing the placental barrier and the
blood-brain barrier. Front Microbiol 2020;11:214. Available from: https://doi.org/10.3389/fmicb.2020.00214.
[58] Colt S, Garcia-Casal MN, Peña-Rosas JP, Finkelstein JL, Rayco-Solon P, Weise Prinzo ZC, et al. Transmission of Zika virus through breast
milk and other breastfeeding-related bodily-fluids: a systematic review. PLoS Negl Trop Dis 2017;11(4):e0005528. Available from: https://doi.
org/10.1371/journal.pntd.0005528.
[59] Quicke KM, Bowen JR, Johnson EL, McDonald CE, Ma H, O’Neal JT, et al. Zika virus infects human placental macrophages. Cell Host
Microbe 2016;20(1):83
90. Available from: https://doi.org/10.1016/j.chom.2016.05.015.
[60] Oliveira Melo AS, Malinger G, Ximenes R, Szejnfeld PO, Alves Sampaio S, Bispo de Filippis AM. Zika virus intrauterine infection causes fetal
brain abnormality and microcephaly: tip of the iceberg? Ultrasound Obstet Gynecol 2016;47(1):6
7. Available from: https://doi.org/10.1002/
uog.15831.
[61] Calvet G, Aguiar RS, Melo AS, Sampaio SA, De Filippis I, Fabri A, et al. Detection and sequencing of Zika virus from amniotic fluid of fetuses
with microcephaly in Brazil: a case study. Lancet Infect Dis 2016;16(6):653
60. Available from: https://doi.org/10.1016/S1473-3099(16)
00095-5.
[62] Cao B, Diamond MS, Mysorekar IU. Maternal-fetal transmission of Zika virus: routes and signals for infection. J Interferon Cytokine Res
2017;37(7):287
94. Available from: https://doi.org/10.1089/jir.2017.0011.
[63] Leung GH, Baird RW, Druce J, Anstey NM. Zika virus infection in Australia following a monkey bite in Indonesia. Southeast Asain J Trop
Med Public Health 2015;46(3):460
4.
[64] WHO Report. ZIKV epidemiology update World Health Organization?; 2019. Available from https://www.who.int/emergencies/diseases/zika/
zika-epidemiology-update-july-2019.pdf.
[65] Broutet N, Krauer F, Riesen M, Khalakdina A, Almiron M, Aldighieri S, et al. Zika virus as a cause of neurologic disorders. N Engl J Med
2016;374(16):1506
9. Available from: https://doi.org/10.1056/NEJMp1602708.
[66] Kleber de Oliveira W, Araujo de franca GV, Carmo EH, Duncan BB, Kuchenbecker RDS, Schmidt MI. Infection-related microcephaly after the
2015 and 2016 Zika virus outbreaks in Brazil: a surveillance-based analysis. Lancet 2017;390:861
70. Available from: https://doi.org/10.1016/
S0140-6736(17)31368-5.
[67] Anderson J. Zika virus. Infect Dis Clin Pract 2017;25(6):289
93. Available from: https://doi.org/10.1097/IPC.0000000000000546.
[68] Zorrilla CD, Garcı́a Garcı́a I, Garcı́a Fragoso L, De La Vega A. Zika virus infection in pregnancy: maternal, fetal, and neonatal considerations.
J Infect Dis 2017;216(suppl_10):S891
6. Available from: https://doi.org/10.1093/infdis/jix448.
[69] Cao-Lormeau VM, Blake A, Mons S, Lastère S, Roche C, Vanhomwegen J, et al. Guillain-Barré syndrome outbreak associated with Zika virus
infection in French Polynesia: a case-control study. Lancet 2016;387(10027):1531
9. Available from: https://doi.org/10.1016/S0140-6736(16)
00562-6.
[70] Walker CL, Merriam AA, Ohuma EO, Dighe MK, Gale Jr M, Rajagopal L, et al. Femur-sparing pattern of abnormal fetal growth in pregnant
women from New York City after maternal Zika virus infection. Am J Obstet Gynecol 2018;219(2) 187-e1.
[71] Sarno M, Sacramento GA, Khouri R, do Rosário MS, Costa F, Archanjo G, et al. Zika virus infection and stillbirths: a case of hydrops fetalis,
hydranencephaly and fetal demise. PLoS Negl Trop Dis 2016;10(2):e0004517.
[72] Schwartz KL, Chan T, Rai N, Murphy KE, Whittle W, Drebot MA, et al. Zika virus infection in a pregnant Canadian traveler with congenital
fetal malformations noted by ultrasonography at 14-weeks gestation. Trop Dis Travel Med Vaccines 2018;4(1):2. Available from: https://doi.
org/10.1186/s40794-018-0062-8.
106 Pandemic Outbreaks in the 21st Century

[73] Ventura CV, Maia M, Bravo-Filho V, Gois AL, Belfrot Jr. R. Zika virus in Brazil and macular atrophy in a child with microcephaly. Lancet
2016;387:228. Available from: https://doi.org/10.1016/S0140-6736(16)00006-4.
[74] Verçosa I, Carneiro P, Verçosa R, Girão R, Ribeiro EM, Pessoa A, Almeida NG, et al. Transient hearing loss in adults associated with Zika
virus infection. Clin Infect Dis 2017;64(5):675
7. Available from: https://doi.org/10.1093/cid/ciw770.
[75] Uncini A, Shahrizaila N, Kuwabara S. Zika virus infection and Guillain-Barré syndrome: a review focused on clinical and electrophysiological
subtypes. J Neurol Neurosurg Psychiatry 2017;88:266
71. Available from: https://doi.org/10.1136/jnnp-2016-314310.
[76] Dirlikov E, Medina NA, Major CG, Munoz-Jordan JL, Luciano CA, Rivera-Garcia B, et al. Acute zika virus infection as a risk factor for
Guillain
Barre syndrome in Puerto Rico. JAMA 2017;318(15):1498
500. Available from: https://doi.org/10.1016/j.chom.2016.04.013.
[77] Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, et al. Zika virus associated with microcephaly. N Engl J Med 2016;374
(10):951
8. Available from: https://doi.org/10.1056/NEJMoa1600651.
[78] Siddique R, Liu Y, Nabi G, Sajjad W, Xue M, Khan S. Zika virus potentiates the development of neurological defects and microcephaly: chal-
lenges and control strategies. Front Neurol 2019;10:319. Available from: https://doi.org/10.3389/fneur.2019.00319.
[79] Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A, Luplertlop N, et al. Biology of Zika virus infection in human skin cells. J Virol
2015;89(17):8880
96. Available from: https://doi.org/10.1128/JVI.00354-15.
[80] Kim JA, Seong RK, Son SW, Shin OS. Insights into ZIKV-mediated innate immune responses in human dermal fibroblasts and epidermal ker-
atinocytes. J Invest Dermatol 2018;139(2):391
9. Available from: https://doi.org/10.1016/j.jid.2018.07.038.
[81] Aagaard KM, Lahon A, Suter MA, Arya RP, Seferovic MD, et al. Primary human placental trophoblasts are permissive for Zika virus (ZIKV)
replication. Sci Rep 2017;7(1):1
14. Available from: https://doi.org/10.1038/srep41389.
[82] Onorati M, Li Z, Liu F, Sousa AM, Nakagawa N, Li M, et al. Zika virus disrupts phospho-TBK1 localization and mitosis inhuman neuroe-
pithelial stem cells and radial glia. Cell Rep 2016;16:2576
92. Available from: https://doi.org/10.1016/j.celrep.2016.08.038.
[83] Bayless NL, Greenberg RS, Swigut T, Wysocka J, Blish CA. Zika virus infection induces cranial neural crest cells to producecytokines at
levels detrimental for neurogenesis. Cell Host Microbe 2016;20:423
8. Available from: https://doi.org/10.1016/j.chom.2016.09.006.
[84] Pagani I, Ghezzi S, Ulisse A, Rubio A, Turrini F, Garavaglia E, et al. Human endometrial stromal cells are highly permissive to productive
infection by Zika virus. Sci Rep 2017;7:44286. Available from: https://doi.org/10.1038/srep44286.
[85] Noronha LD, Zanluca C, Azevedo ML, Luz KG, Santos CN. Zika virus damages the human placental barrier and presents marked fetal neuro-
tropism. Mem Inst Oswaldo Cruz 2016;111(5):287
93. Available from: https://doi.org/10.1590/0074-02760160085.
[86] Retallack H, Lullo ED, Arias C, Knopp KA, Laurie MT, Sandoval-Espinosa C, et al. Zika virus in the developing human brain. Proc Natl
Acad Sci U S A 2016;113(50):14408
13. Available from: https://doi.org/10.1073/pnas.1618029113.
[87] Mead PS, Duggal NK, Hook SA, Delorey M, Fischer M, Olzenak McGuire D, et al. Zika virus shedding in semen of symptomatic infected
men. N Engl J Med 2018;378(15):1377
85. Available from: https://doi.org/10.1056/NEJMoa1711038.
[88] Merfeld E, Ben-Avi L, Kennon M, Cerveny KL. Potential mechanisms of Zika-linked microcephaly. Wiley Interdiscip Rev Dev Biol 2017;6
(4):e273. Available from: https://doi.org/10.1002/wdev.273.
[89] Olagnier D, Muscolini M, Coyne CB, Diamond MS, Hiscott J. Mechanisms of Zika virus infection and neuropathogenesis. DNA Cell Biol
2016;35:367
72. Available from: https://doi.org/10.1089/dna.2016.3404.
[90] Persaud M, Martinez-Lopez A, Buffone C, Porcelli SA, Diaz-Griffero F. Infection by Zika viruses requires the transmembrane protein AXL,
endocytosis and low pH. Virology 2018;518:301
12. Available from: https://doi.org/10.1016/j.virol.2018.03.009.
[91] Meertens L, Labeau A, Dejarnac O, Cipriani S, Sinigaglia L, Bonnet-Madin L, et al. Axl mediates ZIKA virus entry in human glial cells and
modulates innate immune responses. Cell Rep 2017;18(2):324
33. Available from: https://doi.org/10.1016/j.celrep.2016.12.045.
[92] Nowakowski TJ, Pollen AA, Di Lullo E, Sandoval-Espinosa C, Bershteynm M, Kriegstein AR. Expression analysis highlights AXL as acandi-
date Zika virus entry receptor in neuralstem cells. Cell Stem Cell 2016;18:591
6. Available from: https://doi.org/10.1016/j.stem.2016.03.012.
[93] Wells MF, Salick MR, Wiskow O, et al. Genetic ablation of AXL does not protect human neural progenitor cells and cerebral organoids from
Zika virus infection. Cell Stem Cell 2016;19(6):703
8. Available from: https://doi.org/10.1016/j.stem.2016.11.011.
[94] Chen J, Yang YF, Yang Y, Zou P, Chen J, He Y, et al. AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon sig-
nalling. Nat Microbiol 2018;3(3):302
9. Available from: https://doi.org/10.1038/s41564-017-0092-4.
[95] Srivastava M, Zhang Y, Chen J, Sirohi D, Miller A, Zhang Y, et al. Chemical proteomics tracks virus entry and uncovers NCAM1 as Zika
virus receptor. Nat Commun 2020;11(1):1
10. Available from: https://doi.org/10.1038/s41467-020-17638-y.
[96] Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-Spaeth G, Duangchinda T, et al. Dengue virus sero-cross-reactivity drives
antibody-dependent enhancement of infection with zika virus. Nat Immunol 2016;17(9):1102
8. Available from: https://doi.org/10.1038/
ni.3515.
[97] Aliota MT, Dudley DM, Newman CM, Mohr EL, Connor DH, et al. Heterologous protection against Asian zika virus challenge in Rhesus
Macaques. PLoS Negl Trop Dis 2016;10(12):e0005168. Available from: https://doi.org/10.1371/journal.pntd.0005168.
[98] Bowen JR, Quicke KM, Maddur MS, O’Neal JT, McDonald CE, et al. Zika virus antagonizes type I interferon responses during infection of
human dendritic cells. PLoS Pathog 2017;13(2):e1006164. Available from: https://doi.org/10.1371/journal.ppat.1006164.
[99] Frumence E, Roche M, Krejbich-Trotot P, El-Kalamouni C, Nativel B, Rondeau P, et al. The south pacific epidemic strain of Zika virus repli-
cates efficiently in human epithelial A549 cells leading to IFN-β production and apoptosis induction. Virology 2016;493:217
26. Available
from: https://doi.org/10.1016/j.virol.2016.03.006.
[100] Hamel R, Liegeois F, Wichit S, Pompon J, et al. Zika virus: epidemiology, clinical features and host-virus interactions. Microbes Infect
2016;18(7
8):441
9. Available from: https://doi.org/10.1016/j.micinf.2016.03.009.
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 107

[101] Mor G. Placental inflammatory response to Zika virus may affect fetal brain development. Am J Reprod Immunol 2016;75:421
2. Available
from: https://doi.org/10.1111/aji.12505.
[102] Serman TM, Gack MU. Evasion of innate and intrinsic antiviral pathways by the Zika virus. Viruses 2019;11(10):970. Available from: https://
doi.org/10.3390/v11100970.
[103] Savidis G, Perreira JM, Portmann JM, Meraner P, Guo Z, Green S, et al. The IFITMs inhibit Zika virus replication. Cell Rep 2016;15
(11):2323
30. Available from: https://doi.org/10.1016/j.celrep.2016.05.074.
[104] Monel B, Compton AA, Bruel T, Amraoui S, Burlaud-Gaillard J, Roy N, et al. Zika virus induces massive cytoplasmic vacuolization and para-
ptosis-like death in infected cells. EMBO J 2017;36(12):1653
68. Available from: https://doi.org/10.15252/embj.201695597.
[105] Kumar A, Hou S, Airo AM, Limonta D, Mancinelli V, Branton W, et al. Zika virus inhibits type-I interferon production and downstream sig-
naling. EMBO Rep 2016;17(12):1766
75. Available from: https://doi.org/10.15252/embr.201642627.
[106] Xia H, Luo H, Shan C, Muruato AE, Nunes BTD, Medeiros DBA, et al. An evolutionary NS1 mutation enhances Zika virus evasion of host
interferon induction. Nat Commun 2018;9(1):414. Available from: https://doi.org/10.1038/s41467-017-02816-2.
[107] Chaudhary V, Yuen KS, Chan JF, Chan CP, Wang PH, Cai JP, et al. Selective activation of type II interferon signaling by Zika virus NS5 pro-
tein. J Virol 2017;91(14). Available from: https://doi.org/10.1128/JVI.00163-17 e00163-17.
[108] Muñoz-Jordán JL, Laurent-Rolle M, Ashour J, Martı́nez-Sobrido L, Ashok M, Lipkin WI, et al. Inhibition of alpha/beta interferon signaling by
the NS4B protein of flaviviruses. J Virol 2005;79(13):8004
13. Available from: https://doi.org/10.1128/JVI.79.13.8004-8013.2005.
[109] Wu Y, Liu Q, Zhou J, Xie W, Chen C, Wang Z, et al. Zika virus evades interferon-mediated antiviral response through the co-operation of
multiple nonstructural proteins in vitro. Cell Discov 2017;3(1):1
14. Available from: https://doi.org/10.1038/celldisc.2017.6.
[110] Grant A, Ponia SS, Tripathi S, Balasubramaniam V, Miorin L, Sourisseau M, et al. Zika virus targets human STAT2 to inhibit type I interferon
signaling. Cell Host Microbe 2016;19(6):882
90. Available from: https://doi.org/10.1016/j.chom.2016.05.009.
[111] Ma J, Ketkar H, Geng T, Lo E, Wang L, Xi J, et al. Zika virus non-structural protein 4A blocks the RLR-MAVS signaling. Front Microbiol
2018;9:1350. Available from: https://doi.org/10.3389/fmicb.2018.01350.
[112] Liang Q, Luo Z, Zeng J, Chen W, Foo SS, Lee SA, et al. Zika virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in human fetal-
neural stem cells to inhibit neurogenesis and induce autophagy. Cell Stem Cell 2016;19(5):663
71. Available from: https://doi.org/10.1016/j.
stem.2016.07.019.
[113] Sirohi D, Chen Z, Sun L, Klose T, Pierson TC, Rossmann MG, et al. The 3.8 Å resolution cryo-EM structure of Zika virus. Science
2016;352:467
70. Available from: https://doi.org/10.1126/science.aaf5316.
[114] Akiyama BM, Laurence HM, Massey AR, Costantino DA, Xie X, Yang Y, et al. Zika virusproduces noncoding RNAs using a multi-
pseudoknot structurethat confounds a cellular exonuclease. Science 2016;354:1148
52. Available from: https://doi.org/10.1126/science.
aah3963.
[115] Adibi JJ, Marques Jr. ETA, Cartus A, Beigi RH. Teratogenic effects of the Zika virus and the role of the placenta. Lancet (London, Engl)
2016;387(10027):1587
90. Available from: https://doi.org/10.1016/s0140-6736(16)00650-4.
[116] Bhatnagar J, Rabeneck DB, Martines RB, Reagan-Steiner S, Ermias Y, et al. Zika virus RNA replication and persistence in brain and placental
tissue. Emerg Infect Dis 2017;23(3):405
14. Available from: https://doi.org/10.3201/eid2303.161499.
[117] Zhang F, Hammack C, Ogden SC, Cheng Y, Lee EM, Wen Z, et al. Molecular signatures associated with ZIKV exposure in human cortical
neural progenitors. Nucleic Acids Res 2016;44(18):8610
20. Available from: https://doi.org/10.1093/nar/gkw765.
[118] Li S, Shi Y, Zheng K, Dai J, Li X, Yuan S, et al. Morphologic and molecular characterization of a strain of Zika virus imported into
Guangdong, China. PLoS One 2017;12(1):e0169256. Available from: https://doi.org/10.1371/journal.pone.0169256.
[119] Ghouzzi VE, Bianchi FT, Molineris I, Mounce BC, Berto GE, Rak M, et al. ZIKA virus elicits P53 activation and genotoxic stress in human
neural progenitors similar to mutations involved in severe forms of genetic microcephaly. Cell Death Dis 2017;7(10):e2440. Available from:
https://doi.org/10.1038/cddis.2016.266.
[120] Shao Q, Herrlinger S, Yang SL, Lai F, Moore JM, Brindley MA, et al. Zika virus infection disrupts neurovascular development and results in
postnatal microcephaly with brain damage. Development 2016;143(22):4127
36. Available from: https://doi.org/10.1242/dev.143768.
[121] Dang J, Tiwari SK, Lichinchi G, Qin Y, Patil VS, Eroshkin AM, et al. Zika virus depletes neural progenitors in human cerebral organoids
through activation of the innate immune receptor TLR3. Cell Stem Cell 2016;19(2):258
65. Available from: https://doi.org/10.1016/j.
stem.2016.04.014.
[122] Tan Z, Zhang W, Sun J, Fu Z, Ke X, Zheng C, et al. ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein
response in neural cells. J Neuroinflammation 2018;15(1):275. Available from: https://doi.org/10.1186/s12974-018-1311-5.
[123] Chang YF, Jiang YS, Li C, Wang Q, Zhang F, et al. Dissecting of the gene networks affected by Zika virus infection in a mouse microcephaly
model. Genom Proteom Bioinform 2019; (in press).
[124] Gabriel E, Ramani A, Karow U, Gottardo M, Natarajan K, Gooi LM, et al. Recent Zika virus isolates induce premature differentiation of neu-
ral progenitors in human brain organoids. Cell Stem Cell 2017;20(3):397
406. Available from: https://doi.org/10.1016/j.stem.2016.12.005 e5.
[125] Wolf B, Diop F, Ferraris P, Wichit S, Busso C, Missé D, et al. Zika virus causes supernumerary foci with centriolar proteins and impaired
spindle positioning. Open Biol 2017;7(1):160231. Available from: https://doi.org/10.1098/rsob.160231.
[126] Souza BSF, Sampaio GLA, Campos GS, Sardi SI, Freitas LAR, Figueira CP, et al. Zika virus infection induces mitosis abnormalities and apo-
ptotic cell death of human neural progenitor cells. Sci Rep 2016;6:39775. Available from: https://doi.org/10.1038/srep39775.
[127] Yoon KJ, Song G, Qian X, Pan J, Xu D, Rho HS, et al. Zika-virus-encoded NS2A disrupts mammalian cortical neurogenesis by degrading
adherensjunction proteins. Cell Stem Cell 2017;21(3):349
58. Available from: https://doi.org/10.1016/j.stem.2017.07.014 e6.
108 Pandemic Outbreaks in the 21st Century

[128] Kleber de Oliveira W, Cortez-Escalante J, De Oliveira WT, et al. Increase in reported prevalence of microcephaly in infants born to women
living in areas with confirmed Zika virus transmission during the first trimester of pregnancy—Brazil, 2015. MMWR Morb Mortal Wkly Rep
2016;65:242
7. Available from: https://doi.org/10.15585/mmwr.mm6509e2.
[129] Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, Yao B, et al. Brain-region-specific organoids using mini-bioreactors for
modeling ZIKV exposure. Cell 2016;165(5):1238
54. Available from: https://doi.org/10.1016/j.cell.2016.04.032.
[130] Ho CY, Ames HM, Tipton A, Vezina G, Liu JS, Scafidi J, et al. Differential neuronal susceptibility and apoptosis in congenital Zika virus
infection. Ann Neurol 2017;82(1):121
7. Available from: https://doi.org/10.1002/ana.24968.
[131] Olmo IG, Carvalho TG, Costa VV, Alves-Silva J, Ferrari CZ, Izidoro-Toledo TC, et al. Zika virus promotes neuronal cell death in a non-cell
autonomous manner by triggering the release of neurotoxic factors. Front Immunol 2017;8:1016. Available from: https://doi.org/10.3389/
fimmu.2017.01016.
[132] Wang J, Liu J, Zhou R, Ding X, Zhang Q, Zhang C, et al. Zika virus infected primary microglia impairs NPCs proliferation and differentiation.
Biochem Biophys Res Commun 2018;497(2):619
25. Available from: https://doi.org/10.1016/j.bbrc.2018.02.118.
[133] Mesci P, Macia A, LaRock CN, Tejwani L, Fernandes IR, Suarez NA, et al. Modeling neuro-immune interactions during Zika virus infection.
Hum Mol Genet 2018;27(1):41
52. Available from: https://doi.org/10.1093/hmg/ddx382.
[134] Chiramel AI, Best SM. Role of autophagy in Zika virus infection and pathogenesis. Virus Res 2017;254:34
40. Available from: https://doi.
org/10.1016/j.virusres.2017.09.006.
[135] Peng H, Liu B, Yves TD, He Y, Wang S, Tang H, et al. Zika virus induces autophagy in human umbilical vein endothelial cells. Viruses
2018;10(5):259. Available from: https://doi.org/10.3390/v10050259.
[136] Amorim R, Temzi A, Griffin BD, Mouland AJ. Zika virus inhibits eIF2 alpha-dependent stress granule assembly. PLOS Negl Trop Dis
2017;11:e0005775.
[137] Roth H, Magg V, Uch F, Mutz P, Klein P, Haneke K, et al. Flavivirus infection uncouples translation suppression from cellular stress rsponses.
mBio 2017;8(1). Available from: https://doi.org/10.1128/mBio.02150-16 e02150-16.
[138] Hou S, Kumar A, Xu Z, Airo AM, Stryapunina I, Wong CP, et al. Zika virus hijacks stress granule proteins and modulates the host stress
response. J Virol 2017;91(16). Available from: https://doi.org/10.1128/JVI.00474-17 e00474-17.
[139] Lennemann NJ, Coyne CB. Dengue and zika viruses subvert reticulophagy by NS2B3-mediated cleavage of FAM134B. Autophagy
2017;13:322
32. Available from: https://doi.org/10.1080/15548627.2016.1265192.
[140] Azevedo RSS, de Sousa JR, Araujo MTF, et al. In situ immune response and mechanisms of cell damage in central nervous system of fatal
cases microcephaly by Zika virus. Sci Rep 2018;8(1):1. Available from: https://doi.org/10.1038/s41598-017-17765-5.
[141] Morrison TE, Diamond MS. Animal models of Zika virus infection, pathogenesis, and immunity. J Virol 2017;91(8):e00009
17. Available
from: https://doi.org/10.1128/JVI.00009-17.
[142] Faheem M, Naseer MI, Rasool M, et al. Molecular genetics of human primary microcephaly: an overview. BMC Med Genomics 2015;8(Suppl
1):S4. Available from: https://doi.org/10.1186/1755-8794-8-S1-S4.
[143] Miner JJ, Diamond MS. Understanding how Zika virus enters and infects neural target cells. Cell Stem Cell 2016;18(5):559
60. Available
from: https://doi.org/10.1016/j.stem.2016.04.009.
[144] Cugola FR, Fernandes IR, Russo FB, Freitas BC, Dias JL, Guimarães KP, et al. The Brazilian Zika virus strain causes birth defects in experi-
mental models. Nature 2016;534(7606):267
71. Available from: https://doi.org/10.1038/nature18296.
[145] McLean E, Bhattarai R, Hughes BW, Mahalingam K, Bagasra O. Computational identification of mutually homologous Zika virus miRNAs
that target microcephaly genes. Libyan J Med 2017;12(1):1304505. Available from: https://doi.org/10.1080/19932820.2017.1304505.
[146] Vermillion M, Lei J, Shabi Y, Baxter VK, Crilly NP, Mclane M, et al. Intrauterine Zika virus infection of pregnant immunocompetent mice
models transplacental transmission and adverse perinatal outcomes. Nat Commun 2017;8:14575. Available from: https://doi.org/10.1038/
ncomms14575.
[147] Tabata T, Petitt M, Puerta-Guardo H, Michlmayr D, Wang C, Fang-Hoover J, et al. Zika virus targets different primary human placental cells,
suggesting two routes for vertical transmission. Cell Host Microbe 2016;20(2):155
66. Available from: https://doi.org/10.1016/j.
chom.2016.07.002.
[148] Kumar A, Singh HN, Pareek V, Raza K, Dantham S, Kumar P, et al. A possible mechanism of Zika virus associated microcephaly: Imperative
role of retinoic acid response element (RARE) consensus eequencerepeats in the viral genome. Front Hum Neurosci 2016;10:403. Available
from: https://doi.org/10.3389/fnhum.2016.00403.
[149] Garcez PP, Stolp HB, Sravanam S, Christoff RR, Ferreira JCCG, Dias AA, Pezzuto P, et al. Zika virus impairs the development of blood ves-
sels in a mouse model of congenital infection. Sci Rep 2018;8(1):12774. Available from: https://doi.org/10.1038/s41598-018-31149-3.
[150] Gladwyn-Ng I, Cordón-Barris L, Alfano C, Creppe C, Couderc T, Morelli G, et al. Stress-induced unfolded protein response contributes to
Zika virus-associated microcephaly. Nat Neurosci 2018;21(1):63
71. Available from: https://doi.org/10.1038/s41593-017-0038-4.
[151] Chang C, Ortiz K, Ansari A, Gershwin ME. The Zika outbreak of the 21st century. J Autoimmun 2016;68:1
13. Available from: https://doi.
org/10.1016/j.jaut.2016.02.006.
[152] Chen Q, Gouilly J, Ferrat YJ, et al. Metabolic reprogramming by Zika virus provokes inflammation in human placenta. Nat Commun
2020;11:2967. Available from: https://doi.org/10.1038/s41467-020-16754-z.
[153] Chimelli L, Melo ASO, Avvad-Portari E, Wiley CA, Camacho AHS, Lopes VS, et al. The spectrum of neuropathological changes associated
with congenital Zika virus infection. Acta Neuropathol 2017;133(6):983
99. Available from: https://doi.org/10.1007/s00401-017-1699-5.
 Molecular mechanisms of Zika fever in inducing birth defects: an update Chapter | 6 109

[154] Mavigner M, Raper J, Kovacs-Balint Z, Gumber S, O’Neal JT, Bhaumik SK, et al. Postnatal Zika virus infection is associated with persistent
abnormalities in brain structure, function, and behavior in infant macaques. Sci Transl Med 2018;10(435):eaao6975. Available from: https://
doi.org/10.1126/scitranslmed.aao6975.
[155] Nem de Oliveira Souza I, Frost PS, França JV, Nascimento-Viana JB, Neris RLS, Freitas L, et al. Acute and chronic neurological conse-
quences of early-life Zika virus infection in mice. Sci Transl Med 2018;10(444):eaar2749. Available from: https://doi.org/10.1126/sci-
translmed.aar2749.
[156] van der Linden V, Filho EL, Lins OG, van der Linden A, AragãoMde F, Brainer-Lima AM, et al. Congenital Zika syndrome with arthrogrypo-
sis: retrospective case series study. BMJ 2016;9(354):i3899. Available from: https://doi.org/10.1136/bmj.i3899.
[157] Martinot AJ, Abbink P, Afacan O, Prohl AK, Bronson R, Hecht JL, et al. Fetal neuropathology in Zika virus-infected pregnant female rhesus
monkeys. Cell 2018;173(5):1111
22. Available from: https://doi.org/10.1016/j.cell.2018.03.019 e10.
[158] Hirsch AJ, Roberts VHJ, Grigsby PL, Haese N, Schabel MC, Wang X, Lo JO, et al. Streblow DN. Zika virus infection in pregnant rhesus
macaques causes placental dysfunction and immunopathology. Nat Commun 2018;9(1):263. Available from: https://doi.org/10.1038/s41467-
017-02499-9.
[159] Nguyen SM, Antony KM, Dudley DM, Kohn S, Simmons HA, Wolfe B, Salamat MS, et al. Highly efficient maternal-fetal Zika virus trans-
mission in pregnant rhesus macaques. PLoS Pathog 2017;13(5):e1006378. Available from: https://doi.org/10.1371/journal.ppat.1006378.
[160] Chiu CY, Martin C, Bouquet S-S, Li J, Yagi T, Tamhankar S, et al. Experimental Zika virus inoculation in a new world monkey model repro-
duces key features of the human infection. Sci Rep 2017;7:17126. Available from: https://doi.org/10.1038/s41598-017-17067-w.
[161] WichgersSchreur PJ, van Keulen L, Anjema D, Kant J, Kortekaas J. Microencephaly in fetal piglets following in utero inoculation of Zika
virus. Emerg Microbes Infect 2018;7(1):42. Available from: https://doi.org/10.1038/s41426-018-0044-y.
[162] Darbellay J, Cox B, Lai K, Delgado-Ortega M, Wheler C, Wilson D, et al. Zika virus causes persistent infection in porcine conceptuses and
may impair health in offspring. EBioMedicine 2017;25:73
86. Available from: https://doi.org/10.1016/j.ebiom.2017.09.021.
[163] Gurung S, Preno AN, Dubaut JP, Nadeau H, Hyatt K, Reuter N, et al. Translational model of Zika virus disease in baboons. J Virol 2018;92
(16). Available from: https://doi.org/10.1128/JVI.00186-18 e00186-18.
[164] Goodfellow FT, Tesla B, Simchick G, Zhao Q, Hodge T, Brindley MA, et al. Zika virus induced mortality and microcephaly in chicken
embryos. Stem Cell Dev 2016;25(22):1691
7. Available from: https://doi.org/10.1089/scd.2016.0231.
[165] de Paula Freitas B, Ventura CV, Maia M, Belfort Jr. R. Zika virus and the eye. Curr Opin Ophthalmol 2017;28(6):595
9. Available from:
https://doi.org/10.1097/ICU.0000000000000420.
[166] Zhao Z, Yang M, Azar SR, Soong L, Weaver SC, et al. Viral retinopathy in experimental models of Zika infection. Investig Ophthalmol Vis
Sci 2017;58:4355
65. Available from: https://doi.org/10.1167/iovs.17-22016.
[167] Fernandez MP, Parra Saad E, Ospina Martinez M, Corchuelo S, Mercado Reyes M, Herrera MJ, et al. Ocular histopathologic features of con-
genital Zika syndrome. JAMA Ophthalmol 2017;135(11):1163
9. Available from: https://doi.org/10.1001/jamaophthalmol.2017.3595.
[168] Zhu S, Luo H, Liu H, Ha Y, Mays ER, Lawrence RE, et al. p38MAPK plays a critical role in induction of a pro-inflammatory phenotype of
retinal Müller cells following Zika virus infection. Antivir Res 2017;145:70
81. Available from: https://doi.org/10.1016/j.
antiviral.2017.07.012.
[169] Agrawal R, Oo HH, Balne PK, Ng L, Tong L, Leo YS. Zika virus and the eye. Ocul Immunol Inflamm 2018;26:654
9. Available from:
https://doi.org/10.1080/09273948.2017.1294184.
[170] Salinas S, Erkilic N, Damodar K, et al. Zika virus efficientlyreplicates in human retinal epithelium and disturbs its permeability. J Virol
2017;91(3). Available from: https://doi.org/10.1128/JVI.02144-16 e02144-16.
[171] Singh PK, Guest JM, Kanwar M, et al. Zika virus infects cells lining the blood-retinal barrier and causes chorioretinal atrophy in mouse eyes.
JCI Insight 2017;2(4):e92340. Available from: https://doi.org/10.1172/jci.insight.92340.
[172] Miner JJ, Sene A, Richner JM, Smith AM, Santeford A, Ban N, et al. Zika virus infection in mice causes panuveitis with shedding of virus in
tears. Cell Rep 2016;16(12):3208
18. Available from: https://doi.org/10.1016/j.celrep.2016.08.079.
[173] Leal MC, Muniz LF, Ferreira TS, et al. Hearing loss in infants with microcephaly and evidence of congenital Zika virus infection-Brazil,
November 2015-May 2016. MMWR Morb Mortal Wkly Rep 2016;65:917
19. Available from: https://doi.org/10.15585/mmwr.mm6534e3.
[174] Shan C, Xia H, Haller SL, Azar SR, Liu Y, Liu J, et al. A Zika virus envelope mutation preceding the 2015 epidemic enhances virulence and
fitness for transmission. Proc Natl Acad Sci U S A 2020;117(33):20190
7. Available from: https://doi.org/10.1073/pnas.2005722117.
[175] Vinhaes ES, Santos LA, Dias L, Andrade NA, Bezerra VH, de Carvalho AT, et al. Transient hearing loss in adults associated with Zika virus
infection. Clin Infect Dis 2017;64(5):675
7. Available from: https://doi.org/10.1093/cid/ciw770.
[176] Julander JG, Siddharthan V, Park AH, Preston E, Mathur P, Bertolio M, et al. Consequences of in utero exposure to Zika virus in offspring of
AG129 mice. Sci Rep 2018;8(1):1
11. Available from: https://doi.org/10.1038/s41598-018-27611-x.
[177] Yee KT, Neupane B, Bai F, Vetter DE. Zika virus infection causes widespread damage to the inner ear. Hearing Res 2020;108000. Available
from: https://doi.org/10.1016/j.heares.2020.108000.
[178] Thawani A, Sammudin NH, Reygaerts HS, Wozniak AN, Munnamalai V, Kuhn RJ, et al. Zika virus can directly infect and damage the audi-
tory and vestibular components of the embryonic chicken inner ear. Dev Dyn 2020;249(7):867
83. Available from: https://doi.org/10.1002/
dvdy.176.
This page intentionally left blank
Chapter 7

Middle East respiratory syndrome:

outbreak response priorities, treatment
strategies, and clinical management
approaches
Kandati Kusuma1,2, Pandeeti Emmanuel Vijay Paul3 and Buddolla Viswanath2
1
Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, India, 2Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr.
Buddolla’s Educational Society, Tirupati, India, 3Albus Eco Projects, LLP, BioNEST, University of Hyderabad, Hyderabad, India

7.1 Introduction
Middle East respiratory syndrome (MERS) is a viral respiratory disease caused by a novel coronavirus (Middle East
respiratory syndrome coronavirus, or MERS-CoV), which was initially discovered in Saudi Arabia in 2012 and it is one
of six known human coronaviruses that cause respiratory disease in humans and, with a mortality rate .35% [1]. It is
the first highly pathogenic human coronavirus to emerge since the global scare caused by the severe acute respiratory
syndrome coronavirus in 2003 [2]. The first human MERS-CoV infection was found in Saudi Arabia, in June 2012 in a
60-year old man who developed renal failure [3]. Analysis revealed the disease is due to a novel virus which was named
Middle East Respiratory Coronavirus [4]. The World Health Organization [5] has confirmed 2279 cases of human infec-
tions with MERS-CoV in 27 countries since 2012; 806 (35%) infected patients have died as of February 13, 2019 [6].
However, Saudi Arabia still has the highest reported MERS-CoV mortality rate where approximately 80% of the cases
have been reported to occur there [7,8]. MERS-CoV remains a high-threat pathogen identified by WHO as a priority
pathogen because it causes severe disease that has a high mortality rate, epidemic potential, and no medical counter-
measures [9]. MERS-CoV belongs to the family Coronaviridae, order Nidovirales. It is one of the recently reported
zoonotic viruses [10]. The family Coronaviridae is classified into four genera (α, β, γ, and δ). Each genus is divided
into lineage subgroups. MERS-CoV belongs to lineage-C of the β coronaviruses [11]. Viral spread has been observed
among healthcare workers and among individuals visiting MERS-CoV-positive patients. The control of some of these
outbreaks has been achieved by the local center for disease control and prevention (CDC). Respiratory tract infections
are the leading cause of mortality in resource-limited settings, accounting for more than 4 million deaths each year
globally [12]. A hypothetical sequence of how humans and DCs (direct contact) lead to the spread of MERS cases is
summarized in Fig. 7.1.
MERS-CoV infection may be implicated in transmission. As an emerging Betacoronavirus, Middle East respiratory
syndrome coronavirus (MERS-CoV) causes illness characterized predominantly by mild-to-severe respiratory com-
plaints, with most patients requiring admission to hospital because of pneumonitis or acute respiratory distress syn-
drome. Old age and the presence of comorbidities or immunosuppression seem to increase the risk of infection and are
associated with severe forms of the disease [14].
Humans are thought to acquire MERS-CoV through contact with camels or camel products [15]. Despite the
increase in the number of cases, the actual incidence of MERS-CoV among hospitalized patients with community-
acquired pneumonia is low [16]. There are reports of the role of asymptomatic individuals in the transmission of
MERS-CoV; however, the exact role is not known [16]. These observations indicate the need for understanding the
human immune response to the virus to guide immunotherapy of severely ill patients and vaccine development and to

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00016-1

© 2021 Elsevier Inc. All rights reserved. 111
112 Pandemic Outbreaks in the 21st Century

FIGURE 7.1 (A) Risks for acquiring MERS-CoV from a DC. This illustration highlights risks that may originate from a droplet transmission compo-
nent (be they larger, heavier wet droplets or the drier, airborne gel-like droplet nuclei) or a direct contact component (within the green circle). No
routes of MERS-CoV acquisition to or between humans have been proven to date [13]. (B) Camel-to-human infections appear to be infrequent, while
human-to-human spread of infection is regularly facilitated by poor IPC in healthcare settings where transmission is amplified, accounting for the bulk
of cases. (C) A hypothetical sequence of how humans and direct contact (DCs) lead to the spread of MERS cases. MERS-CoV, Middle East respiratory
syndrome coronavirus; DC, dendritic cells.

develop additional tools for determining the prevalence of the infection [17]. MERS-CoV causes acute, highly lethal
pneumonia and renal dysfunction with various clinical symptoms, including but not restricted to fever, cough, sore
throat, myalgia, chest pain, diarrhea, vomiting, and abdominal pain [18]. This chapter focuses on the current informa-
tion of MERS-CoV, epidemiology, and spreading of MERS-CoV virus with reference to the virus structure and life
cycle, molecular mechanisms of pathogenesis, and immune responses to MERS infection. We also look at the initial
and postinfection manifestations of MERS and its future prospects as well.

7.2 Epidemiology
Molecular clock dating of epidemiologically unlinked human MERS-CoV isolates estimated their divergence from a com-
mon ancestor in mid-2011, with a cluster of isolates from the eastern parts of the Arabian Peninsula diverging in late
2012. These findings suggest that the reported MERS-CoV diversity in human beings is the result of several independent,
geographically structured, zoonotic events from an unknown reservoir in the Middle East [19,20]. A primary MERS-CoV
infection is defined by WHO as laboratory-confirmed MERS-CoV infection that has no direct epidemiological link to a
human MERS-CoV infection and was acquired outside of a healthcare facility presumably from direct or indirect contact
 Middle East respiratory syndrome Chapter | 7 113

with the reservoir host, dromedary camels. A secondary MERS-CoV infection is defined by WHO as a laboratory-
confirmed MERS-CoV infection with a direct epidemiological link to an individual with confirmed or probable MERS-
CoV infection [9]. MERS-CoV cases continued to be reported from the community and hospitals across the Arabian
Peninsula. As of July 31, 2019, 2458 laboratory-confirmed MERS cases were reported to WHO of which there were 848
deaths (34% mortality) [15]. Approximately 80% of human cases have been reported in Saudi Arabia. Twenty-seven
countries have reported cases of MERS [21]. Countries in or near the Arabian Peninsula that report MERS cases are
Bahrain, Iran, Jordan, Kuwait, Lebanon, Oman, Qatar, Saudi Arabia, UAE, and Yemen. Cases identified outside the
Middle East are usually in travelers who were infected in the Middle East and then traveled to areas outside the Middle
East. Countries outside the Arabian Peninsula that have reported travel-associated MERS cases are Algeria, Austria,
China, Egypt, France, Germany, Greece, Italy, Malaysia, Netherlands, Philippines, Republic of Korea, Thailand, Tunisia,
Turkey, United Kingdom, and the United States [15]. Serologic surveys subsequently conducted in several countries in the
Arabian Peninsula and Africa identified high rates of MERS-CoV
specific antibodies in dromedary camels [22,23].
Furthermore, MERS-CoV infection in dromedary camels was definitively proven by the detection of virus and virus
sequences in respiratory specimens, feces, and milk collected from camels in Qatar [14,24], Oman [25], Saudi Arabia
[22,26
28], and Egypt [29].

7.3 Ecology and spreading of MERS-CoV virus

MERS-CoV is a virus transferred to humans from infected dromedary camels. It is a zoonotic virus, meaning it is trans-
mitted between animals and people, and it is contractible through direct or indirect contact with infected animals.
MERS-CoV has been identified in dromedaries in several countries in the Middle East, Africa, and South Asia. In total,
27 countries have reported cases since 2012, leading to 858 known deaths due to the infection and related complications
[5]. For MERS-CoV, however, increasing evidence indicates that both camels and humans may play intermediate roles,
as both disease victims and reservoirs for further transmission [30]. Transmission appears to be entirely from humans to
humans [3]. Contact of various types with camels, such as consumption of raw milk, butchering and cleaning meat, and
visiting live animals, has been identified as a significant risk factor, such that camels are seen as a significant reservoir
host for MERS-CoV in its transmission to humans [31].
Cases of MERS-CoV can be found in countries like America, UK, France, Tunisia, Italy, Malaysia, Philippines,
Greece, Egypt, the Netherlands, Algeria, Austria, Turkey, and so on whose citizens have traveled to the Middle East
[3]. On May 20, 2014, a man at the age of 68 was the first to be diagnosed with MERS-CoV in Korea. He traveled to
Bahrain, Saudi Arabia, and Qatar for 16 days. On May 4, 2015, this patient entered Korea, and febrile sense and respi-
ratory symptoms appeared on May 11. He visited Clinic A on the day and was admitted to Hospital B from May 15 to
17. Since the symptoms worsened, he visited Clinic C on May 18, and finally he was transferred to a university hospital
in Seoul on May 18. On May 20, it was confirmed that he was suffering from MERS-CoV. After finding out about the
disease, his family members and medical staff who had been exposed to the virus were isolated. By June 9, 2015, two
medical staffs in the Clinics A and C, one medical staff in the Hospital B, one patient and his wife who was together
with the index case in the same room and 35 of admitted patients in the same ward and their family members visiting
the same ward with the index case in the Hospital B were confirmed to have been infected with MERS-CoV. After
then, several tertiary cases were identified in the Hospital B or other hospitals that secondary patients were transferred
from the Hospital B. A total of 108 people were infected, and nine (8.3%) of them died by June 10, 2015. The ecology
and transmission of MERS-CoV are pictured in Fig. 7.2.
MERS-CoV might have originally spread from bats to camels and others, as yet unidentified, intermediate hosts.
The virus has circulated in camel populations in Africa and the Arabian Peninsula for at least 20 years. In 2012 MERS-
CoV spread to human populations, with camels the most likely source. Several possible routes of spread from camels to
humans exist. MERS-CoV is believed to be transmitted among human beings by droplet, contact, and perhaps airborne
spread. MERS manifests in people in various ways, ranging from asymptomatic to fulminant infections. Patients with
underlying diseases such as diabetes or kidney or liver disease or who are immunocompromised develop more severe
diseases and have a higher mortality rate after infection [32].

7.4 Virus structure and life cycle

The Middle East respiratory syndrome (MERS) is a highly lethal respiratory disease caused by a novel single-stranded,
positive-sense RNA Betacoronavirus (MERS-CoV) [32]. MERS-CoV belongs to the genus Betacoronavirus of the
Coronaviridae family [33]. It is an enveloped, single-stranded, positive-sense RNA virus with a helical capsid structure.
114 Pandemic Outbreaks in the 21st Century

FIGURE 7.2 Ecology and transmission of MERS-CoV. MERS-CoV, Middle East respiratory syndrome coronavirus. Reprinted with permission from
Elsevier, Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet 2015;386(9997):995–1007.

The genome of MERS-CoV is around 30 kb (30,119 nt) long one of the largest among positive-sense RNA viruses and
encodes four structural proteins (spike, envelope, membrane, and nucleocapsid) and 16 nonstructural proteins depicted
in Fig. 7.3 [34]. The immunogenic MERS-CoV proteins include spike (S), membrane (M), and envelope (E). Among
them, the S protein mediates viral entry into the host cells [35]. The virion surface is covered with the spike glycopro-
tein, a 149 kDa glycoprotein that extends outward to create a crown-like appearance. The spike glycoprotein is critical
for binding the host-cell receptor, dipeptidyl peptidase 4 (DPP-4), to initiate infection [36].
For MERS-CoV infection of humans, the primary receptor is a multifunctional cell surface protein, DPP4 (also
known as CD26) [37] which is widely expressed on epithelial cells in the kidney, alveoli, small intestine, liver, and
prostate, and on activated leukocytes (Widagdo et al., 2016). To enter host cells, MERS-CoV attaches to its recep-
tor, DPP4. The S glycoprotein is a typical type I membrane glycoprotein consisting of a globular S1 domain at the
N-terminal, followed by a membrane-proximal S2 domain and a transmembrane domain [39]. The S1 domain med-
iates viral attachment and contains the receptor binding domain (RBD), which determines the host range and cellu-
lar tropism for MERS-CoV [40
42].
Binding between RBD and the cell receptor (DPP4) triggers the conformational change of S glycoprotein to form a
prehairpin intermediate of S2, in which the hydrophobic HR1 is exposed and the fusion peptide inserts into the target
 Middle East respiratory syndrome Chapter | 7 115

FIGURE 7.3 General structure and life cycle of MERS-CoV. (A) Cartoon model structure of MERS-CoV. (B) Membrane fusion mechanism for
MERS-CoV spike glycoprotein. MERS-CoV, Middle East respiratory syndrome coronavirus.

cell membrane. This transient S2 intermediate then refolds with HR2 into a stabilized trimer of hairpins, also called six-
helix bundle structure, bringing the target cell membrane into close proximity of the viral envelope and resulting in the
completion of the fusion process [43]. Protease cleavage of the S protein is then required for virus
cell fusion and
release of genomic RNA into the cytoplasm. Viral RNA transcription and replication occur on double-membrane vesi-
cles and other membranous structures, which are derived from the endoplasmic reticulum. Transcription of the seven
subgenomic mRNAs occurs via negative-strand subgenomic RNA intermediates. Subgenomic RNAs are 30 coterminally
nested and are joined to a common leader encoded at the 50 end of the genome. Viral RNA is encapsulated in the N pro-
tein and transported to the endoplasmic reticulum
Golgi intermediate compartment, the site of assembly. Viral RNA
encapsidated in the N protein then buds into vesicles lined with the S, M, and E proteins. Vesicles are then transported
to the cell surface before release [32]. MERS-CoV binds to its cellular receptor DPP4 via the S protein, which is pro-
cessed by host proteases to expose a fusion peptide. The viral genome is then released into the cytoplasm, where it is
translated on host ribosomes into rep1A and rep1B proteins. The polyprotein is cleaved by two viral-encoded proteases,
encoded by NSP3 and NSP5. Proteins involved in the genome and subgenome replication and transcription include
nsp12 [the RNA-dependent RNA polymerase (RdRP)] and two associated proteins, NSP7 and NSP8 [44].

7.5 Molecular mechanisms of pathogenesis

Dromedary camels are infected with the virus and are believed to be the most likely source of animal-to-human trans-
mission [45]. After intratracheal inoculation of MERS-CoV in nonhuman primates, the virus infects bronchial epithelial
cells through DPP-4 before spreading to lung parenchymal cells, including type I and type II alveolar pneumocytes and
endothelial cells. Viral entry is facilitated by another cell-surface protein, carcinoembryonic antigen
related cell-
adhesion molecule 5, which is also expressed in lung tissue. Inflammatory signaling molecules that are released by
infected cells, alveolar macrophages, and neutrophils recruited to infected tissue have been detected in infected patients
116 Pandemic Outbreaks in the 21st Century

and animal models. A host antiviral type I and type III interferon response occurs, with systemic release of pro-
inflammatory cytokines and chemokines [46
48].
The virus may spread into the circulation, possibly from lung parenchyma or through infected endothelial cells. In
humans, a high viral copy number has been detected in the lower respiratory tract, including tracheal aspirates and
bronchoalveolar lavage specimens, as well as in peripheral blood. In advanced disease, diffuse alveolar damage is seen,
with extensive hemorrhagic edema and hyaline membrane deposition. CXCL10 denotes C-X-C motif chemokine 10,
IL—interleukin, IL-1RA—IL-1 receptor antagonist, IFN—interferon, and MCP—monocyte chemotactic protein. In one
study conducted in Saudi Arabia, the rate of MERS-CoV seropositivity was 15 times as high in shepherds and 23 times
as high in slaughterhouse workers as in the general population [49]. There is high sequence homology between the
viruses from sporadic cases in humans and the implicated dromedaries. Although the routes of transmission remain
unclear, they appear to include contact with infectious nasal or other bodily secretions and possibly the consumption of
raw dromedary products (e.g., unpasteurized milk). Although dromedary-to-human transmission of MERS-CoV is now
well recognized, direct exposure to dromedaries has been documented in only 40% of primary cases [50].
Transmission of MERS-CoV between dromedary camels and humans has been documented in several countries.
Human-to-human transmission in health care settings accounts for the majority of reported cases to date, although
human-to-human transmission in household settings has also been identified [36].

7.6 Immune responses to MERS infection

The virus replicates in macrophages and dendritic cells (DC), it induces the production of pro-inflammatory cytokines.
Infection of human T cells with MERS-CoV induces both intrinsic and extrinsic apoptotic pathways, causing the sup-
pression of immune responses [51]. Neutrophils are considered the first line of the innate immune response. Studies on
MERS-CoV infection revealed that neutrophil chemoattractant chemokine IL-8 is highly expressed in the lower respira-
tory tract of the patient [52]. IL-8 plays an essential role in recruitment, activation, and accumulation of neutrophil in
the site of infection and subsequently induce the formation of neutrophil extracellular traps (NETs). NETs directly
cause inflammation and increase the secretion of IL-8, resulting in the further recruitment of neutrophils to the site of
infection [53].
The launch response of the immune system to the invading of a microorganism such as a virus is directly related to
the host sensing of the target organism and its linked constituents like uncapped viral RNA or the cellular stress
response and consequent biological changes or damages due to infection [54]. This response could be primarily con-
ducted by germline-encoded pattern recognition receptors such as Toll-like receptors or Retinoic acid-inducible gene I-
like from the components receptors (RLRs) that enable to detect pathogen-associated molecular patterns originated of a
virus or its replication intermediates, promoting the initial antiviral signaling cascades in response to the infection [55].
MERS-CoV could infect airway epithelial cells, inducing the responses of pro-inflammatory cytokines like IL-1β, IL-6,
IL-8, and IFNs significantly but delayed. Though MERS-CoV is able to be replicated in either naı̈ve or activated DCs
and monocyte-macrophages, activated T cells can only support the MERS-CoV replication [56]. The higher amount of
these factors in the serum of MERS-infected patients is correlated with increased numbers of monocyte and neutrophil
in the peripheral blood cells and lungs, showing the possible function of these cells in the pathology of the lungs [57].
Importantly, among all the functional/nonfunctional structural proteins of MERS-CoV, the S protein is the principal
antigenic component that induces antibodies to block virus-binding, stimulate host immune responses, fuse or neutralize
antibodies, and/or protect the immune system against virus infection. Therefore the S protein has been selected as a sig-
nificant target for the development of vaccines [58].

7.7 MERS—initial and postinfection manifestations

The clinical manifestations of MERS are nonspecific, which include runny nose, sore throat, low-grade fever, and myal-
gia, before the viremia becomes detectable [36,59]. In case of severe disease, progression to acute respiratory distress
may occur. Extrapulmonary manifestations including gastrointestinal symptoms and acute kidney failure have been
reported in case of severe illness, as well as neurological manifestations [36]. The initial and postinfection manifesta-
tions associated with MERS-CoV-infection were observed to be related to neurological manifestations presenting neuro-
logical symptoms. Different neurological symptoms were reported from the studies on the patients infected with
MERS-CoV. Patients with ataxia, vomiting, confusion were known to have acute disseminated encephalomyelitis or
less probably encephalitis, whereas patients with unresponsiveness, hypotensive with left-sided facial paralysis showed
acute bilaterally nonocclusive stroke probably due to MERS-CoV-vasculopathy, low Glasgow Coma Scale, and fever
 Middle East respiratory syndrome Chapter | 7 117

were also found to be associated with encephalopathy [45]. Patients with right frontal lobe intracerebral hemorrhage
showed symptoms of severe headache, nausea and vomiting, decreased level of consciousness and Glasgow Coma
Scale; Weakness in both legs and inability to walk with numbness and tingling in stocking distribution were observed
in critical illness polyneuropathy patients [60]. The above-mentioned neurological symptoms were more prominent in
patients with comorbidities like hypertension, dyslipidemia, diabetes, peripheral vascular disease, chronic kidney dis-
ease, and ischemic heart disease [61].

7.8 Outbreak response priorities

Patients with MERS should be placed in negative pressure rooms or in rooms in which room exhaust is filtered through
high-efficiency particulate air filters. Airborne precautions with at least six air changes per hour should be applied in
treatment rooms when performing aerosol-generating procedures [62,63]. These recommendations are evidence-based
and have proven to be effective in hospitals in affected countries. Camels infected with MERS-CoV can develop rhinitis
or show no signs of infection and might shed the virus through nasal and eye discharge and feces. The virus can also be
found in raw milk from infected camels. MERS-CoV is stable in camel breast milk for extended periods of time [64].
Thus pasteurization or cooking is recommended to destroy the virus. Camel farm workers, slaughterhouse workers, mar-
ket workers, veterinarians, and those handling camels at racing facilities should practice good personal hygiene, includ-
ing frequent hand washing after touching animals, avoiding touching eyes, nose, or mouth with hands, and avoiding
contact with sick animals. Consideration should also be given to wearing protective gowns and gloves while handling
animals, especially if camels have signs of upper respiratory tract disease [65].

7.9 Diagnostics
Diagnosis of MERS-CoV is a major concern in most diagnostic laboratories. Detecting the virus in respiratory tract
samples remains the gold standard in diagnosing MERS-CoV infection. Several samples can be obtained from the respi-
ratory system that can be used for diagnosing MERS-CoV infection. These include tracheal aspirates, nasopharyngeal
swabs, and sputum. Tracheal aspirates and bronchoalveolar lavage samples (lower respiratory samples) yielded signifi-
cantly higher viral copies than nasopharyngeal and sputum samples [27,28]. Lower respiratory tract specimens such as
bronchoalveolar lavage fluid, sputum, and tracheal aspirates contain the highest viral loads (VLs) [27,28,66,67]. They
should be collected whenever possible.
MERS can be confirmed by detection of viral nucleic acid or by serology. Analyzing whole blood and plasma also
yielded a positive viral genome [66]. Real-time polymerase chain reaction (RT-PCR) is the mainstay for the diagnosis
of MERS-CoV. The presence of viral nucleic acid can be confirmed by positive real-time reverse transcription PCR
[32]. RT-PCR has limitations, including a long turnaround time and lack of common measurements and correlations
with VL. It is recommended to screen for MERS-CoV using RT-PCR of the upstream of envelope gene (upE) followed
by confirmation of the presence of one of the following genes; open reading frame 1A, 1B genes, or nucleocapsid (N)
gene. Scientists are looking to implement viral sequencing on all negative samples by RT-PCR and they believe that
can be exposed to another level of testing using sequencing of the RdRp gene or N gene and in this case, a positive
result is diagnostic. It is also very important to maintain a continuous and random sequencing for MERS-CoV samples
to be able to pick early viral mutations [68].

7.10 Vaccines
In vaccine production, a major limiting factor in designing comprehensive delivery systems for aerosol transmissible
diseases is the enhancement of efficacy and easy vaccine administration [69,70]. Although monoclonal antibodies show
promising antiviral effects in cell culture and animal models against MERS-CoV infection, their roles are still limited
in large-scale disease prevention [39]. Vaccines still remain the best choice for MERS-CoV prevention. Vaccines
against MERS-CoV thus far developed in the laboratory can be categorized as those based on viral vectors, such as ade-
novirus and Modified Vaccinia virus Ankara (MVA), or those based on recombinant viral proteins, DNAs, nanoparti-
cles, and recombinant virus (Du et al., 2015). Two vaccine candidates, GLS-5300 and MERS001, have entered human
clinical trials. The vaccine GLS-5300 was the first to be tested in healthy human volunteers. It is a DNA plasmid encod-
ing the MERS-CoV S glycoprotein, requiring two to three injections delivered by electroporation [72].
The phase I clinical trial was started in 2016 at the Walter Reed Army Institute, and another phase I/II clinical trial
is being conducted in Korea to test dosage safety and immunogenicity. Another vaccine candidate, MERS001, is a
118 Pandemic Outbreaks in the 21st Century

replication-deficient chimpanzee adenovirus (ChAdOx1) containing the MERS-CoV S glycoprotein antigen [73,74].
This vaccine only requires one-time administration of 5 3 109
5 3 1010 virus particles via intramuscular route, and the
local adverse events, as well as immunogenicity, will be evaluated in phase I clinical trial conducted at the University
of Oxford. In addition, one more candidate vaccine has been tested in dromedary camels either for potential human use
or straight into veterinary use. It explores a MVA as a vector to express MERS-CoV S glycoprotein [75]. The regimen
involves immunization through intranasal as well as intramuscular routes twice at a 4-week interval. The vaccinated
camels demonstrated a significant reduction of excreted infectious virus and viral RNA transcripts in vaccinated ani-
mals upon the MERS-CoV challenge.
Protection against MERS-CoV infection correlated with the presence of serum neutralizing antibodies to MERS-
CoV. The remaining vaccine candidates are all in the stages of preclinical or laboratory development and invariably tar-
get the S glycoprotein or RBD critical for viral entry [39]. High levels of an immunologically active drug may lead to
inflammatory and excessive immunological responses. Therefore safety precautions and new formulations may be
needed to reduce the side effects [35]. Novavax, on June 6, 2013, announced that it has successfully produced a vaccine
candidate designed to provide protection against the recently emerging MERS-CoV. The vaccine candidate was made
using Novavax nanoparticle vaccine technology and is based on the major surface spike (S) protein.

7.11 Treatment strategies

The latency period of MERS-CoV is known to be between 2 and 14 days (median 5.4 days). From the development of
the disease to the patient’s admission, it takes 4 days and the period that people die from the disease takes 11.5 days
[76,77]. In the first stage, flu-like symptoms such as fever, coughing, chilling, myalgia, and arthralgia are observed.
After this, the respiratory difficulty is added. This quickly progresses to pneumonia [14]. A part (30%) of the patients
complains of bowel symptoms like vomiting and diarrhea [78]. In the absence of specific antiviral therapy and lack of
knowledge of viral kinetics, clinical management of MERS largely depends on supportive treatment and prevention of
complications. Lung-protective ventilatory strategies for ARDS, inotropic support, antimicrobial therapy for co-
infections, and renal replacement therapy for acute renal failure have been used [10,66,76,77]. Some patients with
severe disease have been treated with systemic corticosteroids [79].
Several agents have shown inhibitory effects against MERS-CoV in cell cultures, including IFNs, ribavirin, cyclo-
sporin A, and mycophenolic acid [46,47,80
82]. A combination of IFNa2b and ribavirin appears to have beneficial
effects in reducing lung injury and inflammation when given to rhesus macaques within 8 h of inoculation with MERS-
CoV [46,47]. This treatment combination was given to several severely ill patients with unfortunately fatal outcomes,
likely because of late administration [83]. Currently, there is insufficient clinical data supporting the routine use of these
agents, and randomized controlled trials are needed if supported by favorable responses in animal models. Convalescent
plasma to be donated by patients who have fully recovered from MERS-CoV infection would be a good treatment
option [78]. Several drugs inhibit MERS-CoV in cell culture, including ciclosporin and mycophenolic acid [80,81].
MERS-CoV-specific peptide fusion inhibitors, which function similarly to the HIV drug enfuvirtide, diminish virus rep-
lication in cultured cells, providing a novel approach to MERS treatment [84].

7.12 Clinical management approaches

7.12.1 Prevention and control of MERS
A high degree of awareness of the possibility of MERS-CoV infection and early isolation of suspected or confirmed
MERS cases with proactive surveillance are crucial to preventing outbreaks. To decrease MERS-CoV human-to-human
transmission and environmental contamination, aerosol-generating procedures (AGPs) should be avoided in crowded
hospital accident and emergency departments and in inpatient medical wards without adequate infection control mea-
sures [85]. Droplet precautions are required for managing patients with confirmed MERS-CoV infection. Wearing a sur-
gical mask within 1
2 m of the patient, and wearing a gown, gloves, mask, and eye protection on entering the patient’s
room, and removing them upon leaving, are important infection control measures. Airborne precautions should be
applied for AGPs such as open suctioning or aspiration of the respiratory tract, intubation, bronchoscopy, or cardiopul-
monary resuscitation. These precautions include wearing a half-mask air-purifying respirator, such as a United States
National Institute for Occupational Safety and Health approved N95 filtering face-piece respirator or a European
Standard-approved FFP2 or FFP3 filtering face-piece respirator. The respirator should fit properly, and all healthcare
 Middle East respiratory syndrome Chapter | 7 119

workers should undergo in-depth training for the proper use, donning and doffing of the respirator, and performing a
user seal check every time the respirator is used.
Cleaning environmental surfaces with water and detergent and applying commonly used disinfectants (such as hypo-
chlorite) is an effective and sufficient procedure [7,8]. Unprotected or inadvertent exposure of healthcare workers to
patients with MERS-CoV should prompt rapid quarantine. When MERS-CoV cases are suspected or diagnosed in the
community and households, educational awareness of MERS-CoV and MERS prevention measures within the home
could reduce further transmission and prevent outbreaks of community clusters. People with comorbidities such as dia-
betes, kidney disease, chronic lung disease, and cancer, or individuals on immunosuppressive treatments are at high risk
of developing severe MERS-CoV disease, thus they should avoid close contact with camels and bats. Early recognition
of MERS-CoV infections, improved compliance with internationally recommended infection control protocols, and
rapid implementation of infection control measures are required to prevent outbreaks of MERS-CoV associated with
health care facilities [86].

7.13 Summary and future prospective

With the advent of modern techniques in virology, immunology, and vaccinology, we have gained substantial insights
into the biology of MERS-CoV, and its pathogenesis with unprecedented speed and accuracy. We are facing a difficult
predicament when it comes to public health challenges in the new era of emerging and reemerging infectious diseases.
On the one hand, the human population is becoming ever mobile and exposed to an increasing number of pathogens.
On the other hand, translating basic discoveries into preventative and treatment applications has been exceedingly slow.
MERS-CoV is a pathogen with epidemic potential that continues to cause sporadic human disease and remains on the
WHO Blueprint 2020 priority list [87]. MERS-CoV endemic and at-risk countries must invest more in surveillance,
public health research, and medical interventions. Early recognition of cases, improved compliance with internationally
recommended infection control protocols, and rapid implementation of infection control measures are required to pre-
vent health care facility
associated outbreaks of MERS-CoV.

References
[1] WHO Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Available from: http://www.who.int/emergencies/mers-cov/en/. [Accessed
23 May 2016].
[2] Chafekar A, Fielding BC. MERS-CoV: understanding the latest human coronavirus threat. Viruses 2018;10(2):93.
[3] Lee J. Better understanding of MERS coronavirus outbreak in Korea. J Korean Med Sci 2015;30(7):835
6.
[4] Al-Omari A, Rabaan AA, Salih S, Al-Tawfiq JA, Memish ZA. MERS coronavirus outbreak: implications for emerging viral infections. Diagn
Microbiol Infect Dis 2019;93(3):265
85.
[5] WHO. 2022. Available from: https://www.who.int/health-topics/middle-east-respiratory-syndrome-coronavirus-mers#tab 5 tab_1.
[6] Mubarak A, Alturaiki W, Hemida MG. Middle East respiratory syndrome coronavirus (MERS-CoV): infection, immunological response, and
vaccine development. J Immunol Res 2019;2019:6491738.
[7] World Health Organization. Countries agree next steps to combat global health threat by MERS-CoV. Geneva, Switzerland: WHO; 2019.
[8] World Health Organization. Infection prevention and control during health care for probable or confirmed cases of Middle East respiratory syn-
drome coronavirus (MERS-CoV) infection: interim guidance: updated October 2019 (No. WHO/MERS/IPC/15.1 Rev. 1). World Health
Organization; 2019b.
[9] Memish ZA, Perlman S, Van Kerkhove MD, Zumla A. Middle East respiratory syndrome. Lancet 2020;395(10229):1063
77.
[10] Zaki AM, Van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in
Saudi Arabia. N Engl J Med 2012;367(19):1814
20.
[11] Chan JF, Lau SK, To KK, Cheng VC, Woo PC, Yuen KY. Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus
causing SARS-like disease. Clin Microbiol Rev 2015;28(2):465
522.
[12] Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age
groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012;380(9859):2095
128.
[13] Mackay IM, Arden KE. Middle East respiratory syndrome: an emerging coronavirus infection tracked by the crowd. Virus Res
2015;202:60
88.
[14] Haagmans BL, Al Dhahiry SH, Reusken CB, Raj VS, Galiano M, Myers R, et al. Middle East respiratory syndrome coronavirus in dromedary
camels: an outbreak investigation. Lancet Infect Dis 2014;14(2):140
5.
[15] Azhar EI, Hui DS, Memish ZA, Drosten C, Zumla A. The middle east respiratory syndrome (MERS). Infect Dis Clin 2019;33(4):891
905.
[16] Al-Tawfiq JA, Auwaerter PG. Healthcare-associated infections: the hallmark of Middle East respiratory syndrome coronavirus with review of
the literature. J Hospital Infect 2019;101(1):20
9.
120 Pandemic Outbreaks in the 21st Century

[17] Zhao J, Alshukairi AN, Baharoon SA, Ahmed WA, Bokhari AA, Nehdi AM, et al. Recovery from the Middle East respiratory syndrome is asso-
ciated with antibody and T cell responses. Sci Immunol 2017;2(14):eaan5393.
[18] Saad M, Omrani AS, Baig K, Bahloul A, Elzein F, Matin MA, et al. Clinical aspects and outcomes of 70 patients with Middle East respiratory
syndrome coronavirus infection: a single-center experience in Saudi Arabia. Int J Infect Dis 2014;29:301
6.
[19] Cotten M, Watson SJ, Kellam P, Al-Rabeeah AA, Makhdoom HQ, Assiri A, et al. Transmission and evolution of the Middle East respiratory
syndrome coronavirus in Saudi Arabia: a descriptive genomic study. Lancet 2013;382(9909):1993
2002.
[20] Drosten C, Seilmaier M, Corman VM, Hartmann W, Scheible G, Sack S, et al. Clinical features and virological analysis of a case of Middle
East respiratory syndrome coronavirus infection. Lancet Infect Dis 2013;13(9):745
51.
[21] WHO. MERS Global summary and assessment of risk; 2019. Available at: https://www.who.int/csr/disease/coronavirus_infections/risk-assess-
ment-august-2018.pdf. [Accessed 21 January 2019.
[22] Alagaili AN, Briese T, Mishra N, Kapoor V, Sameroff SC, de Wit E, et al. Middle East respiratory syndrome coronavirus infection in drome-
dary camels in Saudi Arabia. MBio 2014;5(2):e00884-14.
[23] Hemida MG, Perera RA, Al Jassim RA, Kayali G, Siu LY, Wang P, et al. Seroepidemiology of Middle East respiratory syndrome (MERS)
coronavirus in Saudi Arabia (1993) and Australia (2014) and characterisation of assay specificity. Eurosurveillance 2014;19(23):20828.
[24] Reusken CB, Farag EA, Jonges M, Godeke GJ, El-Sayed AM, Pas SD, et al. Middle East respiratory syndrome coronavirus (MERS-CoV) RNA
and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014. Eurosurveillance 2014;19
(23):20829.
[25] Nowotny N, Kolodziejek J. Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels, Oman, 2013. Eurosurveillance
2014;19(16):20781.
[26] Hemida MG, Chu DK, Poon LL, Perera RA, Alhammadi MA, Ng HY, et al. MERS coronavirus in dromedary camel herd, Saudi Arabia. Emerg
Infect Dis 2014;20(7):1231.
[27] Memish ZA, Al-Tawfiq JA, Makhdoom HQ, Assiri A, Alhakeem RF, Albarrak A, et al. Respiratory tract samples, viral load, and genome frac-
tion yield in patients with Middle East respiratory syndrome. J Infect Dis 2014;210(10):1590
4.
[28] Memish ZA, Cotten M, Meyer B, Watson SJ, Alsahafi AJ, Al Rabeeah AA, et al. Human infection with MERS coronavirus after exposure to
infected camels, Saudi Arabia, 2013. Emerg Infect Dis 2014;20(6):1012.
[29] Chu DK, Poon LL, Gomaa MM, Shehata MM, Perera RA, Zeid DA, et al. ). MERS coronaviruses in dromedary camels, Egypt. Emerg Infect
Dis 2014;20(6):1049.
[30] Reeves T, Samy AM, Peterson AT. MERS-CoV geography and ecology in the Middle East: analyses of reported camel exposures and a prelim-
inary risk map. BMC Res Notes 2015;8(1):1
7.
[31] Khalafalla AI, Lu X, Al-Mubarak AI, Dalab AHS, Al-Busadah KA, Erdman DD. MERS-CoV in the upper respiratory tract and lungs of drome-
dary camels, Saudi Arabia, 2013
2014. Emerg Infect Dis 2015;21(7):1153.
[32] Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet 2015;386(9997):995
1007.
[33] Buzon MJ, Seiss K, Weiss R, Brass AL, Rosenberg ES, Pereyra F, et al. ). Inhibition of HIV-1 integration in ex vivo-infected CD4 T cells from
elite controllers. J Virol 2011;85(18):9646
50.
[34] Van Boheemen S, de Graaf M, Lauber C, Bestebroer TM, Raj VS, Zaki AM, et al. Genomic characterization of a newly discovered coronavirus
associated with acute respiratory distress syndrome in humans. MBio 2012;3(6):e00473-12.
[35] Kato T, Takami Y, Deo VK, Park EY. Preparation of virus-like particle mimetic nanovesicles displaying the S protein of Middle East respira-
tory syndrome coronavirus using insect cells. J Biotechnol 2019;306:177
84.
[36] Arabi YM, Balkhy HH, Hayden FG, Bouchama A, Luke T, Baillie JK, et al. Middle East respiratory syndrome. N Engl J Med 2017;376
(6):584
94.
[37] Meyerholz DK, Lambertz AM, McCray Jr PB. Dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the Middle
East respiratory syndrome. Am J Pathol 2016;186(1):78
86.
[38] Widagdo, W., Raj, V. S., Schipper, D., Kolijn, K., Van Leenders, G. J., Bosch, et al. Differential expression of the Middle East respiratory syn-
drome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels. J Virol 2016;90(9):4838
4842.
[39] Xu J, Jia W, Wang P, Zhang S, Shi X, Wang X, et al. Antibodies and vaccines against Middle East respiratory syndrome coronavirus. Emerg
Microbes Infect 2019;8(1):841
56.
[40] Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, et al. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor
CD26. Nature 2013;500(7461):227
31.
[41] Mou H, Raj VS, Van Kuppeveld FJ, Rottier PJ, Haagmans BL, Bosch BJ. The receptor binding domain of the new Middle East respiratory syn-
drome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies. J Virol 2013;87
(16):9379
83.
[42] Wang N, Shi X, Jiang L, Zhang S, Wang D, Tong P, et al. Structure of MERS-CoV spike receptor-binding domain complexed with human
receptor DPP4. Cell Res 2013;23(8):986
93.
[43] Molaei S, Dadkhah M, Asghariazar V, Karami C, Safarzadeh E. The immune response and immune evasion characteristics in SARS-CoV,
MERS-CoV, and SARS-CoV-2: vaccine design strategies. Int Immunopharmacol 2020;92:107051.
[44] Kirchdoerfer RN, Ward AB. Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat Commun 2019;10(1):1
9.
[45] Arabi YM, Harthi A, Hussein J, Bouchama A, Johani S, Hajeer AH, et al. Severe neurologic syndrome associated with Middle East respiratory
syndrome corona virus (MERS-CoV). Infection 2015;43(4):495
501.
 Middle East respiratory syndrome Chapter | 7 121

[46] Falzarano D, De Wit E, Martellaro C, Callison J, Munster VJ, Feldmann H. Inhibition of novel β coronavirus replication by a combination of
interferon-α2b and ribavirin. Sci Rep 2013;3(1):1
6.
[47] Falzarano D, De Wit E, Rasmussen AL, Feldmann F, Okumura A, Scott DP, et al. Treatment with interferon-α2b and ribavirin improves out-
come in MERS-CoV
infected rhesus macaques. Nat Med 2013;19(10):1313
17.
[48] Faure E, Poissy J, Goffard A, Fournier C, Kipnis E, Titecat M, et al. Distinct immune response in two MERS-CoV-infected patients: can we go
from bench to bedside? PLoS One 2014;9(2):e88716.
[49] Müller MA, Meyer B, Corman VM, Al-Masri M, Turkestani A, Ritz D, et al. Presence of Middle East respiratory syndrome coronavirus antibo-
dies in Saudi Arabia: a nationwide, cross-sectional, serological study. Lancet Infect Dis 2015;15(5):559
64.
[50] Alraddadi BM, Watson JT, Almarashi A, Abedi GR, Turkistani A, Sadran M, et al. Risk factors for primary Middle East respiratory syndrome
coronavirus illness in humans, Saudi Arabia, 2014. Emerg Infect Dis 2016;22(1):49.
[51] Chan RW, Hemida MG, Kayali G, Chu DK, Poon LL, Alnaeem A, et al. Tropism and replication of Middle East respiratory syndrome corona-
virus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study. Lancet Respir Med 2014;2(10):813
22.
[52] Alosaimi B, Hamed ME, Naeem A, Alsharef AA, AlQahtani SY, AlDosari KM, et al. MERS-CoV infection is associated with downregulation
of genes encoding Th1 and Th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract.
Cytokine 2020;126:154895.
[53] Nakamura H, Yoshimura K, McElvaney NG, Crystal RG. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic
fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest 1992;89(5):1478
84.
[54] Braciale TJ, Hahn YS. Immunity to viruses. Immunol Rev 2013;255(1):5.
[55] Totura AL, Baric RS. SARS coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. Curr Opin Virol
2012;2(3):264
75.
[56] Lau SK, Lau CC, Chan KH, Li CP, Chen H, Jin DY, et al. Delayed induction of proinflammatory cytokines and suppression of innate antiviral
response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment. J Gen Virol 2013;94(12):2679
90.
[57] Channappanavar R, Perlman S. Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology.
Semin Immunopathol 2017;39(5):529
39.
[58] Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, et al. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses 2019;11(1):59.
[59] Bradley BT, Bryan A. Emerging respiratory infections: the infectious disease pathology of SARS, MERS, pandemic influenza, and Legionella.
Semin Diagn Pathol 2019;36(3):152
9.
[60] Algahtani H, Subahi A, Shirah B. Neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review
of the literature. Case Rep Neurol Med 2016;2016:3502683.
[61] Verstrepen K, Baisier L, Cauwer HD. Neurological manifestations of COVID-19, SARS and MERS. Acta Neurol Belgica 2020. Available
from: https://doi.org/10.1007/s13760-020-01412-4.
[62] Drosten C, Muth D, Corman VM, Hussain R, Al Masri M, HajOmar W, et al. An observational, laboratory-based study of outbreaks of middle
East respiratory syndrome coronavirus in Jeddah and Riyadh, kingdom of Saudi Arabia, 2014. Clin Infect Dis 2015;60(3):369
77.
[63] World Health Organization. Infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care: WHO
interim guidelines (No. WHO/CDS/EPR/2007.6). Geneva: World Health Organization; 2007.
[64] Van Doremalen N, Bushmaker T, Karesh WB, Munster VJ. Stability of Middle East respiratory syndrome coronavirus in milk. Emerg Infect
Dis 2014;20(7):1263.
[65] WHO Update on MERS-CoV Transmission from Animals to Humans (2022), and Interim Recommendations for At-risk Groups; 2014.
Available from: http://www.who.int/csr/disease/coronavirus_infections/MERS_CoV_RA_20140613.pdf?ua 5 1 [Accessed 16 February 2015].
[66] Guery B, Poissy J, El Mansouf L, Séjourné C, Ettahar N, Lemaire XMERS-CoV Study Group. Clinical features and viral diagnosis of two cases
of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission. Lancet 2013;381(9885):2265
72.
[67] Poissy J, Goffard A, Parmentier-Decrucq E, Favory R, Kauv M, Kipnis E, et al. Kinetics and pattern of viral excretion in biological specimens
of two MERS-CoV cases. J Clin Virol 2014;61(2):275
8.
[68] Al Johani S, Hajeer AH. MERS-CoV diagnosis: an update. J Infect Public Health 2016;9(3):216
19.
[69] Kim YS, Son A, Kim J, Kwon SB, Kim MH, Kim P, et al. Chaperna-mediated assembly of ferritin-based Middle East respiratory syndrome-
coronavirus nanoparticles. Front Immunol 2018;9:1093.
[70] Zhang RZ, Tsai SS, Chao HR, Shieh YC, Chuang KP. Innovation of a new virus-like nanoparticle vaccine system for the specific aerosol rela-
tive disease. Aerosol Air Qual Res 2016;16(10):2421
7.
[71] Du, L., Jiang, S. Middle East respiratory syndrome: current status and future prospects for vaccine development. Expert Opin Biol Ther
2015;15(11):1647
1651.
[72] Muthumani K, Falzarano D, Reuschel EL, Tingey C, Flingai S, Villarreal DO, et al. A synthetic consensus anti
spike protein DNA vaccine
induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates. Sci Transl Med 2015;7(301)
301ra132.
[73] Alharbi NK, Padron-Regalado E, Thompson CP, Kupke A, Wells D, Sloan MA, et al. ChAdOx1 and MVA based vaccine candidates against
MERS-CoV elicit neutralising antibodies and cellular immune responses in mice. Vaccine 2017;35(30):3780
8.
[74] Munster VJ, Wells D, Lambe T, Wright D, Fischer RJ, Bushmaker T, et al. Protective efficacy of a novel simian adenovirus vaccine against
lethal MERS-CoV challenge in a transgenic human DPP4 mouse model. NPJ Vaccines 2017;2:28. Available from: https://doi.org/10.1038/
s41541-017-0029-1.
122 Pandemic Outbreaks in the 21st Century

[75] Haagmans BL, Van Den Brand JM, Raj VS, Volz A, Wohlsein P, Smits SL, et al. An orthopoxvirus-based vaccine reduces virus excretion after
MERS-CoV infection in dromedary camels. Science 2016;351(6268):77
81.
[76] Assiri A, Al-Tawfiq JA, Al-Rabeeah AA, Al-Rabiah FA, Al-Hajjar S, Al-Barrak A, et al. Epidemiological, demographic, and clinical character-
istics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study. Lancet Infect Dis 2013;13
(9):752
61.
[77] Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, et al. Hospital outbreak of Middle East respiratory syndrome corona-
virus. N Engl J Med 2013;369(5):407
16.
[78] Hui DS, Memish ZA, Zumla A. Severe acute respiratory syndrome versus the Middle East respiratory syndrome. Curr Opin Pulmonary Med
2014;20(3):233
41.
[79] WHO MERS-CoV Research Group. State of knowledge and data gaps of Middle East respiratory syndrome coronavirus (MERS-CoV) in
humans. PLoS Curr 2013;5.
[80] Chan JF, Chan KH, Kao RY, To KK, Zheng BJ, Li CP, et al. Broad-spectrum antivirals for the emerging Middle East respiratory syndrome
coronavirus. J Infect 2013;67(6):606
16.
[81] De Wilde AH, Raj VS, Oudshoorn D, Bestebroer TM, van Nieuwkoop S, Limpens RW, et al. MERS-coronavirus replication induces severe
in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment. J Gen Virol 2013;94(Pt 8):1749.
[82] Josset L, Menachery VD, Gralinski LE, Agnihothram S, Sova P, Carter VS, et al. Cell host response to infection with novel human coronavirus
EMC predicts potential antivirals and important differences with SARS coronavirus. MBio 2013;4(3).
[83] Al-Tawfiq JA, Momattin H, Dib J, Memish ZA. Ribavirin and interferon therapy in patients infected with the Middle East respiratory syndrome
coronavirus: an observational study. Int J Infect Dis 2014;20:42
6.
[84] Lu L, Liu Q, Zhu Y, Chan KH, Qin L, Li Y, et al. Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor.
Nat Commun 2014;5(1):1
12.
[85] Hui DS, Azhar EI, Kim YJ, Memish ZA, Oh MD, Zumla A. Middle East respiratory syndrome coronavirus: risk factors and determinants of pri-
mary, household, and nosocomial transmission. Lancet Infect Dis 2018;18(8):e217
27.
[86] Zumla A, Hui DS. Infection control and MERS-CoV in health-care workers. Lancet 2014;383(9932):1869
71.
[87] World Health Organization. A research and development Blueprint for action to prevent epidemics; 2018. Available from: http://www.who.int/
csr/research-and-development/en.
Chapter 8

Advances in vaccination to combat

pandemic outbreaks
Subramanyam Dasari
Indiana University School of Medicine, Bloomington, IN, United States

8.1 Introduction
Emerging infectious diseases represent a significant and growing cause of morbidity and mortality. Historically, the
global burden of disease has been disproportionately borne throughout the world especially in developing countries [1].
In the realm of infectious diseases, epidemic spreads beyond the countries which makes the worst-case scenario called
as a pandemic [2]. Massive increase in the global population, as well as the extreme increase of contact between people
due to the urbanization, is highly favorable for global spreading of pandemic diseases. The pandemic risk is further
increased by the global climate change that influences the pathogen-bearing vectors, promoting infections with a range
of vector-borne diseases. The pandemics would occur with larger cities, more exotic trade routes, and increased contact
with different populations of people, animals, and ecosystems. These macrotrends are having a profound impact on the
spread of infectious diseases. The occurrence of pandemic outbreaks in the past decades has clearly demonstrated the
reality of global pandemic threats [3].
Antonine Plague (CE 165) was possibly an early appearance of smallpox and measles that initially began with the
Huns. The Greek physician and writer Galen described the plague as “great” and of long duration, and mentioned fever,
diarrhea, and pharyngitis as well as a skin eruption, sometimes dry and sometimes pustular, that appeared on the ninth
day of the illness. The historian William H. McNeill claims another plague called Plague of Cyprian (251
c.270)
which is mostly related to the measles. The severe devastation to the European population from the two plagues may
indicate that people had no previous exposure to either disease, which brought immunity to survivors. Some historians
also believe that both outbreaks involved smallpox [4].
The Plague of Justinian is generally considered as the first historical plague pandemic (CE 541
549) caused by the bac-
terium Yersinia pestis. The disease afflicted the entire Mediterranean Basin, Europe, and the Near East. In 2013 researchers
confirmed earlier speculation that the cause of the Plague of Justinian was Y. pestis, the same bacterium responsible for the
Black Death (1347
51) [5]. Based upon DNA analysis of bones found in graves, the type of plague was characterized by
enlarged lymphatic gland and is carried by rats and spread by fleas. It was very probable that the other two types of plague,
pneumonic and septicemic, were also present [6]. First plague pandemic was one of the deadliest pandemics in history,
resulting in the deaths of an estimated 25
100 million people during two centuries of recurrence.
Cholera Pandemic, the word cholera means a bilious disease and is originally derived from the Greek term “chole”
or bile which means a nonspecific that has been used previously for various gastrointestinal diseases [7]. During the
17th century, cholera was known as severe summer diarrhea and prior to the discovery of the cholera etiologic agent in
the 19th century. Epidemic cholera first appeared in India and may have been endemic there causing outbreaks as far
back as the 16th century. In 1817 a virulent outbreak struck in Eastern India in the state of Bengal and rapidly spread to
other parts of the subcontinent. After spreading to Persia, the Middle East, and Southeast Asia, it reached the
Mediterranean in 1821 and died down, ending the first pandemic.
Primarily, John Snow a British physician explained that the contamination of the drinking water with human excreta is
associated with cholera outbreak. Later in 1854, Filippo Pacini (1812
83) [8] an anatomist, and then in 1883, Robert Koch
(1843
1910) the German bacteriologist, discovered “Vibrio cholerae” the responsible microbial agent of cholera [9]. Vibrio
cholera bacterium primarily afflicts the digestive tract. It is extremely acute, with a gestation period of 1
2 days, and upon

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00002-1

© 2021 Elsevier Inc. All rights reserved. 123
124 Pandemic Outbreaks in the 21st Century

onset, causes severe diarrhea and sometimes vomiting, both leading to dehydration that can cause death in a matter of hours.
Those who are malnourished or already have intestinal parasites can be especially at high risk of death. The cure is relatively
simple: oral or intravenous hydration is administered as quickly as possible. Historically, cholera has been remembered as
one of the diseases in the 19th century and continued into the 20th century, until the present time. Seven major pandemics of
cholera took place during the 19th and 20th centuries causing many people to suffer [7,10].
The influenza (1918
19) pandemic was the deadliest events in recorded human history, killing an estimated
50
100 million persons, this is more than the Great World War I victims [11]. It was caused by the Avian origin of an
influenza A virus of the H1N1 subtype and it spread worldwide during 1918
19. In the United States, it was first iden-
tified in military personnel in spring 1918. It is estimated that about one-third of the world’s population became infected
with this virus. The number of deaths was estimated to be at least 50 million worldwide. The high mortality in healthy
people, including those in the 20
40-year age group, was a unique feature of this pandemic [12].
In 1957 another flu pandemic called Asian flu (1957
58) caused by influenza A virus subtype H2N2 was originated
from China (Guizhou), which infects at least one million people worldwide. This novel type of strain was developed by
the recombination of Avian influenza (probably from geese) and human influenza viruses [13]. Currently, licensed sea-
sonal influenza vaccines are specific for predefined viral strains and are unable to protect against a future pandemic.
Hence, new vaccine technologies able to induce broad protection against influenza A viruses are urgently required.

8.2 Human immunodeficiency virus/acquired immunodeficiency syndrome

Acquired immunodeficiency syndrome (AIDS) is a chronic, potentially life-threatening infection caused by the human
immunodeficiency virus (HIV). HIV/AIDS damages the immune system and interferes with body’s ability to fight
infection and disease. HIV/AIDS is considered a global pandemic by some authors [14]. However, the WHO currently
uses the term “global epidemic” to describe HIV. According to WHO statistics, as of 2018, approximately 37.9 million
people were infected with HIV globally. There are no specific drugs to treat HIV/AIDS but medications can dramati-
cally slow the progression of the disease. Despite the development of effective highly active antiretroviral therapy
(HAART), drugs are cost-intensive and access to therapy remains problematic in resource-limited settings in which
most infections occur. Development of a direly needed vaccine against HIV has proven extremely difficult and identifi-
cation of a suitable method for generating such a vaccine remains the focus of research [15].
Even after more than three decades of HIV, there are no licensed HIV preventative or therapeutic vaccines.
Recently, novel strategies are being developed to prevent and treat HIV-1. Nonefficacious preventative vaccine
approaches include bivalent recombinant gp120 alone, Adenovirus 5 (Ad5) vector HIV vaccine, and the DNA prime/
Ad5 boost vaccine regimen. However, the ALVAC-HIV prime/AIDSVAX B/E gp120 boost regimen showed 31.2%
efficacy at 3.5 years and is being investigated as clade C constructs with an additional boost. Monoclonal antibodies for
passive immunization or treatment show promise, with VRC01 entering advanced clinical trials [16].

8.3 Dengue virus

Initially, only nine countries had experienced the dengue epidemics, then dramatically spread worldwide, and entered
100 countries including Africa, America, Eastern Mediterranean, South-East Asia, and western pacific. The largest
number of dengue cases ever reported globally was in 2019. All regions were affected, and dengue transmission was
recorded in Afghanistan for the first time. In 2020 dengue continued affecting several countries, with reports of increas-
ing number of cases in Bangladesh, Brazil, Cook Islands, Ecuador, India, Indonesia, Maldives, Mauritania, Mayotte
(Fr), Nepal, Singapore, Sri Lanka, Sudan, Thailand, Timor-Leste, and Yemen [17]. Initially, in 1920s development of
the DENV vaccine began based on the attenuating DENV in blood with ox-bile or grinding DENV-infected Aedes
aegypti mosquitoes. In 1952 Sabin and Schlesinger developed an attenuated strain of DENV1 in the mouse brain. This
vaccine was protective in 16 volunteers subjected to the bite of infected mosquitoes [18]. The first dengue vaccine,
Dengvaxia (CYD-TDV), developed by Sanofi Pasteur was licensed in December 2015 and has now been approved by
regulatory authorities in B20 countries [19].

8.4 Chikungunya virus

Chikungunya is a mosquito-borne viral disease first described during an outbreak in southern Tanzania in 1952. It is an
RNA virus that belongs to the alphavirus genus of the family Togaviridae. The name “chikungunya” is derived from a
word in the Kimakonde language, meaning, “to become contorted,” and describes the stooped appearance of sufferers
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 125

with joint pain (arthralgia). Chikungunya virus (CHIKV) was first identified in Tanzania in 1952 and for the following
B50 years was isolated and caused occasional outbreaks in Africa and Asia. Since 2004, chikungunya has been spread
rapidly and been identified in over 60 countries throughout Asia, Africa, Europe, and the Americas [20].
There are currently no licensed vaccines or therapies. A randomized, placebo-controlled, phase 2 clinical trial to
assess the vaccine VRC-CHKVLP059
00-VP (CHIKV VLP) was underway [21].

8.5 Zika virus

Zika virus (ZIKV) is a newly emergent relative of the Flaviviridae family and is linked to dengue (DENV) and chikun-
gunya (CHIVKV). ZIKV is one of the rising pathogens promptly surpassing geographical borders. ZIKV infection was
characterized by mild disease with fever, headache, rash, arthralgia, and conjunctivitis, with exceptional reports of an
association with Guillain
Barre syndrome and microcephaly [22].
ZIKV was first identified in Uganda in 1947 in monkeys and later it was identified in humans in 1952 in Uganda
and the United Republic of Tanzania. Further outbreaks of ZIKV disease have been recorded in Africa, America, Asia,
and the Pacific. Sporadic cases of human ZIKV infections were found across Africa and Asia, from 1960s to 1980s.
Contemporary outbreaks of ZIKV appeared on Yap Island in the Federated States of Micronesia in 2007 and then fol-
lowed by an epidemic in French Polynesia in 2013 [23]. Both of these events were associated with a high prevalence of
infection, with greater than 11% of people on the islands presenting with ZIKV-associated symptoms [23,24]. The next
ZIKV outbreak began in northeastern Brazil in late 2014, which was rapidly spread to many other countries including
America in 2015 and 2016 [25,26]. Recent studies on flaviviruses have shown that antibody responses against the
ZIKV viral E protein can serve as protection in animals and humans [27,28]. The historical evidence on the efficacy of
the vaccines from yellow fever, Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses in preventing
infection and epidemics suggests that an effective vaccine targeting all strains of ZIKV should be feasible, amino acid
variability between E proteins of the two lineages [29]. In terms of prioritization, prepubescent children, men, and
women of childbearing age living within or traveling to endemic areas might be priority recipients in a ZIKV vaccina-
tion campaign [30], JEV, and TBEV viruses.

8.6 Severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) was the first severe and readily transmissible new disease that emerged in
the 21st century. It is a viral respiratory disease/illness caused by a coronavirus called SARS-associated coronavirus
(SARS-CoV). SARS was first reported in 2002 in China and the outbreak was spread to other countries including North
America, South America, Europe, and Asia [31]. SARS is an airborne virus and can spread through small droplets of
saliva in a similar way to the cold and influenza (Fig. 8.1).

8.7 Ebola viral disease

Recent outbreak of the Ebola virus (EBOV) in West Africa can be described as the most severe public health emer-
gency in modern times with mortality rate of up to 74% [32]. The initial source of the outbreak appeared to be in a
small village in southern Guinea in 2013 and the WHO notified an outbreak of Ebola viral disease (EVD) in Guinea in
2014. EBOV is zoonotic filovirus, covered of envelope, nonsegmented negative-stranded RNA. Animal reservoir of the
EBOV is considered the fruit bat. Transmission to humans required contact with animal tissues or body fluids. EBOV
is usually transmitted through direct mucous membrane or percutaneous exposure to infected body fluids [33].
Clinical presentation of EVD is very uncharacteristic and can mimic many tropical diseases such as malaria, typhoid
fever, cholera, so to establish diagnosis the epidemiological data must be strongly taken into consideration. EBOV
causes a severe hemorrhagic fever, which is often a fatal illness affecting humans and other primates. Usually the
EBOV is transmitted through direct mucous membrane or percutaneous exposure to infected body fluids, such as blood,
stool, and vomits. The average EVD case fatality rate is around 50%. Case fatality rates have varied from 25% to 90%
in past outbreaks [32]. The first outbreak occurred in Central Africa, and the second outbreak was the largest and most
complex Ebola outbreak that appeared in West Africa around 2014
16 [34]. There were more cases and deaths in this
outbreak than all others combined. It has also spread between countries, starting in Guinea then moving across land bor-
ders to Sierra Leone and Liberia.
There is no specific treatment available for EBOV, and several experimental therapies are under development but
not fully tested in humans. However, recently FDA has approved some experimental treatments for emergency use in
126 Pandemic Outbreaks in the 21st Century

FIGURE 8.1 Some of the major pandemics that have occurred over time along with the death cases globally.

patients with Ebola infection. One oral nucleotide analog called brincidofovir, which is a modified version of cidofovir,
is used as treatment for EBOV. In vitro data suggest its activity against Ebola and recently FDA approved it for Phase
2 clinical study [35].
Since Ebola is highly contagious and the possibility of its aerosol route of infection is not definitely excluded it is
crucial to reduce the risk of human-to-human transmission. Isolation of the infected patients, protective clothes and
equipment, control protocols, proper waste and sample management are essential to protect medical personnel and pre-
vent the spreading of the infection [36].

8.8 Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome (MERS) is an illness caused by another type of coronavirus. MERS patients devel-
oped severe respiratory illness with symptoms of fever, cough, and shortness of breath. About three or four out of every
10 patients reported with MERS have died. The first cases of this coronavirus infection that appeared in Saudi Arabia
(Jeddah) were reported in 2012; after this outbreak, coronavirus continued to spread overseas to many countries in
Asia, Africa, Europe, and America [37].
According to the European Centre for Disease Prevention and Control, this outbreak, mostly occurred in Middle
Eastern countries, including those in the Gulf region (Saudi Arabia, Qatar, United Arab Emirates, Oman, Bahrain,
Kuwait, and Iraq), as well as Jordan, Syria, Lebanon, Palestine, and Egypt [38,39].
MERS-CoV is also a zoonotic virus; however, the zoonotic transmission that occurs is not clear yet. International
epidemiological studies suggested that bats might be carriers of MERS-CoV. These studies have tested bats mainly for
the 329-bp fragment of RdRp using blood, fecal, and oral samples [40]. The median incubation period for MERS-CoV
is 5
12 days, and adults are mostly affected. However, several pediatric cases are also reported in Saudi Arabia.
Nearly, 87% of patients show flu-like symptoms, like fever and cough, chills, to more severe symptoms, including
shortness of breath in 48% of patients and respiratory failure, resulting in the requirement for intubation and ventilation.
The treatment for typical coronavirus infection is supportive therapy, in deeding administration of antipyretics and
analgesics, maintenance of hydration, respiratory support by ventilation or extracorporeal membrane oxygenation, and
treatment with antibiotics in the case of bacterial superinfections. However, such treatments may not be sufficient for
MERS-CoV infections, which may be more severe. Ribavirin and interferon-alpha have been shown to have synergistic
effects and are more beneficial when started early. Additionally, mycophenolic acid has been shown to be efficacious
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 127

and can be used as a monotherapy; however, initial clinical trials included few patients, and further studies are neces-
sary. Although several companies are attempting to develop MERS-CoV vaccines, none is available yet. Improving our
understanding of viral antibodies will facilitate the design of appropriate and efficacious vaccines.

8.9 Human coronavirus

Coronaviruses are named for the crown-like spikes on their surface. There are four main subgroupings of coronaviruses,
known as alpha, beta, gamma, and delta. Human coronaviruses were first identified in the mid-1960s. The seven coro-
naviruses that can infect people are: 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus),
HKU1 (beta coronavirus); the other three human coronaviruses are MERS-CoV (the beta coronavirus that causes
MERS), SARS-CoV (the beta coronavirus that causes SARS), and SARS-CoV-2 [the novel coronavirus that causes
coronavirus disease 2019 (COVID-19)].
Coronaviruses typically cause respiratory and enteric infections in both animals and humans and were considered
relatively benign to humans before the outbreak in 2002 and 2003 in China caused by severe acute respiratory syn-
drome (SARS-CoV) [41
43]. In 2013 another pathogenic coronavirus with a clinical picture reminiscent of SARS was
isolated in patients presenting with pneumonia in the Middle Eastern countries called Middle East respiratory syndrome
coronavirus (MERS-CoV), [37]. After that, in December 2019 a novel coronavirus (2019-nCoV) has emerged in China
and has turned into a global health concern [37].
In December 2019 a group of patients with pneumonia, caused by a newly identified β-coronavirus, occurred in
Wuhan, China. This coronavirus was initially named the 2019-novel coronavirus by WHO in January 2020 and then
named the disease as COVID-19. The COVID-19 is a β-coronavirus with enveloped nonsegmented positive-sense RNA
virus (subgenus sarbecovirus, Orthocoronavirinae subfamily) [44]. COVID-19 is postulated to have emerged from ani-
mals, although its exact source is not clear. Based on viral genome sequencing results and evolutionary analysis, bat
has been suspected as natural host of virus origin, and SARS-CoV-2 might be transmitted from bats to infect humans
via unknown intermediate hosts. Now it is clear that SARS-CoV-2 could use angiotensin-converting enzyme 2, the
same receptor as SARS-CoV to infect humans [45]. As an emerging acute respiratory infectious disease, COVID-19 pri-
marily spreads through the respiratory tract, by droplets, respiratory secretions, and direct contact [46] for a low infec-
tive dose [47]. Recent epidemiological studies reported that the incubation period is 1
14 days, mostly 3
7 days. In
addition, the COVID-19 is more transmissible during the latency period [48]. It is highly transmissible in humans, espe-
cially in the elderly and people with underlying diseases. The median age of patients is 47
59 years, and 41.9%

45.7% of patients were females [46,49,50]. COVID-19 patients presented certainly similar symptoms, such as fever,
malaise, and cough [51]. Most adults or children with SARS-CoV-2 infection presented with mild flu-like symptoms
and a few patients are in critical condition and rapidly develop acute respiratory distress syndrome, respiratory failure,
multiple organ failure, even deaths [52].
Globally, there have been 50,676,072 confirmed cases of COVID-19, including 1,261,075 deaths, reported to WHO
as of 10 November 2020.

8.10 Evolution of vaccine technologies

For more than a century conventional vaccines have been developed, based on the Pasteur principles by isolating, inac-
tivating, and injecting the microorganisms causing the diseases or a portion of it [53]. During the last three decades,
several conventional vaccines have been developed and successfully decreased the burden of a number of infectious dis-
eases. For the first time, at the end of the 18th century Edward Jenner used the infected cowpox materials to immunize
the smallpox and introduced the term “vaccine” [54].
In the 1970s the development of genetic engineering especially the expression of proteins in plasmids and the
ability to sequence DNA leads to develop the first recombinant vaccine, the hepatitis B vaccine [55]. Recombinant
technology enables the target antigen to be produced outside of the parent organism, which is much easier to handle
the infectious agents or potentially toxic components. As a result, the quantity of antigen produced, the vaccine’s
safety, and the purity of the product are improved; efficacy is increased, and potential side effects are minimized.
Next major progress in the evolution of vaccine technologies was the development of adjuvant treatment in the
1980s. Adjuvants are used to improve the presentation of an antigen to the immune system or to enhance its immuno-
genicity. Mineral salts calcium phosphate and alum are the only adjuvants currently approved in the United States for
concomitant use with vaccines [56].
128 Pandemic Outbreaks in the 21st Century

By the end of the 20th century, novel discoveries including protein conjugation to capsular polysaccharides and
the advent of methods to engineer recombinant DNA led to the development of vaccines for prevention of bacterial
pneumonia and meningitis, hepatitis B, and the recent development of the human papillomavirus vaccine [57
59].
Vaccines have now led to the eradication of smallpox, near eradication of polio, and prevention of untold millions
of deaths from infectious diseases each year. However, several vaccines are not available for diseases that cause
significant global morbidity and mortality. Because these vaccines needed to be more complex in their pathogene-
sis, exhibit extensive variability, or have evolved immune evasion mechanisms to prevent the immune system
(Table 8.1).

8.11 Box 1: ideal characteristics of a vaccine

1. Ideal vaccine shows an impeccable safety profile in all populations (irrespective of races and ages).
2. Elicits a high level of stable efficacy.
3. Mostly requires only a single dose (two doses to confer protection called booster dose).
4. Stimulates protection within 1
2 weeks of administration.
5. Easily administrable, like orally, nasally or transcutaneously or with a needle-free injection device.
6. Manufactured in large scale and relatively less cost effective.

8.12 Box 2: strategies for the development of vaccines

1. Viral vector-based vaccines.
2. Conjugate vaccines.
3. Attenuation of known pathogens by inactivation of specific genes.
4. Bacterial live vector vaccines.
5. Subunit vaccines.
6. Reverse vaccinology’ (genomics-based vaccines).
7. Nonliving antigen delivery systems (such as liposomes, proteosomes, virus-like particles, virosomes, and
microspheres).
8. DNA vaccines and replicons.
9. “Heterologous” prime-boost vaccination strategies.
10. Powerful but well-tolerated adjuvants to enhance immune responses to vaccines.
11. Needle-free administration of vaccines.

TABLE 8.1 Major global infections prevented by vaccines.

Bacterial infections Viral infections

Cholera Influenza
Diphtheria Hepatitis A and B
Haemophilus influenzae Human papilloma virus
Meningococcal meningitis Japanese encephalitis
Plague Measles and Mumps
Pneumococcal pneumonia Polio
Tetanus Rabies
Tuberculosis Rotavirus diarrhea
Typhoid fever Rubella
Smallpox and yellow fever

The level of efficacy for the vaccines noted here varies in different populations and
regions of the world.
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 129

8.13 Viral vector-based vaccines

Viral vectors are promising tools for the development of novel vaccines and vaccination, and they rely on the
delivery of one or more antigens encoded by an unrelated, modified virus. This technology employs either live
(replicating but often attenuated) or nonreplicating vectors. The concept of viral vector was introduced in 1972, by
Jackson et al., using recombinant DNA technology from SV40 virus by genetic engineering [60]. Later in 1982
Moss et al. reported the use of vaccinia virus as a transient gene expression vector [61]. A variety of viruses were
used as vaccine vectors by engineering them to encode for heterologous antigens that are shuttled into the host
cells by the vector. Upon delivery, antigens are expressed, and the host can induce immune responses against the
respective target pathogen [62].
Viral vector-based vaccines present advantages over traditional vaccines in that they can enhance a broad range of
immunogenicity without an adjuvant and induce a robust cytotoxic T lymphocyte response to eliminate virus-infected
cells.
A wide range of different viruses has been used for constructing viral vector-based vaccines. These vectors include
adenoviruses, adeno-associated viruses (AAV), togaviruses (Semliki Forest virus), paramyxoviruses (measles virus,
Newcastle disease virus, or human parainfluenza virus), rhabdoviruses (vesicular stomatitis virus), and poxviruses
(Modified vaccinia Ankara, MVA).

8.14 Adenovirus vectors

Adenovirus vectors are the most used vectors for vaccine. Both preclinical and clinical studies suggest that the adenovi-
rus vector-based vaccines have shown protective efficacy against a wide variety of infectious diseases. Adenoviruses
are nonenveloped viruses with icosahedral capsid and a linear double-strand genome with 30
40 kb size. Adenovirus
vaccines can be constructed based on early transcripts 1A and B, as replication-competent or replication-defective vec-
tors [2]. Adenovirus vectors can stably express inserts of up to 8 kb and the vector is manufactured in mammalian cell
culture system (HEK 293 cells) [63]. Ad vector and recombinant Ad vector-based vaccines have been examined in clin-
ical trials against influenza [64], and HIV-1 [65]. Very recently, a group of scientists evaluated the safety and immuno-
genicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: in phase 1/2, single blind, randomized controlled
trial. The ChAdOx1 nCoV-19 vaccine (AZD1222) consists of the replication-deficient simian adenovirus vector
ChAdOx1, containing the full-length structural surface glycoprotein (spike protein) of SARS-CoV-2, with a tissue plas-
minogen activator leader sequence. ChAdOx1 nCoV-19 expresses a codon-optimized coding sequence for the spike pro-
tein (GenBank accession number MN908947) [66].

8.15 Poxviruses as vaccine vector

Vaccinia virus is a large member of the poxvirus family, with a linear, double-stranded DNA genome approximately
190 kb in length. Because of its large size, the viral genome has a high capacity for the insertion of foreign gene or tar-
get gene (approximately 25 kb). The virus is traditionally used for the smallpox vaccine, and its efficacy and safety
have been demonstrated. During vaccine development, highly attenuated vaccinia virus strains have been generated,
including replication-competent (LC16m8) and replication-deficient (NYVAC, ALVAC, TROVAC, and MVA) strains.

8.16 Frontrunners in COVID-19 vaccine race

Leaving in its wake more than 12 million infections, over 550,000 deaths, and an economic toll in the trillions of
dollars to date, the COVID-19 pandemic has devastated the most vulnerable in our society [67]. A vaccine is
urgently needed to prevent COVID-19 and thereby stem complications and deaths resulting from transmission of
the disease. Several companies are in the race to develop a safe and effective vaccine candidate to meet both medi-
cal need and the potential payday. According to WHO, total of 49 vaccine candidates are at the stage of clinical
trials in humans by mid-November. Thirteen of them are at the advanced stage of “clinical phase 3,” in which vac-
cine’s effectiveness is tested on large scale, generally on thousands of peoples globally. BioNTech/Pfizer and
Moderna are the two frontrunners in the vaccine’s development and have the United States-FDA approval on both
sides of the Atlantic.
130 Pandemic Outbreaks in the 21st Century

8.17 Vector-based vaccines come to the fore in the COVID-19 pandemic

Recently, Feng-Cai Zhu reported on its randomized controlled trial of Phase II COVID-19 vaccine that uses nonre-
plicating adenovirus serotype 5 (Ad5). During this randomized trial, they mainly assess the immunogenicity and
safety of the vaccine candidate. The phase II results indicate that the Ad5-vectored COVID-19 vaccine has a suffi-
cient safety profile with mild transient adverse events. Single dose of immunization induces rapid onset of immune
responses within 14 days and significant humoral and cellular immune responses within 28 days in majority of the
recipients. Receptor binding domain (RBD) and neutralizing antibody responses are also in peak level at day 28
(95%) [68]. Another group of scientists, Corbett et al. [69] synthesized an mRNA encoding prefusion-stabilized
SARS-CoV-2 S-2P protein in vitro and given to the nonhuman primate. The mRNA was purified by oligo-dT affin-
ity purification and encapsulated in a lipid nanoparticle through a modified ethanol-drop nanoprecipitation process
[70]. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens
were assessed by polymerase chain reaction, and antibody and T-cell responses were assessed before upper- and
lower-airway challenge with SARS-CoV-2. Viral replication was not detectable in BAL fluid by day 2 after chal-
lenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of
the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral
genome or antigen was noted in lungs of animals in either vaccine group. The results from mRNA-1273 vaccina-
tion induced robust S-specific antibody responses, targeting both the RBD and the N-terminal subdomains of S1
with potent neutralizing capacity. Antibodies are the primary mechanism of protection for this vaccine because of
the rapid reduction in viral replication within 24
48 h after challenge and the detection of antibodies in BAL fluid.
Vaccination also induces type 1 helper T-cell (Th1)
biased CD4 T-cell responses and low or undetectable Th2 or
CD8 T-cell responses.
Another group of scientists from Russia developed a heterologous COVID-19 vaccine based on Adenovirus vector.
The vaccine consisting of a recombinant adenovirus type 26 (rAd26) vector and a recombinant adenovirus type 5
(rAd5) vector, both carrying the gene for SARS-CoV-2 spike glycoprotein (rAd26-S and rAd5-S) [71]. The two (both
frozen and formulated vaccine) open and nonrandomized phase 1 and 2 studies indicated that no serious adverse events
were detected. All participants produced antibodies to SARS-CoV-2 glycoprotein. At the 42 days of vaccination, IgG
(RBD) titers were 14,703 with the frozen formulation and 11,143 with the lyophilized formulation, and neutralizing
antibodies were 49  25 with the frozen formulation and 45  95 with the lyophilized formulation, with a 100% of sero-
conversion rate. Cell-mediated responses were detected in all participants at day 28, with median cell proliferation of
both frozen and lyophilized formulation [71].
Adenovirus vectors can induce robust and durable neutralizing antibody responses after a single immunization
[72,73]. A single-shot of vaccine would have important logistical and practical advantages compared with a two-dose
vaccine for mass vaccination campaigns and control of the pandemics like SARS-CoV. A series of Ad26 vectors that

TABLE 8.2 Seven Ad26 vectors were produced that expressed SARS-CoV-2 S protein variants.

S. SARS-CoV-2 S protein variants References

no.
1 Tissue plasminogen activator (tPA) leader sequence with full-length S (tPA.S) [75]
2 tPA leader sequence with full-length S and mutation of the furin cleavage site and two proline-stabilizing [76,77]
mutations (tPA.S.PP)
3 Wild-type leader sequence with native full-length S (S) [78]
4 Wild-type leader sequence with S and deletion of the cytoplasmic tail (S.dCT) [78]
5 Tandem tPA and wild-type leader sequences with full-length S (tPA.WT.S) [75]
6 wild-type leader sequence with S with deletion of the transmembrane region and cytoplasmic tail reflecting [77]
the soluble ectodomain, with mutation of the furin cleavage site, proline-stabilizing mutations, and a fold on
trimerization domain (S.dTM.PP)
7 Wild-type leader sequence with full-length S and mutation of the furin cleavage site and proline-stabilizing
mutations (S.PP)
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 131

TABLE 8.3 Vaccine candidates specific to SARS-CoV-2, several clinical trials are underway testing for both safety and
efficacy.

S. Developer(s) Principle method of Clinical evidence Status References

no. vaccine
1 Moderna and the mRNAs for the SARS- Vaccine is safe and Phase 1, Phase 2, and [69]
United States CoV-2 spike protein elicits higher levels of Phase 3 clinical trials
government SARS-CoV-2 are underway across
antibodiesPhase 3 trial the United States
suggest that it is nearly
95% protective
2 CanSino Biologics and Nonreplicating Vaccine elicits an Phase 3 trials have [68]
the Academy of adenovirus 5 (Ad5) immune response, begun in Russia and
Military Medical vector carrying the either a T cell response Pakistan
Sciences gene for the SARS-CoV- or an antibody
2 spike protein response
3 University of Oxford Adenovirus vaccine Vaccine is safe and Clinical trials at [79]
and AstraZeneca vector (ChAdOx1) elicits strong antibody various stages are
carrying the gene for and T cell immune underway around the
the SARS-CoV-2 spike responses, and globe and in mid-
protein preliminary results from October, the FDA
a Phase 2Preliminary authorized the restart
results from the Phase 3 of the United States
study showed the trial
vaccine to be 70%
effective on average
4 Sinovac Biotech China, Inactivated SARS-CoV- The vaccine is safe and A Phase 1/2 clinical [80]
Brazil, Bangladesh, and 2 elicits an antibody- trial is underway in
Indonesia based immune China, and Phase 3
response, although trials are underway in
antibody levels were Bangladesh, Indonesia,
lower than in patients and Turkey
who have been
infected and recovered
5 Wuhan Institute of Inactivated SARS-CoV- Vaccine tested positive A Phase 1/2 clinical [81]
Biological Products and 2 for antibodies against trial is underway in
China National SARS-CoV-2 China. A Phase 3 trial
Pharmaceutical Group is underway in UAE
(Sinopharm) China and
United Arab Emirates
(UAE)
6 Beijing Institute of Inactivated SARS-CoV- Two-dose Phase 1/2 clinical trial [82,83]
Biological Products and 2 (BBIBP-CorV) immunization with underway in China
China National 2 μg per dose BBIBP-
Pharmaceutical Group CorV efficiently
(Sinopharm) China protects rhesus
macaques and induces
high levels of
neutralizing antibodies
titers in animal
modelsIt is genetically
stable and seems to be
safe in animals
7 BioNTech and Pfizer Lipid-nanoparticle Preliminary results from The companies [84,85]
International formulated mRNA the early-stage trials announced in early
vaccine that encodes suggest that the vaccine October that the
the trimerized receptor- is safe and elicits vaccine was starting

(Continued )
132 Pandemic Outbreaks in the 21st Century

TABLE 8.3 (Continued)

S. Developer(s) Principle method of Clinical evidence Status References

no. vaccine
binding domain (RBD) higher levels of SARS- the process of rolling
of the glycoprotein CoV-2 antibodies than submission with the
spike for SARS-CoV-2 infection with the European Medicines
(BNT162b1) virusThe vaccine Agency, and in
generated T cell November, they
responses in addition to announced that they
antibody responses were asking the United
States FDA for
Emergency Use
Authorization
8 Novavax Australia and Nanoparticles carrying Vaccine candidate to A Phase 1 and 2 [86]
South Africa antigens derived from be safe and elicit clinical trial is
the SARS-CoV-2 spike neutralizing antibody underway in Australia
protein (NVX- levels greater than and South AfricaA
CoV2373) those provided by Phase 3 trial has begun
treatment with COVID- in the United Kingdom
19 convalescent serum
9 Gamaleya Research Adenovirus vector Vaccine has a good Phase 1/2 clinical trials [71]
Institute of displaying the SARS- safety profile and are underway in Russia
Epidemiology and CoV-2 spike protein on induced strong humoral to test liquid and
Microbiology, Health its surface (two and cellular immune powder forms of the
Ministry of the Russian components, a responses in vaccine. Approved for
Federation, Acellena recombinant participantsThe first phase 3 clinical trials
Contract Drug adenovirus type 26 interim data analysis of are underway in
Research and (rAd26) vector and a the Sputnik V vaccine various countries
Development Russia recombinant against COVID-19
(Sputnik V vaccine) adenovirus type 5 phase 3 clinical trials in
(rAd5) vector, both the Russian federation
carrying the gene for demonstrated 92%
SARS-CoV-2 spike efficacy
glycoprotein)
10 Janssen International Nonreplicating Vaccine protected Early-stage clinical [74]
adenovirus 26 (Ad26) monkeys against SARS- trials are underway in
vectors with CoV-2 infectionSingle the United States,
undisclosed genetic dose of the vaccine to Belgium, and Japan,
material of SARS-CoV- be safe and to elicit and a Phase 2 trial is
2 neutralizing antibodies ongoing in Spain,
in nearly all study Germany, and the
participants Netherlands.
Meanwhile, an
international Phase 3
trial is underway in the
United States, Mexico,
several South
American countries,
the Philippines, South
Africa, and Ukraine.
11 Inovio Pharmaceuticals Spike Results of Phase 1 trial A Phase 1/2 is also No
USA protein
encoding suggested that the underway in various published
DNA molecules vaccine was safe and countries including the data
through the skin spurred immune USA and South Korea
(synthetic DNA vaccine responses in 94% of
for COVID-19) the 36 participants
analyzed

(Continued )
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 133

TABLE 8.3 (Continued)

S. Developer(s) Principle method of Clinical evidence Status References

no. vaccine
12 Shenzhen Geno- This is a synthetic mini- Cells using lentivirus Clinical trial phase 1/2 No
Immune Medical gene-based vaccine vectors that it has used is underway in China published
Institute (LV- called LV-SMENP_DC, to develop CAR-T cell for the dendritic cell data
SMENP_DC) China which equates to therapies as well as and T cell
based
lentivirus (disabled gene therapies vaccines, and a Phase
HIV) used to deliver 1 trial is underway for
viral proteins and a vaccine using
immune modularity artificial antigen-
genes that trigger presenting cells
dendritic cells
13 Bharat Biotech India Inactivated SARS-CoV- In guinea pigs and Phase 1/2 trial is No
2 mice the vaccine was underway in India. The published
safe and elicited an country’s regulatory data
immune response agency approved the
vaccine for emergency
use at the beginning of
January
14 Clover The vaccine delivers The Trimer-Tag Phase 1 clinical trial is No
Biopharmaceuticals pieces of the SARS- platform used is the underway clinical in published
Australia CoV-2 spike protein basis for other viral Australia data
vaccines in
development
15 Inovio Pharmaceuticals Administers spike Mice and guinea pigs Phase 1 and Phase 2/3 No
United States and protein
encoding mounted immune clinical trials are published
South Korea DNA molecules responses against the ongoing in the United data
through the skin virus, according to a States. A Phase 1/2
recent preprint, and study is also underway
they announced in South Korea
interim results from the
Phase 1 trial
16 Institute of Medical Inactivated SARS-CoV- Data from the Phase 1 Phase 1/2 clinical trial No
Biology at Chinese 2 trial, the vaccine is safe is underway in China published
Academy of Medical and elicits an immune data
Sciences, West China response, although
Second University levels of neutralizing
Hospital, Yunnan antibodies started to
Center for Disease drop after just 2 weeks
Control and Prevention
China
17 CureVac Belgium and RNA vaccine; details The preliminary data Phase 1 trial is No
Germany not disclosed from the ongoing Phase underway in Belgium published
1 trial that showed the and Germany; data
vaccine candidate company says it could
elicits levels of manufacture 10 million
neutralizing antibodies doses by that time
comparable to levels
seen in people who
have recovered from
serious COVID-19
illness and appears to
trigger the production of
SARS-CoV-2
fighting T
cells
18 Aivita Biomedical US A patient’s own Antigen-carrying A Phase 1/2 trial has No
dendritic cells are dendritic cells triggered been approved to published
modified to carry a response in the same begin in California data
SARS-CoV-2 antigens patient’s lymphocytes
and then reinfused in vitro

(Continued )
134 Pandemic Outbreaks in the 21st Century

TABLE 8.3 (Continued)

S. Developer(s) Principle method of Clinical evidence Status References

no. vaccine
19 Genexine South Korea DNA encoding the The vaccine was Phase 1/2 trial is No
SARS-CoV-2 spike shown to produce underway in South published
protein neutralizing antibodies Korea data
in nonhuman primates
20 Vaxine, Medytox Recombinant SARS- Vaxine developed an Phase 1 trial approved No
Australia CoV-2 spike protein experimental swine flu to begin in Australia published
plus a polysaccharide vaccine during the data
adjuvant 2009 pandemic
21 Zydus Cadila India Engineered DNA In a preclinical study, Phase 1/2 trial is No
plasmid encoding a the vaccine neutralized underway in India published
SARS-CoV-2 antigen SARS-CoV-2 in a virus data
neutralization assay

encode different variants of the SARS-CoV-2 spike (S) protein were developed and their immunogenicity and protec-
tive efficacy against the SARS-CoV-2 challenge in rhesus macaques were evaluated [74]. They produced seven Ad26
vectors that expressed SARS-CoV-2 S variants that reflected different leader sequences, antigen forms, and stabilization
mutations as shown in Table 8.2.
The optimal Ad26 vaccine induced robust neutralizing antibody responses and provided complete or near-complete
protection in bronchoalveolar lavage and nasal swabs after the SARS-CoV-2 challenge. Titers of vaccine-elicited neu-
tralizing antibodies correlated with protective efficacy, suggesting an immune correlate of protection. These data dem-
onstrate robust single-shot vaccine protection against SARS-CoV-2 in nonhuman primates. These findings suggest that
the serum antibody titers may prove a useful correlate of protection for the COVID-19 vaccine. However, CD4 1 and
CD8 1 intracellular cytokine staining responses did not correlate with protection (data not shown).
Table 8.3 shows that various vaccine candidates specific to SARS-CoV-2, and the status of their clinical trials
including principle method for developing the vaccine and the safety and efficacy of the clinical trials.

8.18 Conclusion
The major pandemics such as HIV, Ebola, Zika, and currently COVID-19 have raised the awareness of global threats to
health of humans posed by newly emerging pathogens and can provide the impetus to prepare new platforms for vac-
cine technologies that can battle the challenges of emerging pandemic situations. New platforms include viral vectors
and nucleic acid-based vaccines provide some of these challenges by representing efficacy and safety-wise. It is also
important that these advanced technologies allow fast vaccine manufacturing to meet the emerging outbreaks. Each vac-
cine technology has its own advantages and disadvantages based on its ability to induce immune responses, safety for
human use, production capacity, and route of administration. Although further studies will be required to fully charac-
terize and develop this vaccination technology in humans, clinical studies conducted so far have yielded overall encour-
aging results in terms of safety, inducing immunogenicity, and providing support for further clinical explorations.
A single technology will not be able to provide a solution for any present or future outbreaks, the combination of
present knowledge, ongoing development, and growing understanding of human immunology and majorly human food
habitats can provide tools to successfully combat emerging global threats.

References
[1] Maslow JN. Challenges and solutions in the development of vaccines against emerging and neglected infectious diseases. Hum Vaccin
Immunother 2019;15(10):2230
4.
[2] Rauch S, Jasny E, Schmidt KE, Petsch B. New vaccine technologies to combat outbreak situations. Front Immunol 2018;9:1963.
[3] Halabowski D, Rzymski P. Taking a lesson from the COVID-19 pandemic: preventing the future outbreaks of viral zoonoses through a multi-
faceted approach. Sci Total Environ 2021;757:143723.
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 135

[4] Furuse Y, Suzuki A, Oshitani H. Origin of measles virus: divergence from rinderpest virus between the 11th and 12th centuries. Virol J
2010;7:52.
[5] Mordechai L, Eisenberg M, Newfield TP, Izdebski A, Kay JE, Poinar H. The Justinianic Plague: an inconsequential pandemic? Proc Natl Acad
Sci USA 2019;116(51):25546
54.
[6] Harbeck M, Seifert L, Hansch S, Wagner DM, Birdsell D, Parise KL, et al. Yersinia pestis DNA from skeletal remains from the 6(th) century
AD reveals insights into Justinianic Plague. PLoS Pathog 2013;9(5):e1003349.
[7] Azizi M, Azizi F. History of cholera outbreaks in Iran during the 19(th) and 20(th) centuries. Middle East J Dig Dis 2010;2(1):51
5.
[8] Bentivoglio M, Pacini P. Filippo Pacini: a determined observer. Brain Res Bull 1995;38(2):161
5.
[9] Gangarosa EJ. The epidemiologic basis of cholera control. Bull Pan Am Health Organ 1974;8(3):189
97.
[10] Hardy A. Cholera, quarantine and the English preventive system, 1850
1895. Med Hist 1993;37(3):250
69.
[11] Morens DM, Fauci AS. The 1918 influenza pandemic: insights for the 21st century. J Infect Dis 2007;195(7):1018
28.
[12] Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918
1920 "Spanish" influenza pandemic. Bull Hist Med 2002;76
(1):105
15.
[13] Jackson C. History lessons: the Asian flu pandemic. Br J Gen Pract 2009;59(565):622
3.
[14] Cohen MS, Hellmann N, Levy JA, DeCock K, Lange J. The spread, treatment, and prevention of HIV-1: evolution of a global pandemic. J Clin
Invest 2008;118(4):1244
54.
[15] Robinson HL. HIV/AIDS vaccines: 2018. Clin Pharmacol Ther 2018;104(6):1062
73.
[16] Gray GE, Laher F, Lazarus E, Ensoli B, Corey L. Approaches to preventative and therapeutic HIV vaccines. Curr Opin Virol 2016;17:104
9.
[17] Halstead SB. Dengue. Lancet 2007;370(9599):1644
52.
[18] McArthur MA, Sztein MB, Edelman R. Dengue vaccines: recent developments, ongoing challenges and current candidates. Expert Rev
Vaccines 2013;12(8):933
53.
[19] Sabchareon A, Wallace D, Sirivichayakul C, Limkittikul K, Chanthavanich P, Suvannadabba S, et al. Protective efficacy of the recombinant,
live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 2012;380
(9853):1559
67.
[20] Weaver SC, Lecuit M. Chikungunya virus and the global spread of a mosquito-borne disease. N Engl J Med 2015;372(13):1231
9.
[21] Chen GL, Coates EE, Plummer SH, Carter CA, Berkowitz N, Conan-Cibotti M, et al. Effect of a Chikungunya virus-like particle vaccine on
safety and tolerability outcomes: a randomized clinical trial. JAMA 2020;323(14):1369
77.
[22] Kazmi SS, Ali W, Bibi N, Nouroz F. A review on Zika virus outbreak, epidemiology, transmission and infection dynamics. J Biol Res
(Thessalon) 2020;27:5.
[23] Musso D, Nilles EJ, Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect 2014;20(10):O595
596.
[24] David LM, Ravishankara AR, Kodros JK, Pierce JR, Venkataraman C, Sadavarte P. Premature mortality due to PM2.5 over India: effect of
atmospheric transport and anthropogenic emissions. Geohealth 2019;3(1):2
10.
[25] Castro LA, Fox SJ, Chen X, Liu K, Bellan SE, Dimitrov NB, et al. Assessing real-time Zika risk in the United States. BMC Infect Dis 2017;17
(1):284.
[26] Fernandez E, Diamond MS. Vaccination strategies against Zika virus. Curr Opin Virol 2017;23:59
67.
[27] Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibo-
dies from individuals infected with West Nile Virus. J Virol 2006;80(14):6982
92.
[28] Vogt MR, Moesker B, Goudsmit J, Jongeneelen M, Austin SK, Oliphant T, et al. Human monoclonal antibodies against West Nile virus
induced by natural infection neutralize at a postattachment step. J Virol 2009;83(13):6494
507.
[29] Haddow AD, Schuh AJ, Yasuda CY, Kasper MR, Heang V, Huy R, et al. Genetic characterization of Zika virus strains: geographic expansion
of the Asian lineage. PLoS Negl Trop Dis 2012;6(2):e1477.
[30] Marston HD, Lurie N, Borio LL, Fauci AS. Considerations for developing a Zika virus vaccine. N Engl J Med 2016;375(13):1209
12.
[31] Feng D, de Vlas SJ, Fang LQ, Han XN, Zhao WJ, Sheng S, et al. The SARS epidemic in mainland China: bringing together all epidemiological
data. Trop Med Int Health 2009;14(Suppl 1):4
13.
[32] Bociaga-Jasik M, Piatek A, Garlicki A. Ebola virus disease—pathogenesis, clinical presentation and management. Folia Med Cracov 2014;54
(3):49
55.
[33] Kanapathipillai R. Ebola virus disease
current knowledge. N Engl J Med 2014;371(13):e18.
[34] Piot P, Muyembe JJ, Edmunds WJ. Ebola in west Africa: from disease outbreak to humanitarian crisis. Lancet Infect Dis 2014;14(11):1034
5.
[35] Maurice J. WHO meeting chooses untried interventions to defeat Ebola. Lancet 2014;384(9948):e45
46.
[36] West TE, von Saint Andre-von Arnim A. Clinical presentation and management of severe Ebola virus disease. Ann Am Thorac Soc 2014;11
(9):1341
50.
[37] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in
Saudi Arabia. N Engl J Med 2012;367(19):1814
20.
[38] Buchholz U, Muller MA, Nitsche A, Sanewski A, Wevering N, Bauer-Balci T, et al. Contact investigation of a case of human novel coronavirus
infection treated in a German hospital, October-November 2012. Euro Surveill 2013;18(8):20406.
[39] Bermingham A, Chand MA, Brown CS, Aarons E, Tong C, Langrish C, et al. Severe respiratory illness caused by a novel coronavirus, in a
patient transferred to the United Kingdom from the Middle East, September 2012. Euro Surveill 2012;17(40):20290.
[40] Al-Osail AM, Al-Wazzah MJ. The history and epidemiology of Middle East respiratory syndrome corona virus. Multidiscip Respir Med
2017;12:20.
136 Pandemic Outbreaks in the 21st Century

[41] Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17(3):181
92.
[42] Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute
respiratory syndrome. N Engl J Med 2003;348(20):1967
76.
[43] Zhong NS, Zheng BJ, Li YM, Poon ZH, Xie KH, Chan PH, et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in
Guangdong, People’s Republic of China, in February, 2003. Lancet 2003;362(9393):1353
8.
[44] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel Coronavirus from patients with pneumonia in China, 2019. N Engl J Med
2020;382(8):727
33.
[45] Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin.
Nature 2020;579(7798):270
3.
[46] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia.
N Engl J Med 2020;382(13):1199
207.
[47] Lee PI, Hsueh PR. Emerging threats from zoonotic coronaviruses-from SARS and MERS to 2019-nCoV. J Microbiol Immunol Infect 2020;53
(3):365
7.
[48] Jin YH, Cai L, Cheng ZS, Cheng H, Deng T, Fan YP, et al. A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus
(2019-nCoV) infected pneumonia (standard version). Mil Med Res 2020;7(1):4.
[49] Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med
2020;382(18):1708
20.
[50] Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected
pneumonia in Wuhan, China. JAMA 2020;323(11):1061
9.
[51] Poutanen SM, Low DE, Henry B, Finkelstein S, Rose D, Green K, et al. Identification of severe acute respiratory syndrome in Canada. N Engl
J Med 2003;348(20):1995
2005.
[52] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Lancet 2020;395(10223):497
506.
[53] Rappuoli R, Pizza M, Del Giudice G, De Gregorio E. Vaccines, new opportunities for a new society. Proc Natl Acad Sci USA 2014;111
(34):12288
93.
[54] Escoula L. [Toxinogenic moulds in silage. VI—Effect of propionic and formic acids on the production of patulin and of byssochlamic acid by
Byssochlamys nivea Westling (author’s transl)]. Ann Rech Vet 1975;6(3):315
24.
[55] Stevens CE, Taylor PE, Tong MJ, Toy PT, Vyas GN, Nair PV, et al. Yeast-recombinant hepatitis B vaccine. Efficacy with hepatitis B immune
globulin in prevention of perinatal hepatitis B virus transmission. JAMA 1987;257(19):2612
16.
[56] Aguilar JC, Rodriguez EG. Vaccine adjuvants revisited. Vaccine 2007;25(19):3752
62.
[57] McNeil C. Who invented the VLP cervical cancer vaccines? J Natl Cancer Inst 2006;98(7):433.
[58] Schneerson R, Barrera O, Sutton A, Robbins JB. Preparation, characterization, and immunogenicity of Haemophilus influenzae type b
polysaccharide-protein conjugates. J Exp Med 1980;152(2):361
76.
[59] Black S, Shinefield H, Fireman B, Lewis E, Ray P, Hansen JR, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conju-
gate vaccine in children. Northern California Kaiser Permanente Vaccine Study Center Group. Pediatr Infect Dis J 2000;19(3):187
95.
[60] Jackson DA, Symons RH, Berg P. Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40
DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci USA 1972;69(10):2904
9.
[61] Mackett M, Smith GL, Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci USA 1982;79
(23):7415
19.
[62] Bouard D, Alazard-Dany D, Cosset FL. Viral vectors: from virology to transgene expression. Br J Pharmacol 2009;157(2):153
65.
[63] Lauer KB, Borrow R, Blanchard TJ. Multivalent and multipathogen viral vector vaccines. Clin Vaccine Immunol 2017;24(1):e00298-16.
[64] Gurwith M, Lock M, Taylor EM, Ishioka G, Alexander J, Mayall T, et al. Safety and immunogenicity of an oral, replicating adenovirus serotype 4
vector vaccine for H5N1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study. Lancet Infect Dis 2013;13(3):238
50.
[65] Kibuuka H, Kimutai R, Maboko L, Sawe F, Schunk MS, Kroidl A, et al. A phase 1/2 study of a multiclade HIV-1 DNA plasmid prime and
recombinant adenovirus serotype 5 boost vaccine in HIV-Uninfected East Africans (RV 172). J Infect Dis 2010;201(4):600
7.
[66] Folegatti PM, Ewer KJ, Aley PK, Angus B, Becker S, Belij-Rammerstorfer S, et al. Safety and immunogenicity of the ChAdOx1 nCoV-19 vac-
cine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet 2020;396(10249):467
78.
[67] Heaton PM. The Covid-19 vaccine-development multiverse. N Engl J Med 2020;383(20):1986
8.
[68] Zhu FC, Li YH, Guan XH, Hou LH, Wang WJ, Li JX, et al. Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vec-
tored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial. Lancet 2020;395(10240):1845
54.
[69] Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, et al. Evaluation of the mRNA-1273 Vaccine against SARS-
CoV-2 in Nonhuman Primates. N Engl J Med 2020;383(16):1544
55.
[70] Hassett KJ, Benenato KE, Jacquinet E, Lee A, Woods A, Yuzhakov O, et al. Optimization of lipid nanoparticles for intramuscular administra-
tion of mRNA vaccines. Mol Ther Nucleic Acids 2019;15:1
11.
[71] Logunov DY, Dolzhikova IV, Zubkova OV, Tukhvatullin AI, Shcheblyakov DV, Dzharullaeva AS, et al. Safety and immunogenicity of an
rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies
from Russia. Lancet 2020;396(10255):887
97.
[72] Abbink P, Larocca RA, De La Barrera RA, Bricault CA, Moseley ET, Boyd M, et al. Protective efficacy of multiple vaccine platforms against
Zika virus challenge in rhesus monkeys. Science 2016;353(6304):1129
32.
 Advances in vaccination to combat pandemic outbreaks Chapter | 8 137

[73] Abbink P, Larocca RA, Visitsunthorn K, Boyd M, De La Barrera RA, Gromowski GD, et al. Durability and correlates of vaccine protection
against Zika virus in rhesus monkeys. Sci Transl Med 2017;9(420):eaao4163.
[74] Mercado NB, Zahn R, Wegmann F, Loos C, Chandrashekar A, Yu J, et al. Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus
macaques. Nature 2020;586(7830):583
8.
[75] Alharbi NK, Padron-Regalado E, Thompson CP, Kupke A, Wells D, Sloan MA, et al. ChAdOx1 and MVA based vaccine candidates against
MERS-CoV elicit neutralising antibodies and cellular immune responses in mice. Vaccine 2017;35(30):3780
8.
[76] Pallesen J, Wang N, Corbett KS, Wrapp D, Kirchdoerfer RN, Turner HL, et al. Immunogenicity and structures of a rationally designed prefu-
sion MERS-CoV spike antigen. Proc Natl Acad Sci U S A 2017;114(35):E7348
57.
[77] Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion confor-
mation. Science 2020;367(6483):1260
3.
[78] Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K, et al. A DNA vaccine induces SARS coronavirus neutralization and pro-
tective immunity in mice. Nature 2004;428(6982):561
4.
[79] Ramasamy MN, Minassian AM, Ewer KJ, Flaxman AL, Folegatti PM, Owens DR, et al. Safety and immunogenicity of ChAdOx1 nCoV-19
vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial.
Lancet 2021;396(10267):1979
93.
[80] Zhang Y, Zeng G, Pan H, Li C, Hu Y, Chu K, et al. Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy
adults aged 18
59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial. Lancet Infect Dis 2020;21(2):181
92.
[81] Xia S, Duan K, Zhang Y, Zhao D, Zhang H, Xie Z, et al. Effect of an inactivated vaccine against SARS-CoV-2 on safety and immunogenicity
outcomes: Interim analysis of 2 randomized clinical trials. JAMA 2020;324(10):951
60.
[82] Wang H, Zhang Y, Huang B, Deng W, Quan Y, Wang W, et al. Development of an inactivated vaccine candidate, BBIBP-CorV, with potent
protection against SARS-CoV-2. Cell 2020;182(3) 713
721.e719.
[83] Xia S, Zhang Y, Wang Y, Wang H, Yang Y, Gao GF, et al. Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBIBP-CorV:
a randomised, double-blind, placebo-controlled, phase 1/2 trial. Lancet Infect Dis 2021;21(1):39
51.
[84] Mulligan MJ, Lyke KE, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults.
Nature 2020;586(7830):589
93.
[85] Sahin U, Muik A, Derhovanessian E, Vogler I, Kranz LM, Vormehr M, et al. COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T
cell responses. Nature 2020;586(7830):594
9.
[86] Keech C, Albert G, Cho I, Robertson A, Reed P, Neal S, et al. Phase 1
2 trial of a SARS-CoV-2 recombinant spike protein nanoparticle vac-
cine. N Engl J Med 2020;383(24):2320
32.
This page intentionally left blank
Chapter 9

Pandemics of the 21st century: lessons

and future perspectives
Hunasanahally Puttaswamygowda Gurushankara
Department of Zoology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Kasaragod, India

9.1 The legacy of an epidemic and pandemic

Epidemic and Pandemic words are used more frequently in the outbreak of infectious disease. Both terms contain-demic
used for disease outbreaks, but they are not the same. Even medical experts and science communicators regularly blur
the distinction between these two words and use them interchangeably or incorrectly altogether. Because each term’s
definition is fluid and changes as diseases become more or less prevalent over time. An epidemic is “an outbreak of an
infectious disease that spreads quickly and affects many individuals within a community, population, or region at the
same time.” The word epidemic is derived from the Greek word epidemios, which means within the country/people of
district/among the people. The World Health Organization (WHO) further specifies epidemics as localized at a region
or community level. Still, the number of those infected in that region is significantly higher than usual. For example,
when COVID-19 was in Wuhan, China, it was an epidemic. Hippocrates (460
477 BCE), the famous physician of
ancient Greece, father of modern medicine, and the first epidemiologist, examined different diseases that afflicted peo-
ple several times of the year. His work was published under a very familiar title today: “Epidemic” about 2500 years
ago. The ancient Greek physician wrote about the “Cough of Perinthus,” possibly many illnesses. The meaning of a
term used to describe one in his country (“epi”: on and “demos”: people); Hippocrates and the cough of perinthus left
us the first legacy of an epidemic. Later, Thomas Lodge (1603) mentioned in a treatise of the plague, An Epidemick
plague, is a common and popular sicknesse, hapning in some region, or countrey, at a certaine time, caused by a cer-
taine indisposition of the aire, or waters of the same region, producing in all sorts of people, the same kind of sicknesse.
The word epidemic began being used frequently later in the 17th century [1].
A pandemic is an epidemic occurring at vast geographic area (worldwide), crossing international boundaries and
usually affecting many people over multiple countries or continents in the Greek pandemos (of all the people), which
itself is from the pan (all, every) and dēmos (people) [1]. The word pandemic was started using in the 19th century. It
can be a type of epidemic; an epidemic is not a pandemic type. As J. A. Allen mentioned in the Boston Medical and
Surgical Journal, September, 5 1832, “Those diseases that have some strong resemblance in their general characters,
and attack many individuals to a large extent of the country the same time termed epidemics. If all, or about all the
inhabitants of a country, be similarly attacked, at or near the same time, with a particular complaint, it is more properly
called a pandemic.” For example, when COVID-19 was in Wuhan, China, it was an epidemic. Due to the geographical
spread, crossing international boundaries and affecting many people over multiple countries or continents turned it into
a pandemic. The WHO defines an epidemic explicitly as “a worldwide spread of a new disease.” WHO officially
declared the COVID-19 outbreak as a pandemic based on the global spread and severity of the disease on March 11,
2020.

9.2 Origin of communicable diseases

Infectious diseases such as malaria, tuberculosis, leprosy, influenza, smallpox, and other contagious diseases first
appeared during humankind’s hunter-gatherer days when they shifted to agricultural life 10,000 years ago.
Microorganisms such as bacteria, viruses, parasites, and so on can be spread, directly or indirectly, from one person to
Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00011-2
© 2021 Elsevier Inc. All rights reserved. 139
140 Pandemic Outbreaks in the 21st Century

another through insect bites that cause these infectious diseases. Ingesting contaminated food, water, or air also causes
contagious diseases. Many emerging infectious diseases are associated with human modification of the environment.
The concurrent advent of agriculture created communities that made human infectious diseases more possible to out-
break. After the origin of agriculture may have changed the transmission system’s human pathogens and the pathogen-
carrying organisms (vectors), it has resulted in novel interactions between humans and wildlife. Animals’ domestication
is providing a stable channel for human’s infection [2]. In five stages, the animal pathogen can transform into a special-
ized specific pathogen to cause diseases to humans [3] (Table 9.1).

9.2.1 Clio-epidemiology to neo-epidemiology

The term “clio-epidemiology” refers to the mining and reanalysis of the rich store of often-neglected historical informa-
tion [4]. Clio-epidemiology defines the practice of studying information from past infectious disease outbreaks for
advice about the present. It emphasizes the importance of local knowledge, data, and the value of age-old detailed infor-
mation. It understands the epidemiology of emerging and reemerging infectious diseases in the past and monitoring
them in the present neo-epidemiological surveillance, research, and policy. It provides information on global pandemics
that happened in the World. Past pandemics teach to manage current and future emerging diseases. There is an invalu-
able treasure of useful historical data that have only just begun to be used to inform our actions. If well noted, the les-
sons might help us avoid repeating the same history of outbreaks today. Recurrent episodes of infectious diseases have
had profound and lasting effects on human societies throughout history. Those global events have powerfully shaped
human civilization’s economic, political, and social aspects [5]. Some of the pandemic outbreaks have well-defined
modern medicine’s basic views, pushing the scientific community to develop modern epidemiological surveillance, pre-
vention, immunization, and specific type of treatments.

9.2.2 The worst diseases outbreaks in history

This chapter outlines the most notable disease outbreaks in human history for understanding the facts to avoid future
pandemics.

TABLE 9.1 Transformation of the animal pathogen into a specific human pathogen.

Stage Transformation process Example(s)

1 A pathogen present in animals but not in humans (pathogen only in Most malarial plasmodia specific to one host
animals) species or a closely related group of host
species
2 A pathogen of animals has been transmitted from animals to humans Anthrax, Nipah, Rabies, and West Nile viruses
(primary infection) but has not been transferring between humans
(secondary infection)
3 Animal pathogens can undergo only a few secondary transmission Ebola, Marburg, and Monkey poxviruses
cycles between humans so that occasional human outbreaks triggered
by a primary infection soon die out (limited attack)
4 A disease in animals has a sylvatic cycle of infecting humans by direct
transmission from the animal host. It also undergoes long sequences of
secondary transmission between humans without animal hosts
(prolonged outbreak)
Based on the primary and secondary transmission of the pathogen, stage
4 is divided into three substages
4a Diseases exist in animals and have a sylvatic cycle of infecting humans Chagas’ disease and Yellow fever
through the animal’s primary transmission
4b Both sylvatic and direct transmissions can be possible Dengue fever in forest areas of West Africa and
Southeast Asia
4c The most significant transmission is between humans Influenza A, Cholera, Typhus, and West African
sleeping sickness
5 Diseases causing pathogens present exclusively in humans (exclusive The agents cause Falciparum Malaria, Measles,
human pathogens) Mumps, Rubella, Smallpox, and Syphilis
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 141

9.2.3 Prehistoric pandemic

About 5000 years ago (3000 BCE), an outbreak wiped out a prehistoric village in China. They stuffed dead bodies inside a
house and burned. No age group was spared. The skeletons of juveniles, young adults, and middle-aged people found inside
the home. This site is now called “Hamin Mangha” in northeastern China. Another prehistoric mass burial happened
roughly the same period at Miaozigou, Northeastern China. Together, these two outbreaks ravaged the entire region [6].

9.2.4 Historic pandemics

The earliest recorded pandemic has happened during the Peloponnesian War in Athens (430 BCE) [7]. The disease
spread through Libya, Ethiopia, and Egypt; it crossed the Athenian walls. Two-thirds of the population died. Suspected
it as typhoid symptoms included fever, thirst, bloody throat and tongue, red skin, and lesions [8].

9.2.5 Plague
The Antonine plague (CE 165) was an early appearance of smallpox in the Huns. They have infected the Germans, who
passed it to the Romans and then returning troops spread it throughout the Roman Empire. Symptoms included fever, sore
throat, diarrhea, and the patient lived long with pus-filled sores [9]. It was continued until about CE 180, claiming Emperor
Marcus Aurelius as one of its victims [10]. Deadliest disease outbreaks to a population for the first time when people lack
immunity. Greek word plaga (strike, blow), the word plague is a polyseme to describe a particular virulent contagious
febrile disease caused by Yersinia pestis. It can refer to any sickness; in Latin, the words are plaga and pestis [11].
Cyprian plague (CE 250), named after the first known victim, St. Cyprian, a bishop of Carthage, described the epi-
demic as signaling the World’s end. Cyprian plague has killed 5000 people a day in Rome alone. This plague’s symp-
toms were diarrhea, vomiting, throat ulcers, fever, and gangrenous hands and feet. City dwellers fled to the village to
escape infection and instead spread the disease. Possibly it was started in Ethiopia. It passed through Northern Africa,
Rome, then to Egypt. Recurring outbreaks over the next three centuries hit the British defense system in CE 444 [12].
Justinian Plague (CE 541) was history’s first recorded real plague pandemic named Justinian; the Byzantine Empire
(Eastern Roman Empire) Justinian contracted the plague himself but did not succumb [13]. It originated in Ethiopia,
then in Egypt, it spread through Palestine and the Byzantine Empire, and then throughout the Mediterranean [14]. The
plague changed the empire’s course and caused massive economic struggle [15]. Recurrences eventually killed about 50
million people over the next two centuries, 26% of the world population. The enlarged lymphatic gland of rats is the
first appearance of bubonic plague and spread by fleas [16].
The second global plague pandemic is known as Black Death (1347
51), which was likely originated in China and
decimated populations in Asia, Europe, Northern Africa, and other regions following the Silk Road [17]. This outbreak
was responsible for the death of one-third of the World’s human population. It reduced the global community from 450
million to below 300 million, with the pandemic killing as many as 150 million humans. Dead bodies were so prevalent
that many remained rotting on the ground and created a constant stench in cities [18]. The 14th century Black Death
pandemic has catalyzed enormous societal, economic, artistic, and cultural reforms. Neighborhoods, towns, were wiped
out or settlements abandoned. Crops could not harvest, traveling and trade became curtailed, and food and manufac-
tured goods became short. Regular divisions between the upper and lower classes broke and led to a new middle class.
The widespread death caused labor shortages across primitive societies and often led to higher wages and cheaper land.
The lack of labor encouraged the innovation of labor-saving technologies, leading to higher productivity [19].
With the overwhelming devastation of the plague, several authorities lost credibility. People started asking questions
on the societal structure, traditions, and religious orthodoxy. Catholics of Europe interpreted that plague was the divine
“punishment for sins.” Those infected minorities or women individuals and groups who were the “gravest sinners
against God.” Jews in Europe were commonly targeted and accused of “poisoning the wells,” and entire communities
were offended and killed. Non-Catholic Christians were also blamed as “heretics” and experienced a similar fate [20].
A similar sentiment prevailed in other parts of the world affected by the plague. In Cairo the sultan put a law prohibit-
ing women from making public appearances as they may tempt men into sin [21]. It prompted fundamental shifts in
peoples’ interactions and experiences with religion, philosophy, and politics.
During the renaissance period, humanism and learning were encouraged. At the time, scientific communities were
not having any idea regarding the cause of the plague illness. The first official report blamed three planets aligned,
causing a “great pestilence in the air” [22]. Until the late 19th century, the Black Death was due to miasma theory, an
interpretation that blamed bad air was causing Black Death [23]. As we know, the aerobic, nonmotile, Gram-negative
142 Pandemic Outbreaks in the 21st Century

bacterium Y. pestis causes the plague. It infects and surpluses rats, mice, and squirrels’ guts, forcing them to vomit con-
centrated bacteria when feeding. Fleas (Xenopsylla cheopis) transmitted the pathogen to humans through the bite of an
infected organism (host)-bubonic plague [24]. Humans to humans can transmit the disease by droplets, leading to a
pneumonic type plague.
The 14th century Black Death pandemic illustrated how infectious disease can be a significant turning point in his-
tory. One of the most significant public health legacies that emerged from the plague pandemic was the concept of
“quarantine,” from the Venetian term “quarantena” meaning 40 days. In the 21st century, how COVID-19 reshapes our
culture and its unexpected influence for generations to come is unknown. Evident economic changes are arising from
this outbreak, as some industries rise, others fall, and some businesses seem likely to disappear forever. COVID-19 pan-
demic may permanently normalize virtual technologies for socializing, business, education, healthcare, religious wor-
ship, and even government and private sectors.
London suffered two terrible disasters in two successive years of the 17th century. In April 1665 and September
1666 an outbreak of plague spread from the poor, overcrowded parish of St. Giles-in-the-field to other parish killed
75,000
100,000 people in London, up to a fifth of London’s population (15%). As increased human death mounted,
and mass graves appeared, thousands of cats and dogs were slaughtered as the possible cause [25]. Whole communities
wiped out and corpses littered the streets like no one left to bury them. Rodents carried the fleas that caused the epi-
demic disease identified as bubonic plague, an infection by the bacterium Y. pestis transmitted through a flea vector.
Bubonic plague as the Black Death the victim’s skin turned black in patches, and inflamed glands or “buboes” in the
groin, combined with uncontrollable vomiting, swollen tongue, and splitting headaches, made it a horrible, agonizing
killer. During the outbreak in 1665, the royal family, most doctors, lawyers, and merchants fled London. They have
also moved court cases from Westminster to Oxford and stopped all trade with London and other plague towns. The
Council of Scotland closed its borders with England. There was no trade with other countries. Many people lost their
jobs, from servants to shoemakers, and this epidemic devastated Londoners’ lives.
London mayor ordered victims to be locked in their homes to avoid disease spread [26]. In the fall of 1666, the out-
break tapered off simultaneously as another destructive event—the Great Fire of London, destroyed much of London’s
center and helped kill off some of the plague bacteria carried black rats and fleas. The bubonic plague was only remem-
bered afterward as the “Great Plague of London” since it was the last widespread outbreak in England [27]. Bubonic
plague was far nastier than the 21st century coronavirus pandemic. Flea bites transmit it around 75% fatal, while in its
lung-to-lung form that figure went up to 95%.
The third bubonic plague, referred to as the modern plague, started in China in 1855 and then spread to Hong Kong
by 1894 and distributed to all inhabited continents. India faced the most substantial casualties (about 10 million deaths),
and the pandemic was an excuse for brutal policies that sparked some revolt against the British rules. The plague was
considered active until 1960 when worldwide casualties dropped to 200 per year, and deaths have continued at a lower
level every year [28] due to the presence of natural plague foci in animals and humans [29].
Three major plague pandemics caused by aerobic, nonmotile, Gram-negative bacterium Y. pestis have killed nearly
200 million people due to its extreme virulence and ease of transmission. Plague outbreaks are still breaking sporadically
in most parts of the world. Approximately 2000 cases of plague are reporting each year to the WHO [30]. Antibiotics are
used for treating plague disease but bacteria are developing antibiotic resistance. Currently, a perfect plague vaccine is not
available. Therefore, prophylactic vaccination against this plague holds the brightest prospect for its long-term prevention.
Creating an ideal plague vaccine is in the pipeline. I hope it can be commercially available in the future.

9.2.6 Leprosy
The earliest written records describing leprosy are from India (600 BCE), spread from there to China (500 BCE), and
carried to Japan. Due to this pandemic in Europe in the Middle Ages numerous leprosy-focused hospitals were built to
accommodate the vast number of victims. Leprosy is a slow-developing bacterial disease that causes sores and deformi-
ties. It is believed to be a punishment from God that ran in families. This belief led to moral judgments and ostraciza-
tion of victims. Now it is known as Hansen’s disease, it still afflicts thousands of people a year and can be fatal if not
treated with antibiotics [31]. Mycobacterium leprae is a bacterium isolated by Armauer Hansen in Norway in 1873,
which is causing leprosy [32]. It multiplies slowly and the incubation period of the disease is on average of 5 years.
Symptoms may occur within 1 year and take as long as 20 years or even more to happen. The disease affects the skin,
peripheral nerves, upper respiratory tract, and eyes. It is curable with multidrug therapy (dapsone, rifampicin, and clofa-
zimine). M. leprae is transmitted via droplets, from the mouth and nose, during close and frequent contact with
untreated cases, and contact with armadillos is a risk factor for leprosy. Leprosy can cause progressive and permanent
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 143

damage to the skin, nerves, limbs, and eyes, if untreated. There were 208,619 new leprosy cases registered globally in
2018 [33]. A team led by Dr. G. P. Talwar at the All India Institute of Medical Sciences (AIIMS) in New Delhi in the
1970s developed an autoclaved Mycobacterium indicus pranii (MIP) vaccine. The vaccine has received approval from
the Drugs Controller General of India (DCGI) and the United States Food and Drug Administration (FDA). At present,
this is the only type of vaccine in the World. Cadila Pharma India manufactures MIP under the brand name
IMMUVAC and is available in the market as an immunomodulator injection [34]. India is home to 60% of the World’s
leprosy patients; on May 7, 2017, India’s Government launched an immunization project to eradicate leprosy from
India [35].

9.2.7 Influenza
The earliest influenza cases might have occurred among Greek soldiers fighting the Peloponnesian War in 430
BCE [36]. The first actual flu pandemic appeared in Asia and quickly spread over trade routes into Europe and North
America in 1580. The exact number of death reported is unknown; at least 8000 deaths in Rome alone. Announced the
health emergency in Europe of early quarantine measures and border checkpoints were formed [37]. The first reference
to “influenza” in scientific literature dates 1650 and derives from the Italian word “influence,” possibly the influence of
misaligned stars [38]. The history of influenza pandemics has been documented more reliably after this date.
The first flu pandemic started in Russia and then spread through Europe, Asia, the United States, and Africa. The
1889
90 flu pandemic is also known as the “Asiatic flu” or “Russian flu.” This pandemic’s suspected pathogen is
influenza A virus subtype H2N2 (A/H2N2) [39]. Russian flu was the last deadly outbreak of the 19th century reported
about 1 million deaths [40].
The avian-borne flu (Influenza A-H1N1), the worst devastating medical disaster in history, is due to a complex
interplay between how the virus works, the immune response, and the social context in which it spread [41]. First flu
cases were observed in Europe in 1918, the United States, and parts of Asia before spread worldwide. At the time, there
were no effective drugs or vaccines to treat this killer flu strain. On May 22, 1918, Madrid’s ABC newspaper reported
that Madrid’s flu outbreak led to the pandemic being called the “Spanish flu.” It arose in a world left vulnerable by the
preceding 4 years of World War I. Malnutrition and overcrowding were common. The flu threat disappeared in 1919
when most of the infected had either developed immunities or died. A unique characteristic of infection was its ten-
dency to kill healthy adults between 20 and 40. It resulted in 50
100 million deaths and around 500 million people
infected worldwide [42]. At the time, scientists have recognized an influenza infection as a bacterium (Haemophilus
influenzae) rather than a virus [43]. Antibiotics and intensive care wards with mechanical ventilators were not available.
Medical and scientific understanding of the flu in 1918 made it difficult to combat.
Public health interventions, including quarantine, face masks, hand hygiene, bans on mass gatherings, closing schools,
worship places, cinema theaters, dance halls, and swimming pools, helped limit the spread. It was forcefully enforced in
some jurisdictions by police officers issuing fines, and at times using weapons. Many countries have imposed maritime
quarantine; the effect of naval quarantine was most striking, with the latter enforcing strict quarantine and experiencing no
deaths. In the United States cities that committed earlier, longer, and more aggressively to public health interventions
(social distancing) saved lives. They emerged economically stronger than those that did not [44] implemented sterilized
operating rooms, surgical procedures, and hand washing in hospitals. Vaccination is the stellar scientific advance born of
outbreaks of infectious diseases. The cultural and political evolution of clothing had another familiar one. The widespread
custom of drinking from the same cup might be spreading contagious diseases. It was discouraged cup-sharing and worked
to ban spitting in public places instead of promoting spittoons cleaned regularly. Although their purpose was social rather
than sanitary, they may have helped contain the spread of disease. The Spanish flu of 1918 boosted public health in less-
developed countries with medicine’s ineffectiveness to prevent the pandemic led to alternative therapies. The outbreak
was unfolding, Philadelphia, United States, threw a parade with 200,000 people marching in support of the world war I
effort; by the end of the week, 4500 people died from the flu. St. Louis shuttered public buildings and curtailed transit;
the flu death rate there was half of Philadelphia’s population [45].
During the 1918
19 Spanish flu pandemic period, public health measures were the only effective weapons against
the disease, as no vaccines or antivirals were available. Public health education, isolation, sanitation, and surveillance
improved our knowledge of influenza transmission and are still implementing today to stop spreading the disease.
Analysis of preserved samples from infected persons who died during the 1918 Spanish flu is a significant step toward
understanding this pandemic [46]. Such knowledge may contribute to discovering new therapeutics and developing pre-
ventive strategies, including insights into the appropriate timing of administering antivirals, thereby providing indica-
tions for preparing for future pandemics.
144 Pandemic Outbreaks in the 21st Century

Asian flu (influenza A H2N2) virus emerged in Guizhou, China, and was reported in Singapore in February 1957. It
sparked a significant outbreak in Hong Kong in April 1957, where infected 250,000 people, and by June 1957, India had
seen over a million cases finally spread rapidly worldwide. Hence, the 1957
58 pandemic is referred to as “Asian flu” [47].
Infections were reported in young children, the elderly, pregnant women, about 2 million deaths worldwide [48]. The recom-
bination (reassortment) of avian influenza A virus (from geese) and human H1N1 influenza viruses resulted in a new influ-
enza A subtype H2N2 virus. This hybrid virus contains polymerase basic1, hemagglutinin (HA), and neuraminidase (NA)
three genes [49]. The oligosaccharide side chains of the influenza virus HA subtypes help facilitate viral escape from
immune response and maintain the influenza virus population in natural reservoirs [50]. The 1957 outbreak was with varia-
tion in susceptibility and course of illness. Some infected individuals experienced symptoms like cough and mild fever;
others experienced pneumonia-like life-threatening complications. Those unaffected by the virus possessed protective antibo-
dies. The rapid development of a vaccine against the H2N2 virus and the availability of antibiotics to treat secondary infec-
tions limited the pandemic’s spread and mortality [51].
The Hong Kong strain of influenza virus A2 H3N2 might have originated in China’s mainland (not sure). It caused
a massive outbreak in Hong Kong and spread rapidly to countries as far as India and Australia and reached the United
States in September 1968 [52]. In the 1960s the human H2N2 strain underwent minor genetic modifications, a process
known as antigenic drift. These slight modifications produced periodic outbreaks. After 10 years of evolution, the 1957
flu virus disappeared, replaced through an antigenic shift by a new influenza A subtype, H3N2. This H3N2 virus con-
tains an avian influenza A virus H3 HA and the N2 NA from the 1957 H2N2 virus. This new human influenza virus
strains with avian HA molecules on its surface because the human population lacks neutralizing antibodies to this
unique glycoprotein, which gave rise to the 1968 Hong Kong flu pandemic [53].
About 1 million deaths were recorded worldwide and were in 65 years and older people. The H3N2 virus circulates
worldwide as a seasonal influenza A virus associated with severe illness in older people undergo normal antigenic drift [54].
Researchers have isolated a closely related H3N2 virus from pigs in the 1990s and suspect that the human H3N2 virus
jumped to pigs; infected animals may show swine flu symptoms. A vaccine was available but not produced early enough to
provide significant protection [55]. Similarity with the 1957
58 Asian flu may have helped protect people from more dis-
ease severity. The influenza vaccine has shown adverse effects. As an influenza virus had a rapid change in its nature, stud-
ies on the effectiveness, risk, and benefit of influenza vaccination are required. Constant search develops a new effective
vaccine against influenza for combating the disease.
The highly pathogenic Asian avian influenza A (H5N1) virus occurs mainly in birds and is highly contagious for
poultry (epizootic). It was first detected in 1996 in Geese of China. The H5N1 virus was first seen in humans in 1997
during a poultry outbreak in Hong Kong and later extended to Africa, Asia, Europe, and the Middle East. By 2000, the
H5N1 virus host range had expanded to domestic ducks, which played a crucial role in the 2003
04 outbreaks’ genesis.
The increasing spread of the H5N1 viruses from existing reservoirs of infection in domestic waterfowl and live bird
markets, leading to more significant environmental contamination, causes millions of poultry and waterfowl death and
infected humans [56]. In 2005 an explosion of H5N1 outbreaks in poultry and migratory bird populations in Asia spread
to Russia and Eastern Europe.
The H5N1virus did not spread from human to human but only among birds and then to humans. Thus, sporadic
human infections with avian H5N1 virus resulting from prolonged and unprotected direct or close contact with infected
sick or dead poultry. Some of the infected humans became severely ill and died. The lack of human-to-human spread
limited avian flu incidence. After the widespread destruction of poultry flocks, the threat weakened [57]. However, the
bird flu virus that remains circulating among poultry has the potential to recombine with human influenza A viruses;
another rare, deadly novel strain of mixed virus (a reassortant) could arise. These novel recombinant influenza viruses
are always changing. It can change so that they may gain the ability to infect humans easily and spread potentially
among humans, causing a pandemic with high rates of illness and death worldwide. So, the H5N1 epizootic continues
to pose a significant public health threat [58].
Therapies have been developed based on the clinical evidence that H5N1 elicits “cytokine storms” that can subsidize
disease pathogenesis [59]. Oseltamivir (Tamiflu) or zanamivir (Relenza) antiviral medicines can reduce the disease’s
severity. However, we must give the medicine within 48 h after symptoms first appear. Flu’s human virus develops
resistance against amantadine and rimantadine, the most commonly used antiviral drugs. Vaccines are the principle of
combating influenza, the FDA has approved a vaccine designed to protect against the avian flu, but the vaccine is not
currently available to the public. The Asian avian H5N1 vaccine is used for preventing virus transmission from human
to human. Those people who contact infected birds may also be given influenza antiviral drugs as a preventive measure
[60]. However, Asian avian H5N1 has not been sustained long, and no community spread of this novel virus has been
recorded. As the pandemic COVID-19 continues worldwide from December 2019, researchers compare influenza
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 145

TABLE 9.2 Comparison between influenza and COVID-19.

Similarities
Disease presentation Respiratory type
Range of illness Asymptomatic/mild to severe, and death
Mode of transmission Contact, fomites, and droplets
Public health measures Hand hygiene and good respiratory etiquette

Differences

Influenza COVID-19
Speed of Shorter incubation period (time from infection to Interval for the COVID-19 virus is 5
6 days
transmission appearance of symptoms) and a shorter serial interval
(time between successive cases) 3 days.
Transmission In the first 3
5 days of illness, or potentially People who can shed SARS-CoV-2 virus 24
48 h before
presymptomatic transmission (transmission of the virus symptom onset
before the appearance of symptoms)
Reproductive Lower number of secondary infections generated from Higher rate 2 and 2.5 persons
number one infected individual
Age group Children are essential drivers of influenza virus Children are less affected than adults. 0
19 age group
transmission in the community children are infected by adults, rather than vice versa
Range of Severe and critical illness is low 80% of infections are mild/asymptomatic, 15% are
symptoms severe infections, and 5% are acute infections
Risk Children, pregnant women, the elderly, those with Older age people. Still, it is not understandable.
underlying chronic medical conditions, and those who
are immunosuppressed
Mortality ,0.1%. However, mortality is, to a small extent, Higher than for influenza. The crude mortality ratio (the
determined by access to and quality of health care number of reported deaths divided by the reported
cases) is between 3% and 4%. The infection mortality
rate (the number of reported deaths divided by the
number of infections) is less in number
Medical Antivirals and vaccines are available; recommended to Licensed vaccines or therapeutics for COVID-19 are
interventions get vaccinated each year to prevent influenza infection available—Covaxin and Covishield

(Table 9.2). Both viruses cause respiratory disease, yet there are significant differences between them and how they
spread. This type of knowledge is essential for implementing public health measures to respond to each virus.

9.2.8 Cholera
Cholera is an ancient acute disease of the gastrointestinal tract caused by a toxicogenic bacterium called Vibrio cholera.
It is a public health problem worldwide in many underprivileged locations. The bacteria typically live in the aquatic
environment, mainly brackish riverine, estuarine, and coastal waters [61]. Cholera spread through the fecal
oral route,
contaminated fluids from an environmental reservoir of varying duration, food, and potentially fly (Housefly: Musca
domestica) and fomites [62]. There are hundreds of strains or serotypes of the cholera bacteria; only two 01 and 0139
serotypes of bacteria are known to cause acute secretory diarrhea. It is endemic in more than 50 countries and causes
large outbreaks [63]. These strains produce the cholera toxin that causes the small intestine’s cell lining to release
increased water quantities, leading to diarrhea and rapid loss of fluids and electrolytes (salts) [64]. The National
Institute of Allergy and Infectious Diseases (NIAID) had estimated that a single diarrhea incident could cause a one-
million-fold increase of bacterial numbers in the environment [65].
Throughout history, populations worldwide have been affected by devastating outbreaks of cholera, although people
were unclear when it was first affected. Early texts have confirmed it from India Sushruta Samhita (500 BCE), Greece
Hippocrates (460
377 BCE), Galen (CE 129
216), and Aretaeus of Cappadocia (CE 100) described an illness that
146 Pandemic Outbreaks in the 21st Century

could have been cholera. Reports indicated that cholera-like sickness was in the Ganges River’s plains in ancient
times [66]. The local people called the disease “moryxy,” which killed the infected person within 8 h of developing
symptoms. The case fatality rate (CFR) is so high that locals struggled to bury all the dead bodies [67,68]. Cholera
spread globally beyond Asia seven times, referred to as cholera pandemics. The first cholera pandemic started in 1817,
and subsequent outbreaks began in 1829, 1852, 1863, 1881, 1889, and 1961, the last one persisting until the present.
The primary hub linking the spread of cholera worldwide during the 19th and early 20th centuries is the Bay of Bengal
[69]. There have been many additional documented cholera outbreaks, such as a 1991
94 outbreak in South America
and the 2016
20 Yemen, the last of which is ongoing [70,71]. Overcrowding, poverty, insufficient water, and sanita-
tion facilities are the risk factors for cholera outbreaks. The epidemiology of cholera in Asia, Africa, and America,
where the disease occurs, continues to evolve [72]. Cholera transmission is preventable through access to potable water,
sanitation, and hygiene (WASH). Cholera pandemic continued in 47 countries have an estimated 2.86 million cases and
95,000 deaths per year [73]. Though improved water and sanitation remain the backbones of cholera prevention efforts,
significant improvements to infrastructure continue to be a goal far out of reach for many of those affected and near-
term interventions, including vaccines. The availability of newer-generation oral vaccines protective against new variant
strain affords a tool to control cholera. Oral cholera vaccine Shanchol (Shantha Biotechnics, India) licensed in India in
2009, prequalified by WHO in 2011, killed whole V. cholera serotypes O1 and O139 [74].

9.2.9 Ebola
Ebola virus (EBOV) is a highly lethal pathogen that has caused the deadliest Ebola virus disease (EVD), formerly
known as Ebola hemorrhagic fever, rare but severe, often fatal in humans (if untreated). The first 2 simultaneous EVD
outbreaks that occurred in different parts of Central Africa appeared in 1976. The first outbreak in the Democratic
Republic of Congo (formerly Zaire) in a village near the Ebola River gave the virus its name. The second outbreak
occurred in Nzara, South Sudan. Initially, these outbreaks were a single event associated with an infected person who
traveled between the two locations. Dr. Peter Piot of Belgium and his colleagues were the first to identify Ebola. Two
genetically diverse viruses, Zaire ebolavirus and Sudan ebolavirus, caused the two outbreaks. Scientists concluded that
the virus came from two sources and spread independently in each affected area [75]. The Filoviridae family contains
three genera: Cuevavirus, Marburgvirus, and Ebolavirus. The genus Ebolavirus includes Zaire, Bundibugyo, Sudan, Tai
Forest, Reston, and Bombali. Filoviruses are enveloped negative-sense single-stranded RNA viruses known to cause
hemorrhagic fever in humans with a case fatality of up to 90%.
The ebolavirus species is responsible for the 1976 Ebola outbreak with 318 cases and 218 deaths for a case fatality
ratio of 88% [76]. Subsequently, the virus has emerged periodically and infected people in several African and other
countries, including the Democratic Republic of Congo, Sudan, Gabon, Cote d’Ivoire, South Africa, Uganda; Congo,
Guinea, Sierra Leone, and Liberia; one of America, and one of Europe, Spain. The 2014
16 Ebola outbreak in West
Africa was the largest one; the virus causing the 1976 DRC outbreak Zaire ebolavirus species is responsible. The epi-
sode started in Guinea in West Africa and then moved across land borders to Sierra Leone and Liberia. There were
11,310 deaths out of nearly 29,000 cases of Ebola [77]. The 2018-ongoing Ebola outbreak in Eastern DRC is highly
complex, adversely affecting public health response activities [78]. The International public health cooperation failed to
protect the public from the disease’s social, economic, and political consequences.
The EBOV existed long before the recorded outbreaks occurred. Factors like human population growth, the encroach-
ment of forest areas (habitat degradation and alteration), and direct interaction with wildlife (Bushmeat consumption) may
have contributed to the spread of the EBOV [79]. EBOV was introduced to the human population through close contact
with infected blood, secretions, reuse of contaminated needles, improper nursing techniques, body fluids of infected animals
such as fruit bats, chimpanzees, gorillas, monkeys, antelope, or porcupines [80]. Most importantly, African fruit bats of the
Pteropodidae family are likely the EBOV’s natural reservoir, and it is involved in the spread of the EBOV. The EBOV liv-
ing in organs, tissues, and blood without causing any illness to the host. It is spreading from host to host or through interme-
diate hosts or as vectors. The most recent EBOV identified the Bombali virus in samples from Sierra Leone’s bats [81].
The Ebola genome and antibodies in bat evidence the bat’s role in transmitting Ebola [82].
In 1989 the Reston ebolavirus discovery in the Philippines’ monkeys revealed that Ebola was no longer confined to
Africa and Asia. It confirmed that the virus spread throughout the monkey population through droplets in the air
(aerosolized transmission); such airborne transmission is not proven to be a significant factor in human outbreaks [82].
No proven treatment is available for EVD; United States company Gilead Sciences, Inc. manufactures the EBOV drug
Remdesivir [83]. Supportive care rehydration with oral or intravenous fluids and treatment of specific symptoms
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 147

improves survival. A trial Ebola vaccine proved highly protective against EVD in Guinea in 2015 [84]. The rVSV-
ZEBOV vaccine has been used in the 2018
19 Ebola outbreak [85].

9.2.10 Lessons learned from Ebola outbreaks

Public health interventions: Based on a better understanding of how the EBOV spreads to reduce transmission, it introduced face
masks, gloves, and gowns for healthcare personnel. Also, the use of disposable equipment, such as needles, was introduced.
International public health cooperation: International public health community played a vital role in the EBOV and
was paramount in ending outbreaks. They educated the local community on how the disease spreads; the hospital was
adequately staffed and stocked with the necessary equipment. Healthcare personnel was trained on disease reporting,
patient case identification, and methods for reducing transmission.
Duration of the outbreak: There were 318 cases of Ebola in 1976 and 280 deaths in an episode that lasted less than
11 weeks. In the West Africa outbreak there were 11,310 deaths out of nearly 29,000 cases, and it lasted more than 2
years, almost 10 times as long as in 1976. In 1976 88% of the death rate was much higher than in the outbreak in
Liberia, Guinea, and Sierra Leone, around 50% [77].
Adopting safe burial practices: During the Ebola outbreak, most transmission events were between family members
of an infected person. Burial ceremonies involving direct contact with the bodies of those who died from the EVD are
among the most dangerous and effective transmission methods. For controlling the pandemic changes in behaviors
related to mourning and burial, adopted safe burial practices.
Future perspectives: Community engagement is key to successfully controlling outbreaks. Reasonable outbreak con-
trol relies on applying a package of interventions. More extensive preparations, including improved screening capabili-
ties, are needed to detect and manage future attacks promptly. Primary prevention can be possible through strengthened
prediction models, detection, response, and control mechanisms. A suitable laboratory service, safe and dignified bur-
ials, social mobilization, and international cooperation and coordination are essential for all countries where Ebola and
new and reemergent pathogens are sure to surface again.

9.2.11 HIV/AIDS
Acquired immunodeficiency syndrome (AIDS) is one of humanity’s more severe, chronic, life-threatening, and complex
health problems. It is initiated by the human immunodeficiency virus (HIV), which destroys a person’s immune system
and can reduce an infected person’s ability to resist opportunistic infections and diseases, resulting in eventual death by
illness. Those infected by HIV encounter fever, headache, and enlarged lymph nodes. When people with HIV do not
get treatment, they typically progress through three stages of illness; acute HIV infection (Stage 1), chronic HIV infec-
tion (Stage 2), and AIDS (Stage 3). HIV carriers become highly infectious through blood and genital fluid, and the dis-
ease destroys T-cells. AIDS is the most advanced (late) stage of HIV infection when the body’s immune system is
damaged by the virus and develops certain cancers, diseases, or other severe long-term clinical manifestations [86]. The
number of their CD4 cells (T-cells) falls below 200 cells mm23 of blood. In the healthy human immune system, CD4
counts are between 500 and 1600 cells mm23 [87].
HIV in humans came from a chimpanzee in West Africa in the late 1800s. The chimpanzee simian immunodeficiency
virus was probably passed to humans when humans hunted these chimpanzees for meat and direct contact with their
infected blood. HIV slowly spread through certain body fluids across Africa and later into other parts of the World [88].
The virus has existed in the United States since the 1970s. In American gay communities in 1981 reported the first clinical
evidence of AIDS [89], and its cause, HIV, was identified in 1983 [90].
Globally 75.7 million people have become infected with HIV, and 32.7 million people have died from AIDS since
the start of this pandemic [91]. HIV continues to be a significant global public health issue. HIV is transmitted through
sexually transmitted infection, contact with infected blood, or from mother to child during pregnancy, childbirth, or
breastfeeding. The human body cannot get rid of HIV; once a person has HIV, they have it for life; a specific drug for
the cure is yet to discover. However, antiretroviral therapy can slow or prevent the progression of the disease. With the
effective advanced treatment, advance to stage 3 is less today than in the early days of HIV infection [92]. HIV preven-
tion, diagnosis, treatment, and care, enabling people living with HIV to lead long and healthy lives. Besides, effective
methods to avoid getting HIV through sex or drug use include preexposure and postexposure prophylaxis [93].
Advances in HIV medicines and public health programs like needle and syringe exchanges and the widespread use of
condoms also held many lessons about the deadly danger of social stigma [94].
148 Pandemic Outbreaks in the 21st Century

TABLE 9.3 Important differences between the 2009 swine flu and COVID-19.

2009 swine flu 2019 COVID-19

Mortality in the age group 80% of the deaths were in people below In all the age group, the highest percentage of
the age of 65 deaths in people ages 65 and older
Immunity Herd immunity is protected Some groups of people have immunity to the
2019-SARS-CoV-2 virus, but herd immunity is
still being researched
Spread Respiratory droplets and airborne Respiratory droplets and oral
fecal route
particles
Symptoms The pneumonia-like respiratory issues: Fever, dry cough, shortness of breath,
fever, cough, headache, body aches, sore headache, sore throat, abdominal pain, and
throat, chills, fatigue, and runny nose diarrhea. The pneumonia-like respiratory issues
Appearance of symptoms Symptoms appeared between 1 and 4 The virus incubation period between 4 and 14
days after the virus infection days; an individual could be carrying the virus
up to 2 weeks before feeling any illness
Contagious Less More
R-nought value 1.46 Between 2 and 2.5
People response Better prepared quite as fast or as Prepared for a pandemic not quite as fast or as
smoothly smoothly
Release virus genetic sequences to On April 24, 2009, 9 days after the initial On January 12, 2020, 5 days after the novel
the public to create diagnostic tests detection of the H1N1 virus coronavirus was isolated, Chinese scientists
and begin developing a vaccine published the virus’s genetic sequence
Declaration of a pandemic by the 11days after the first case of confirmed 11days after the first case of COVID-19
United States swine flu
Diagnosis of virus infection Capable of diagnosing H1N1 in all the Significantly diagnosing with the RT-PCR and
labs that could prevent and treat influenza antigen tests
Media of communication
First pandemic in the current era of social
media. The misinformation about the disease
has spread faster than the virus
The advanced technological age is
Many pharmaceutical companies have
the speed at which research and developed effective vaccines
vaccine development

9.2.12 Swine flu

A new strain of novel influenza A H1N1 originated in Mexico and was first detected in America in April 2009.
H1N1 is a combined virus from pigs, birds, and humans that cause disease in humans [95]. The virus quickly spread
globally, affecting children and adults under 65 who lacked immunity to H1N1. It causes a respiratory infection in
humans, referred to as swine flu [96]. Because many people worldwide got sick, the WHO declared the swine flu out-
break a pandemic on June 11, 2009, and termed it “(H1N1) 2009 pandemic.” Globally, an estimated
151,700
575,400 people died from the swine flu pandemic [97]. The 2009 H1N1flu pandemic was the second one in
history, the first being the 1918 Spanish flu, the most deadly outbreak in history. The 2009 swine flu pandemic lasted
19 months, from January 2009 to August 2010 [98]. The WHO officially named “A(H1N1)pdm09” for the novel
virus H1N1 after the declaration of pandemic end on August 10, 2010 [99]. The oseltamivir or zanamivir antiviral
drugs are used for treating people when they first experience flu symptoms [100]. Pandemrix and Celvapan vaccines
have been developed to protect against the virus that causes swine flu. The United States began administering a
newly approved H1N1 vaccine on October 5, 2009, started the vaccination. The flu vaccine now helps to protect
against swine flu. The H1N1 and H3N2 influenza virus strains that cause H1N1 flu (swine flu) are included in the flu
vaccine for 2020
21 [101] (Table 9.3).
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 149

9.2.13 Zika
Zika is a dormant, little-known virus found in rhesus monkeys of Uganda. The recorded earliest known outbreak was in
Micronesia (subregion of Oceania) in 2007. Then identified the virus in Brazil in 2015. It is transmitted through a mos-
quito (Aedes aegypti) and sexually. Mosquitoes that carry the Zika virus and well survived in warm and humid climates.
South America, Central America, and parts of the southern United States prime areas for the virus to thrive and out-
break happens. Zika virus infections in adults or children causing a mild illness include flat pinkish rash, bloodshot
eyes, fever, joint pain, and headaches, resembling dengue symptoms. Zika can cause Guillain
Barre syndrome in
adults, and children cause severe microcephalia in unborn children of infected mothers [102]. There were 2400 congeni-
tal disabilities in Brazil and 29 infant deaths due to suspected Zika infection recorded in 2015 [103]. It is a significant
public health concern in the United States, as there is no vaccine. The only reliable way to avoid the offspring’s risk is
to avoid areas where the Zika virus identified or postpone pregnancy should travel to or live in affected areas is
unavoidable. The Zika virus pandemic is continuing in the present day.

9.2.14 Coronaviruses
SARS (Severe Acute Respiratory Syndrome): It is a contagious and potentially fatal respiratory illness caused by a
SARS-associated coronavirus (SARS-CoV or SARS-CoV-1) [104]. It happened from 2002 to 2003, but the disease is
no longer circulating. SARS-CoV is a strain of coronavirus that causes the common cold. Previously, these viruses had
never been dangerous to humans. SARS-CoV is related to SARS-CoV-2, the virus that causes COVID-19 infection.
SARS was a zoonotic disease; SARS-CoV is an animal virus evolved from an animal reservoir, bats, that spread to
civet cats and first infected humans in the Guangdong province of southern China in November 2002 [105]. Then
quickly moved to Hong Kong and other countries in North America, South America, Europe, and Asia [106,107].
Transmission of SARS-CoV is primarily from human to human occurred in the health care setting, in the absence of
adequate control measures. It appears to have emerged mainly during the second week of illness, which corresponds to
the peak of virus excretion in respiratory secretions and stool. Severe disease cases start to deteriorate clinically. A total
of 8098 people worldwide had viral respiratory illnesses with SARS during the 2003 outbreak; 774 died with a CFR of
11% [108]. Patients under 24 years of age were least likely to die (,1%); those 65 and older people were most likely
to die (over 55%) [109]. As with Middle East Respiratory Syndrome (MERS) and COVID-19, SARS resulted in signifi-
cantly more male deaths than females.
SARS symptoms are respiratory problems, dry cough, fever of 100.5  F (38 C) or higher, head, body aches, diar-
rhea, and shivering (rigors). No specific symptoms for this disease; dry cough, shortness of breath, and diarrhea are
present in the first and second week of illness. Severe cases often evolve rapidly develop pneumonia, progressing to
respiratory distress so severe that a mechanical respirator is needed. SARS is fatal in some cases, usually due to respira-
tory failure; other possible complications include heart and liver failure [110]. People older than 60 years, especially
those with underlying health conditions such as diabetes or hepatitis, are at the highest risk [111].
As of the year 2020, there is no cure or protective vaccine for SARS. There is currently no proven antiviral therapy
for SARS. The drugs like ribavirin, lopinavir, ritonavir, and type I interferon are used to treat SARS [112]. Many health
experts recommended administering corticosteroids in patients with severe disease and O2 saturation of ,90% [113].
In general, people at the most significant risk of SARS are those who have had direct, close contact with someone
who’s infected, such as family members and health care workers. The respiratory droplets from coughs and sneezes
were spreading the virus. SARS showed how quickly infection could spread in a highly mobile and interconnected
world. On the other hand, the implementation of international collaborative effort allowed health experts to contain the
disease’s spread quickly. Particularly infection control practices such as quarantine proved useful, and the virus con-
tained. There has been no known transmission of SARS anywhere in the World. The global outbreak came to an end
and has not reappeared since 2004. As of 2020, SARS was eradicated in humans, but as the virus also infects animals,
it may reemerge again in the future. International health professionals saw SARS as a wake-up call to improve outbreak
responses and lessons from the pandemic to keep diseases like H1N1, Ebola, and Zika under control. Experimental vac-
cines are under development; precautionary measure is following as per the WHO guidelines.
Middle East Respiratory Syndrome (MERS): The Middle East respiratory syndrome coronavirus (MERS-CoV)
causes viral respiratory illness that is new to humans. It was first reported in June 2012 in Jeddah, Saudi Arabia, and
has since spread to several other countries or near the Arabian Peninsula. Still, travel-associated cases are seen in coun-
tries outside the Arabian Peninsula [114]. Most people infected with MERS-CoV symptoms may range from none
(asymptomatic) to mild respiratory symptoms and severe respiratory illness, including fever, cough, shortness of breath,
150 Pandemic Outbreaks in the 21st Century

and death. The virus causes more severe diseases in older people with weakened immune systems and chronic health
problems such as renal disease, cancer, chronic lung disease, and diabetes [115]. The 35% of the patients reported with
MERS-CoV infection have died with a CFR as high as 40% [114].
The MERS-CoV has a close relationship with two bat-CoVs (HKU4 and HKU5), and camelids (Camelus dromedar-
ies) serve as intermediaries between infected vespertilionid bats and humans, that is, camel-to-human and human-to-
camel transmission. Consuming raw or undercooked animal products, including camel milk and meat, carries a high
risk of infection that might cause humans disease. MERS-CoV’s human-to-human transmission is limited; MERS-CoV
is less contagious than its SARS cousin [114]. There were two further MERS-CoV outbreaks: South Korea in 2015 and
Saudi Arabia in 2018. There are intermittent MERS-CoV infections that continue to this day, but the episodes are usu-
ally well contained [116]. Vaccine or specific treatment is not available; several MERS-CoV vaccines and drugs are in
the pipeline [117].

9.2.15 COVID-19
The novel human coronavirus pandemic, known as COVID-19, was first reported in Wuhan, Hubei Province, China, in
late December 2019. The earliest date of symptom onset was December 1, 2019. These patients’ symptoms, including
fever, sickness, dry cough, and dyspnea, were diagnosed as viral pneumonia [118,119]. Initially, it was called “Wuhan
pneumonia” by the media because of the area and pneumonia-like symptoms. Whole-genome sequencing data confirmed
that the causative agent is a novel coronavirus; it is the seventh member of the coronaviridae family to infect humans
[120]. The WHO provisionally called the new virus 2019 novel coronavirus (2019-nCoV) on January 12, 2020. Then offi-
cially named this infectious coronavirus disease 2019 (COVID-19) on February 12, 2020. The International Committee on
Taxonomy of Viruses (ICTV) has called this virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [121].
On March 11, 2020, the WHO finally assessed that COVID-19 is spreading freely across the World as a global threat;
hence it was officially designated as a pandemic outbreak. SARS-CoV-2 is a spillover of an animal coronavirus and later
adapted the ability of human-to-human transmission. The virus is highly contagious; it rapidly spreads worldwide and con-
tinuously evolves in the human population [122]. Worldwide 86.7 million COVID-19 cases were confirmed, with 1.87
million deaths were recorded on January 6, 2021 [123]. Currently, a specific vaccine is available for COVID-19. To con-
trol the disease symptoms, recommended using the antiviral drug Remdesivir or the steroid Dexamethasone [124].

9.2.16 Lessons
The history of pandemics informed us (Table 9.4) that infectious diseases have shaped human history. Some of those
lessons are applied in the 21st century to fight this new global pandemic have given below.
Quarantine: Isolation of the sick person is a measure against leprosy, plague, and so on; during the outbreak of the
Black Death (14th century), its literal form has emerged. In 1377 at the seaport of Ragusa (Dubrovnik, Croatia), the
authorities imposed a trentina (Italian word: trenta—the number thirty), requiring ships to remain at anchor for 30 days;
for land travelers, the isolation period was 40 days quarantina (Italian quaranta, or 40). In many pandemics quarantine,
the only way to prevent pathogen spread was to isolate people. Some communities did that and managed well; others
did not and suffered high death rates. That lesson for us now; if we do not learn from it, we could suffer repeatedly.
Testing, contact tracing, isolation of infected, and precautionary self-isolation of contacts are critical in reducing the
number of new cases during the pandemics.
Social distancing: The history of pandemics showed that nonpharmaceutical interventions, a traditional term for
social distancing practices like closing schools and banning large public gatherings, could prevent infectious diseases
related to deaths. The pandemic took a lesser toll on cities that implemented these interventions earlier and for more
extended periods. In Mexico where social distancing was in effect during the 2009 influenza A(H1N1) pandemic when
those practices were relaxed, cases went back up. They pulled back again that reduced cases. Social distancing and
hygienic practices can help delay community transmission.
Personal protective measures: 17th-century beaky-plague-protection till the recent COVID-19 face masks used as indi-
vidual protective measures. Significant hygienic—sanitary improvements—hand washing was introduced in the past pan-
demics. A better understanding of how the pathogen spreads to reduce transmission, infection prevention, and control
measures includes hand hygiene, personal protective equipment (PPE), and waste management materials. WHO recom-
mended the use of PPE in health care and community settings. PPE includes gloves, medical masks, goggles or a face
shield, and gowns, as well as for specific procedures, air-purifying respirators (i.e., N95 or FFP2 standard or equivalent),
and aprons.
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 151

TABLE 9.4 A history of notable world disease outbreaks.

Timeline Outbreaks
430 BCE Athens (the earliest recorded outbreak)
G Happened during the Peloponnesian War
G Two-thirds of the population died

CE 165 Antonine Plague

G Smallpox that began with the Huns

G Infected the Germans, Romans, and the Roman Empire-Emperor Marcus Aurelius

CE 250 Cyprian Plague

G Named after the first known victim, the Cyprian bishop of Carthage

G An outbreak in Ethiopia, Northern Africa, Rome, Egypt, and northward

G Recurring outbreaks occurred over the next three centuries

CE 541
543 Justinian Plague

G The first recorded real plague pandemic
G Named after the emperor of the Byzantine Empire

G Originated in Ethiopia than in Egypt, it spread through Palestine and the Byzantine Empire, and the

Mediterranean
G Caused a massive economic struggle
G Killed about 100 million people, 26% of the world population

G First appearance of bubonic plague

11th century Leprosy

G Europe in the middle Ages

G A slow-developing bacteria that causes sores and deformities

G Punishment from God that ran in families

G It is known as Hansen’s disease

1334
50 Black Death

G The second plague pandemic in the World
G 30
50 million deaths

G Bubonic plague

G Originated in China and spread to Europe along trade routes

1580 First influenza

G The first actual flu pandemic

G Unknown number of deaths

G Started in Asia and quickly spread over trade routes into Europe and North America

G Announced the health emergence in Europe of early quarantine measures and border checkpoints

1665 The Great Plague of London

G Bubonic Plague killed 75,000
100,000 people in London, up to a fifth of London’s population

G Great Fire of London destroyed much of London’s center and helped kill off some of the vectors that carried the

plague bacillus
1860
1903 The Modern Plague
G The third plague pandemic globalized Y. pestis
G About 10 million deaths

G Bubonic plague

G It started in China and then spread to Hong Kong by 1894

1889
90 Russian Flu

G About 1 million deaths

G Influenza A
G First recorded in Russia, and then spread through Europe, Asia, and reached the United States

1918
20 Spanish Flu

G The first flu pandemic was announced in Madrid, Spain
G 50
100 million deaths

G Influenza A (H1N1)

G Infected over 500 million people worldwide

1957
58 Asian Flu

(Continued )
152 Pandemic Outbreaks in the 21st Century

TABLE 9.4 (Continued)

Timeline Outbreaks
G About 2 million deaths
G Influenza A (H2N2)
G Originated in China and spread to Singapore, Hong Kong, and the United States
1968
70 i
G About 1 million deaths
G Influenza A (H3N2)
G Started in China and Hong Kong and then spread through Asia, Australia, Europe, and the USA
1976 Ebola
G First recorded outbreak of the Ebola virus

G 280 deaths
G It is contained within Zaire (now DRC)

1981 (Ongoing) HIV/AIDS

G About 32 million deaths

G Human immunodeficiency virus (HIV) develops into acquired immunodeficiency syndrome (AIDS)

G In the early 20th Century, it originated in West Africa

G Discovered in the United States in 1981, it has spread globally

1996 (Ongoing) Asian Avian Flu

G Avian influenza (H5N1) infected poultry, wild birds and spread to humans

G Originated in geese in China, detected in humans in Hong Kong, and later spread in Africa, Asia, Europe, and

the Middle East

G Mutated forms of the H5N1 virus spread potentially among humans and cause a pandemic

G H5N1 epizootic continues to pose a significant public health threat

2003 SARS (Severe Acute Respiratory Syndrome)

G 774 deaths

G Viral respiratory illness (Coronavirus)

G Reported outbreaks in China and then quickly moved to Hong Kong and other countries

2009
10 Swine flu

G Origin of the outbreak in Mexico and the United States in 2009

G More than 200,000 deaths

G Influenza A (H1N1)

2012 Middle East Respiratory Syndrome (MERS)

G First reported in 2012 in Jeddah, Saudi Arabia

G Middle East respiratory syndrome coronavirus (MERS-CoV) causes viral respiratory illness
G Approximately 35% of reported patients with MERS-CoV infection have died with a case fatality rate as high as

40%
2014 Ebola
G Second outbreak, more than 11,000 deaths

G Over 28,000 cases of Ebola

G The worst-hit countries were Liberia, Sierra, Leone, and Guinea in West Africa

2015 (Ongoing) Zika

G Spread through mosquitoes and also sexually transmitted

G Attack infants still in the womb and cause congenital disabilities

G Reported in South America and Central America

2019
21 COVID-19
(Ongoing) G First reported in Wuhan, China, in late December 2019

G More than 1.87 million deaths

G Respiratory illness symptoms

Coronanomics: Throughout the centuries, pandemics have highlighted the clear and persistent divide between socio-
economic classes, xenophobia, and pervasive fear of the invisible enemy of the pathogen. Governments have often
failed to support citizens through pandemics’ social and economic effects, from mandatory quarantines, travel restric-
tions, closing educational institutions, workplaces, and local businesses. These crises also expose social inequality in
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 153

the past and current pandemics—the COVID-19 pandemic and its economic impacts globally, throwing many into
recession and possibly economic depression. Precise economic changes are arising from the COVID-19 outbreak, as
some industries rise, others fall, and some businesses seem likely to disappear forever. Many countries are boarding on
overcoming the COVID-19 crisis as governments step up to serve or save the business sector. To encourage public
cooperation, institute measures like economic relief for workers who might lose wages and offer assistance like grocery
deliveries, there would be better compliance with self-quarantine standards. How COVID-19 can reshape our societal
culture and its unexpected influence for generations to come is unknown.
Technology and innovation: The pandemic leads to a fundamental rethink of where and how we work. Social dis-
tancing enforces video conferencing, virtual classrooms and telemedicine, digital commerce, and so on. Accelerate the
development of next-generation remote working technologies, such as improved virtual reality. Governments deploy
surveillance technologies to track those infected and identify those who came into contact with them. Researchers are
employing bioinformatics, artificial intelligence, and synthetic biology in drug and vaccine development. 3D print venti-
lators, masks, hands-free door openers, and other critical equipment technology and innovations launched to battle the
global pandemics. The impact on society is as inevitable as they are hard to predict; behavioral economics might prove
useful in responding to in-person connections over social media, remote shopping (online). That is undoubtedly one of
the major themes of pandemics, past and present. COVID-19 may permanently standardize virtual technologies for
socializing, business, education, healthcare, religious worship, and even government and private sectors.
Zoonosis: A disease transmitted from animals (vector) to people or an illness (pathogen) that generally exists in ani-
mals and can infect humans. There are multitudes of zoonotic diseases. Some examples include Zoonotic influenza,
Salmonellosis, West Nile virus, Plague, Coronaviruses, Rabies, Brucellosis, and Lyme disease. From pandemic history,
one can understand that animals are the reservoir/incubators of pandemic disease pathogens. Zoonoses may appear sud-
denly and relatively virulent, as illustrated by the HIV, AIDS epidemic, and the coronavirus responsible for the out-
break of SARS and COVID-19. Researchers are studying how animals are developing immunity against pathogens and
which factors make them ideal viral incubators.
Prediction of pathogen transmission: Several well-established methodologies, including risk factor analysis, risk
modeling, and dynamic modeling, are used to make predictions of an infectious disease outbreak in the future, how the
disease spread, and how to control it. Pandemic forecasts at the national and international level steer the policy to
implement appropriate intervention strategies. The early stage of the outbreak predicts the possible time point of the
exponential transmission and alerts the public health system.
Inoculation (variolation) works as Convalescent Plasma Therapy (CPT): Smallpox’s history holds a unique medical
place. To date, the only human disease to have been eradicated by vaccination in the year 2016 [125]. The smallpox
pandemic swept across North America from 1775 to 1782; war soldiers took an unusual approach to protect themselves
from the virus is known as variolation. They took virus-loaded material (variola major) from an infected person’s small-
pox pustule, carved an incision into the flesh of a healthy soldier, and rubbed it in. From the inoculation of virus-
infected person pustule, healthy soldiers developed immunity against the virus, and they did not catch smallpox [126].
Edward Jenner (1976), who himself had been variolated a child taking lesion material from a woman who had cowpox,
rubbed it into the wound of an 8-year-old boy. When he later attempted to infect the boy with smallpox, no disease
developed—Variolation as a natural precursor to discovering a vaccine. The smallpox vaccine, introduced by Edward
Jenner, was the first successful vaccine developed based on his observation that milkmaids who previously had caught
cowpox did not catch smallpox.
Inoculated vaccinia is protecting against the inoculated variola virus that becomes the principle of vaccination. The
concept of vaccination, named from the Latin word cow, or vacca, was born [127]. Fast forward to today, variolation
has come full circle, as researchers explore the idea of using “convalescent plasma” as a treatment. CPT, an old-school
weapon against deadly illnesses, gives coronavirus patients a dose of virus-fighting antibodies.
A vaccine has yet to materialize precisely. As the pandemic continues to take a crushing toll, doctors resort to a
century-old treatment that has helped manage previous pandemics: taking antibodies from those who have recov-
ered and giving them to the sick. It is known as convalescent plasma therapy, or “survivors’ blood.” Plasma, the
liquid component of blood, contains antibodies; collecting plasma from someone that has “convalesced” or recov-
ered from an illness might provide a much-needed boost to the immune system of someone struggling with corona-
virus. In the past CPT has been a weapon against the 1918 flu, polio, measles, rabies, hepatitis B, and Ebola, with
the evidence that polyclonal neutralizing antibodies can reduce viremia duration. CPT has proven to be a winning
and logistically feasible therapeutic strategy. Recent large outbreaks of viral diseases for which effective antivirals
or vaccines are still lacking have renewed the CPT’s interest as a life-saving treatment. Some promise in treating
coronaviruses, particularly when given to a patient early in their illness [128,129]. The COVID-19 pandemic has
154 Pandemic Outbreaks in the 21st Century

led to CPT’s scaling up to unprecedented levels [130]. It has been evidenced that many patients who received
plasma therapy improved [131].
Pandemic names: Pandemics were named based on geographic or animal origins of the infectious diseases, for
example, Antonine Plague, Cyprian Plague, Justinian Plague, The Great Plague of London, Russian Flu, Spanish Flu,
Asian flu, Hong Kong Flu. There are several well-known examples of fuzziness in naming. The 1918 influenza pan-
demic was called the Spanish flu in the United States, even though it did not originate in Spain. Chickenpox is a misno-
mer. It is caused by the varicella virus, has nothing to do with chickens. Germans misnamed German measles.
Germany’s only reference is that the disease, caused by the rubella virus, was first described by German physicians.
The coronavirus that causes the disease has been known provisionally as 2019-nCoV. Disease caused by the novel coro-
navirus officially has a name: COVID-19. The WHO chose the name based on the type of virus and the year saw the
first cases. Co and Vi come from coronavirus, with D meaning disease and 19 standing for 2019, the year. For naming
new viruses, the ICTV is responsible. It has called the novel coronavirus SARS-CoV-2. The SARS-CoV-2 is a close
cousin of the SARS coronavirus caused by the SARS outbreak of 2002
03.
The functional naming convention provides a standard format to use for any future coronavirus outbreaks. If there will
be a new Coronavirus outbreak in 2023, the disease’s name can be COVID-23. In selecting COVID-19 as the disease’s
name, the WHO name-givers steered clear linking the episode to China or Wuhan’s city, where the first identified the ill-
ness. However, origin sites have been used to determine new viruses, such a namesake now seen as demeaning. Some
experts have regret naming the infection caused by a different coronavirus, the MERS. Naming matters to prevent the use
of other terms that can be inaccurate or branding. Viruses and the disease cause do not have related names HIV and AIDS.
An exact name could stop using the many of which, like the Wuhan virus or Wu Flu, linked the virus to the city.
Pandemics to specific age group: The influenza epidemic of 1918 was most likely to hit the young and healthy, fall-
ing people ages 15
45 with swift lethality. They got sick so rapidly, some dropped in the streets, noting that their faces
often turned bluish-red due to lack of oxygen. The patients’ robust immune system is part of the problem, unleashing a
torrent of virus-fighting molecules called cytokines that latched onto lung tissue causing lethal damage. While the
demographics of coronavirus are very different, it is hitting older populations. The immune compromised the most chal-
lenging behavior in the young and healthy individual. Some reports that immune responses called “cytokine storms” are
a likely cause of the collateral damage occurring in younger patients. Precisely the same thing happened in 1918.
Healthy immune systems overwhelmed the other organs of the body, especially the lungs. That realization is sparking
new ways of thinking about treatments for COVID-19.
Blame the sick: We are very prone to blaming people who get sick. It has happened throughout the history of pan-
demics. Cholera that hit from the 1830s to 1860s, white protestants shunned Irish immigrants as the menace’s vectors.
In the 1950s polio swept the nation, targeted African Americans and the poor. In the 1980s placed blame on lesbian,
gay, bisexual, and transgender (LGBT) people for spreading HIV-AIDS. In contrast, the WHO in 1980 announced that
the eradication of smallpox was officially the first and only human infectious disease. How was that removed? Through
collaboration. Today, we can learn and act upon the fact that global cooperation and knowledge sharing can shut ongo-
ing and future pandemics.
End of pandemics: In comparison to past pandemics, we also started tackling this horrific coronavirus. Our public
health systems, scientific inventions, and medical supplies are far better than in the past. The first pandemic of this
scope is where we know the type of pathogen is from the beginning. Hopefully, herd immunity building and collabora-
tive work develop treatments and a vaccine with social distancing in place. Now time for us to pay attention and learn
what this illness reveals about us to take that knowledge forward. Every generation in the past century challenged a
great pandemic. Each one teaches us valuable lessons about microbes’ ecology and how they interact with human socie-
ties. The history of pandemics can also tell us about ourselves. The fighting epidemic disease can bring us together. But
epidemics can also separate us, turning our fellow citizens into potential threats and creating fear of the other. But if the
past is the preface, we are awful at using the past as a preamble. We regretted the vital lessons we had failed to learn
from disease before episodes. In recent, many others have been taking note of how plagues of swine flu and the like
have somehow failed to yield more pandemic preparedness. Those who do not remember the past are condemned to
repeat it. Unfortunately, many countries ignored the precautionary principle to protect healthcare workers and the pub-
lic. Looking forward, how pandemics end or fail to complete. This is especially true as politicians start to discuss how
to relax restrictions and get people back to work. Knowing when to declare victory shall be tricky; looking to SARS,
we can see what happens if we do that too soon. In that outbreak precautions such as disease surveillance and restric-
tions on hospital visits were relaxed, the state of emergency formally ended 4 days. After that, just a week later, the sec-
ond wave of SARS infection killed and sickened many people. The stakes are even higher in this pandemic. If we do
not hit the history books for guidance now, reality can hit us harder.
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 155

9.2.17 Future perspectives

Research and Development: Historical data can offer valuable insights that are highly relevant to today’s disease out-
break. The irony is that now, almost all prehistoric to modern times worst pandemic in history; still, we have not under-
stood many fundamental aspects of infectious diseases. We must rely on empirical results, almost centuries-old data.
Understanding the potential origin and evolution of the novel human emerging infectious diseases is critical for studies
of pathogenicity, design useful interventions, drug and vaccine development, and innovate the technology to produce
the necessary drug/vaccines in bulk quantity rapidly.
Emerging Zoonotic diseases database: Animals are the reservoir/incubators of pandemic diseases pathogen, act as
vectors. The evaluation of animal coronaviruses’ zoonotic potential and genomic sequencing of closely related viruses
silently circulates in bats is required. COVID-19 highlights the importance of bats as a reservoir for new viruses causing
infection in humans. It can also be used as an excellent model to design studies and strategies to prevent the future
emergence of new zoonotic diseases. To characterize the viral genome of different animal coronaviruses look for the
viral evolution and adaptation to their natural hosts. To predict the interspecies transmission can help plan specific sur-
veillance programs and act quickly in an outbreak. Extensive research in finding experimental models for emerging
zoonotic viral diseases is mandatory.
Protecting the nature and conservation of wildlife: From the history of pandemics, it is clear that novel zoonotic dis-
eases are emerging at an alarming rate. A growing body of research points to a direct link between the destruction of
nature and disease outbreaks spotlighting the role of protecting and restoring nature in preventing future pandemics.
But human activity—deforestation, intensive and polluting agriculture, and unsafe management and consumption of
wildlife and natural resources have contributed to the origin of infectious and vector-borne diseases. Human impact on
the environment increases the risk of emerging infectious diseases in humans, over 60% of which originate from ani-
mals, mainly from wildlife. The risk of zoonotic diseases is determined by two key factors: (1) the trade of high-risk
live wild animals and their meat, (2) unsustainable food systems operating the large-scale conversion of land for agri-
culture. COVID-19 is the latest zoonotic pandemics. SARS, MERS, and Ebola have exposed the grave dangers of
exploiting high-risk wildlife and encroaching nature. They are tackling high-risk wildlife trade, deforestation, and frag-
mentation, despite numerous interventions to address these issues. Urgent action is necessary to reduce future pan-
demics’ risk through systemic changes that create a more sustainable relationship with nature. Governments must
establish effective legislation addressing wildlife trade, protecting habitats, and reducing the wildlife
livestock
human
interface to prevent future zoonoses.
One Health approach: From the history of pandemics, infectious diseases in people are zoonotic. The nation
strengthens its capabilities to prevent and respond to these diseases using a One Health approach. This approach recog-
nizes the connection between people, animals, plants, and their shared environment and appeals to specialists in human,
animal, and environmental health who work together to achieve the best health outcomes for all.

References
[1] Last JM, editor. A dictionary of epidemiology. 4th edition New York: Oxford University Press; 2001.
[2] Pearce-Duvet JM. The origin of human pathogens: evaluating the role of agriculture and domestic animals in the evolution of human disease.
Biol Rev Camb Philos Soc 2006;81(3):369
82.
[3] Wolfe ND, Dunavan CP, Diamond J. Origins of major human infectious diseases. Nature 2007;447(7142):279
83.
[4] Olson DR, Simonsen L, Edelson PJ, Morse SS. Epidemiological evidence of an early wave of the 1918 influenza pandemic in New York City.
Proc Natl Acad Sci U S A 2005;102(31):11059
63.
[5] Huremović D. Brief history of pandemics (pandemics throughout history). Psychiatry Pandemics 2019;7
35.
[6] Jarus O. 20 of the worst epidemics and pandemics in history, All about history March 20; 2020.
[7] Crawley R. Thucydides, history of the Peloponnesian War, Book 2, Chapter VII. Digireads.com Publishing; 2017. p. 89
100. ISBN-10:
1420956418.
[8] Littman RJ. The plague of Athens: epidemiology and paleopathology. Mt Sinai J Med 2009;76(5):456
67.
[9] Laes C. Disability in antiquity (Rewriting antiquity). London; New York: Routledge; 2017. p. 490. ISBN: 9781138814851.
[10] Fears JR. The plague under Marcus Aurelius and the decline and fall of the Roman Empire. Infect Dis Clin N Am 2004;18(1):65
77.
[11] Merriam-Webster, Plague. Merriam-Webster.com. 2018 Nov. 10.
[12] Schaff P. “Fathers of the third century: Hippolytus, Cyprian, Caius, Novatian, Appendix,” Translated from Cyprian wrote in Latin in a work
called “De mortalitate.” Christian Classics Ethereal Library; 1885.
[13] Rosen W. Justinian’s flea: plague, empire, and the birth of Europe. New York: Viking Adult, Hardcover; 2007.
[14] Horgan J. Justinian’s Plague (541
542 CE). Ancient history encyclopedia; 2014. Available from: https://www.ancient.eu/article/782/.
[15] McCormick M. Tracking mass death during the fall of Rome’s empire (I). J Roman Archaeol 2015;28:325
57.
156 Pandemic Outbreaks in the 21st Century

[16] Mordechai L, Eisenberg M. Rejecting catastrophe: the case of the Justinianic Plague. Past Present 2019;244:3
50.
[17] Green M. Taking “pandemic” seriously: making the Black Death global. Medieval Globe 2014;1:27
61.
[18] DeWitte SN. Mortality risk and survival in the aftermath of the medieval Black Death. PLoS One 2014;9(5):e96513.
[19] Scheidel W. Chapter 10: The black death. The great leveler: violence and the history of inequality from the stone age to the twenty-first cen-
tury. Princeton: Princeton University Press; 2017. p. 291
313.
[20] Gottfried RS. Black death. Simon and Schuster; 2010. p. 74. ISBN: 978-1-4391-1846-7.
[21] Byrne JP. The black death. Westport: Greenwood Press; 2004. p. 108.
[22] Horrox R. Black death. Manchester University Press; 1994. p. 159. ISBN 978-0-7190-3498-5.
[23] Halliday S. Death and miasma in Victorian London: an obstinate belief. BMJ 2001;323(7327):1469
71.
[24] Eisen RJ, Gage KL. Adaptive strategies of Yersinia pestis to persist during inter-epizootic and epizootic periods. Vet Res 2009;40(2):1.
[25] Shrewsbury JFD. A history of Bubonic Plague in the British Isles. Cambridge: Cambridge University Press,; 1970.
[26] Defoe D. Journal of the plague year. London: Penguin; 1978.
[27] Langdon-Davis J. The plague and fire of London. Jonathan Cape, London: Jackdaw Publications; 1963.
[28] Walløe L. Medieval and modern bubonic plague: some clinical continuities. Med Hist Suppl 2008;27:59
73.
[29] Stenseth NC, Atshabar BB, Begon M, Belmain SR, Bertherat E, Carniel E, et al. Plague: past, present, and future. PLoS Med. 2008;5(1):e3.
[30] Sun W. Plague vaccines: status and future. Adv Exp Med Biol 2016;918:313
60.
[31] Eichman P. The history, biology and medical aspects of leprosy. Am Biol Teach 1999;61(7):490
5.
[32] Vogelsang TM. Gerhard Henrik Armauer Hansen. Int J Lepr 1978;46(3):257
332.
[33] WHO. Global leprosy update, 2018: moving towards a leprosy-free world. Weekly Epidemiological Record. WER No 35/36, 2019; 2019 94:
389
412.
[34] Talwar GP, Gupta JC. Launching of immunization with the vaccine Mycobacterium Indicus Pranii for eradication of page 4 of 5 leprosy in
India. Int J Vaccine Res 2017;2(3):1
5.
[35] Rao PN, Suneetha S. Current situation of leprosy in india and its future implications. Indian Dermatol Online J 2018;9(2):83
9.
[36] Potter C. Chronicle of influenza pandemics. In: Nicholson KG, Webster RF, Hay AJ, editors. Textbook of influenza. Oxford, UK: Blackwell
Science Ltd.; 1998.
[37] Potter CW. A history of influenza. J Appl Microbiol 2001;91(4):572
9.
[38] Broxmeyer L. Bird flu, influenza and 1918: the case for Avian mutant tuberculosis. Med Hypotheses 2006;67(5):1006
15.
[39] Hilleman Maurice R. Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control. Vaccine 2002;20
(25
26):3068
87.
[40] Gregg MB, Hinman AR, Craven RB. The Russian flu. Its history and implications for this year’s influenza season. JAMA 1978;240(21):2260
3.
[41] Spinney L. The Spanish flu: an interdisciplinary problem. Lancet 2018;392(10164):2552.
[42] Trilla A, Trilla G, Daer C. The 1918 “Spanish flu” in Spain. Clin Infect Dis 2008;47(5):668
73.
[43] Taubenberger JK, Hultin JV, Morens DM. Discovery and characterization of the 1918 pandemic influenza virus in historical context. Antivir
Ther 2007;12(4 Pt B):581
91.
[44] Martini M, Gazzaniga V, Bragazzi NL, Barberis I. The Spanish Influenza Pandemic: a lesson from history 100 years after 1918. J Prev Med
Hyg 2019;60(1):E64
7.
[45] Jones MM. The American Red Cross and local response to the 1918 influenza pandemic: a four-city case study. Public Health Rep 2010;125
(Suppl 3):92
104.
[46] Reid AH, Taubenberger JK, Fanning TG. Evidence of an absence: the genetic origins of the 1918 pandemic influenza virus. Nat Rev Microbiol
2004;2(11):909
14.
[47] Burnet FM. Asian influenza. Triangle 1959;4(1):9
13.
[48] Honigsbaum M. Revisiting the 1957 and 1968 influenza pandemics. Lancet 2020;395(10240):1824
6.
[49] Schafer JR, Kawaoka Y, Bean WJ, Suss J, Senne D, Webster RG. Origin of the pandemic 1957 H2 influenza A virus and the persistence of its
possible progenitors in the avian reservoir. Virology 1993;194:781
8.
[50] Igarashi M, Ito K, Kida H, Takada A. Genetically destined potentials for N-linked glycosylation of influenza virus hemagglutinin. Virology
2008;376(2):323
9.
[51] Zost SJ, Wu NC, Hensley SE, Wilson IA. Immunodominance and antigenic variation of influenza virus hemagglutinin: implications for design
of universal vaccine immunogens. J Infect Dis 2019;219(Suppl_1):S38
45.
[52] Cockburn WC, Delon PJ, Ferreira W. Origin and progress of the 1968
69 Hong Kong influenza epidemic. Bull World Health Organ 1969;41
(3):345
8.
[53] Scholtissek C. Molecular epidemiology of influenza. Arch Virol Suppl 1997;13:99
103.
[54] Viboud C, Grais RF, Lafont BA, Miller MA, Simonsen L, Multinational Influenza Seasonal Mortality Study Group. Multinational impact of the
1968 Hong Kong influenza pandemic: evidence for a smoldering pandemic. J Infect Dis 2005;192(2):233
48.
[55] CDC. 1968 Pandemic (H3N2 virus). Centers for Disease Control and Prevention. Archived from the original on 30 January 2019. Retrieved 22
August 2020; 2019.
[56] Sims LD, Domenech J, Benigno C, Kahn S, Kamata A, Lubroth J, et al. Origin and evolution of highly pathogenic H5N1 avian influenza in
Asia. Vet Rec 2005;157(6):159
64.
[57] Lycett SJ, Duchatel F, Digard P. A brief history of bird flu. Philos Trans R Soc Lond B Biol Sci 2019;374(1775):20180257.
 Pandemics of the 21st century: lessons and future perspectives Chapter | 9 157

[58] Lu Y, Landreth S, Gaba A, Hlasny M, Liu G, Huang Y, et al. In vivo characterization of Avian influenza A (H5N1) and (H7N9) viruses iso-
lated from Canadian travelers. Viruses 2019;11(2):193.
[59] Swayne DE. Principles for vaccine protection in chickens and domestic waterfowl against avian influenza: emphasis on Asian H5N1 high path-
ogenicity avian influenza. Ann N Y Acad Sci 2006;1081:174
81.
[60] Sanz I, Rojo S, Tamames S, Eiros JM, Ortiz de Lejarazu R. Heterologous humoral response against H5N1, H7N3, and H9N2 Avian influenza
viruses after seasonal vaccination in a European elderly population. Vaccines (Basel) 2017;5(3):17.
[61] Almagro-Moreno S, Taylor RK. Cholera Environmental reservoirs and impact on disease transmission. Microbiol Spectrum 2013;1(2).
[62] Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. Lancet 2004;363(9404):223
33.
[63] Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. Cholera. Lancet 2012;379(9835):2466
76.
[64] Cava F. Biology of Vibrio cholera. Editorial overview. Int Microbiol 2017;20(3):105.
[65] NIAID. Cholera biology and genetics. National Institute of Allergy and Infectious Diseases; 2011.
[66] History.com editors. Cholera; 2020. Available from: https://www.history.com/topics/inventions/history-of-cholera. [Accessed 24 October 2020].
[67] Lee K. The global dimensions of cholera. Global Change Hum Health 2001;2:6
17.
[68] Hayes JN. Epidemics and pandemics: their impacts on human history. Santa Barbara, CA: ABC-CLIO; 2005. p. 214
9.
[69] Moore S, Thomson N, Mutreja A, Piarroux R. Widespread epidemic cholera caused by a restricted subset of Vibrio cholerae clones. Clin
Microbiol Infect 2014;20(5):373
9.
[70] Hu D, Liu B, Feng L, Ding P, Guo X, Wang M, et al. Origins of the current seventh cholera pandemic. Proc Natl Acad Sci U S A 2016;113
(48):E7730
9.
[71] Rabaan AA. Cholera: an overview with reference to the Yemen epidemic. Front Med 2019;13(2):213
28.
[72] Deen J, Mengel MA, Clemens JD. Epidemiology of cholera. Vaccine 2020;38(Suppl 1):A31
40.
[73] Legros D, Partners of the Global Task Force on Cholera Control. Global cholera epidemiology: opportunities to reduce the burden of cholera
by 2030. J Infect Dis 2018;218(suppl3):S137
40.
[74] Wierzba TF. Oral cholera vaccines and their impact on the global burden of disease. Hum Vaccin Immunother 2019;15(6):1294
301.
[75] Pigott DC. Hemorrhagic fever viruses. Crit Care Clin 2005;21 765
783 vii.
[76] Kuhn JH, Becker S, Ebihara H, et al. Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and
virus abbreviations. Arch Virol 2010;155:2083
103.
[77] Breman JG, David LH, Graham L, Joseph BM, Malonga M, Frederick AM, et al. Discovery and description of Ebola Zaire Virus in 1976 and
relevance to the West African epidemic during 2013
2016. J Infect Dis 2016;214(suppl_3):S93
101.
[78] Languon S, Quaye O. Filovirus disease outbreaks: a chronological overview. Virology (Auckl) 2019;10 1178122X19849927.
[79] Alexander KA, Sanderson CE, Marathe M, Lewis BL, Rivers CM, Shaman J, et al. What factors might have led to the emergence of Ebola in
West Africa? PLoS Negl Trop Dis 2015;9(6):e0003652.
[80] Amundsen S. Historical analysis of the Ebola virus: prospective implications for primary care nursing today. Clin Excellence Nurse Pract
1998;2(6):343
51.
[81] Goldstein T, Anthony SJ, Gbakima A, et al. The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses. Nat
Microbiol 2018;3:1084
9.
[82] Baseler L, Chertow DS, Johnson KM, Feldmann H, Morens DM. The pathogenesis of Ebola virus disease. Annu Rev Pathol 2017;12:387
418.
[83] Tchesnokov EP, Feng JY, Porter DP, Götte M. Mechanism of inhibition of Ebola virus RNA-dependent RNA polymerase by Remdesivir.
Viruses 2019;11(4):326.
[84] Henao-Restrepo AM, Preziosi MP, Wood D, Moorthy V, Kieny MP, WHO Ebola Research, Development Team. On a path to accelerate access
to Ebola vaccines: the WHO’s research and development efforts during the 2014
2016 Ebola epidemics in West Africa. Curr Opin Virol
2016;17:138
44.
[85] Meyer M, Malherbe DC, Bukreyev A. Can Ebola virus vaccines have universal immune correlates of protection? Trends Microbiol. 2019;27
(1):8
16.
[86] Mann JM. AIDS-A global perspective. West J Med 1987;147(6):693.
[87] Okoye AA, Picker LJ. CD4(1) T-cell depletion in HIV infection: mechanisms of immunological failure. Immunol Rev 2013;254(1):54
64.
[88] Case K. Nomenclature: human immunodeficiency virus. Ann Intern Med 1986;105(1):133.
[89] Hymes K.B., Cheung T., Greene J.B., Prose N.S., Marcus A., Ballard H., William D.C., Laubenstein L.J. Kaposi’s sarcoma in homosexual
men-a report of eight cases. Lancet. 1981;2(8247):598
600.
[90] Barré-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk
for acquired immune deficiency syndrome (AIDS). Science 1983;220(4599):868
71.
[91] UNAIDS. United Nations Programme on AIDS: Global HIV and AIDS statistics-2020 fact sheet.
[92] Mateo-Urdiales A, Johnson S, Smith R, Nachega JB, Eshun-Wilson I. Rapid initiation of antiretroviral therapy for people living with HIV.
Cochrane Database Syst Rev 2019;6(6):CD012962.
[93] Desai S, Tavoschi L, Sullivan AK, Combs L, Raben D, Delpech V, et al. HIV testing strategies employed in health care settings in the
European Union/European Economic Area (EU/EEA): evidence from a systematic review. HIV Med 2020;21(3):163
79.
[94] Piot P, Quinn TC. Response to the AIDS pandemic—a global health model. N Engl J Med 2013;368(23):2210
18.
[95] Trifonov V, Khiabanian H, Rabadan R. Geographic dependence, surveillance, and origins of the 2009 influenza A (H1N1) virus. N Engl J Med
2009;361(2):115
19.
158 Pandemic Outbreaks in the 21st Century

[96] Bautista E, Chotpitayasunondh T, Gao Z, Harper SA, Shaw M, Uyeki TM, et al. Clinical aspects of pandemic 2009 influenza A (H1N1) virus
infection. N Engl J Med 2010;362(18):1708
19.
[97] Kelly H, Peck HA, Laurie KL, Wu P, Nishiura H, Cowling BJ. The age-specific cumulative incidence of infection with pandemic influenza
H1N1 2009 was similar in various countries prior to vaccination. PLoS One 2011;6(8):e21828.
[98] Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 2009;459(7249):931
9.
[99] WHO. “Standardization of terminology of the pandemic A(H1N1)2009 virus.” World Health Organization (WHO). Retrieved 31 March 2020; 2010.
[100] WHO. “Recommended use of antivirals.” Global Alert and Response (GAR). Geneva: World Health Organization (WHO). 21 August 2009.
Archived from the original on 4 November 2009. Retrieved 6 November 2020.
[101] Ma W. Swine influenza virus: current status and challenge. Virus Res 2020;288:198118.
[102] Kindhauser MK, Allen T, Frank V, Santhanaa RS, Dye C. Zika: the origin and spread of a mosquito-borne virus. Bull World Health Organ
2016;94(9):675
86. Available from: https://doi.org/10.2471/BLT.16.171082.
[103] Albuquerque MFPM, Souza WV, Araújo TVB, Braga MC, Miranda Filho DB, Ximenes RAA, et al. The microcephaly epidemic and Zika
virus: building knowledge in epidemiology. Cad Saude Public 2018;34(10):e00069018.
[104] Kuiken T, Fouchier RA, Schutten M, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet
2003;362:263
70.
[105] Guan Y, Zheng BJ, He YQ, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.
Science 2003;302:276
8.
[106] Lee N, Hui DS, Wu A, et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003;348:1986
94.
[107] Cheng VC, Lau SK, Woo PC, Yuen KY. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection.
Clin Microbiol Rev 2007;20(4):660
94.
[108] Chan-Yeung Moira, Xu Rui-Heng. SARS: epidemiology. Respirology 2003;8(s1):S9
14.
[109] Monaghan Karen J. SARS: down but still a threat. United States: National Academies Press; 2004.
[110] Woodhead M, Ewig S, Torres A. Severe acute respiratory syndrome (SARS). Eur Respir J 2003;21:739
40.
[111] Michael DC, Susan MP, Mona RL, Matthew PM, Donald E. Low, severe acute respiratory syndrome. Clin Infect Dis 2004;38(10):1420
7.
[112] Stockman LJ, Bellamy R, Garner P. SARS: systematic review of treatment effects. PLoS Med 2006;3(9):e343.
[113] Lim WS, Anderson SR, Read RC. Hospital management of adults with severe acute respiratory syndrome (SARS) if SARS re-emerges—
updated 10 February 2004. J Infect 2004;49(1):1
7.
[114] Rasmussen SA, Watson AK, Swerdlow DL. Middle east respiratory syndrome (MERS). Microbiol Spectr 2016;4(3).
[115] Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet 2015;386(9997):995
1007.
[116] Willman M, Kobasa D, Kindrachuk J. A comparative analysis of factors influencing two outbreaks of middle eastern respiratory syndrome
(MERS) in Saudi Arabia and South Korea. Viruses 2019;11(12):1119.
[117] Mubarak A, Alturaiki W, Hemida MG. Middle east respiratory syndrome coronavirus (MERS-CoV): infection, immunological response, and
vaccine development. J Immunol Res 2019;2019:6491738.
[118] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet
2020;395:497
506.
[119] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J. A novel coronavirus from patients with pneumonia in China 2019. N Engl J Med
2020;382:727
33.
[120] Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG. A new coronavirus associated with human respiratory disease in China. Nature
2020;579:265
9.
[121] ICTV, Coronaviridae Study Group of the International Committee on Taxonomy of V. The species severe acute respiratory syndrome-related
coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020;5:536
44.
[122] Liu YC, Kuo RL, Shih SR. COVID-19: The first documented coronavirus pandemic in history. Biomed J 2020;S2319
4170(20):30044
5.
[123] WHO. Coronavirus disease (COVID-19) pandemic. Numbers at glance. 31 July, 2020.
[124] Davis MR, McCreary EK, Pogue JM. That escalated quickly: remdesivir’s place in therapy for COVID-19. Infect Dis Ther 2020. Available
from: https://doi.org/10.1007/s40121-020-00318-1.
[125] Henderson D. Smallpox: the death of a disease. New York: Prometheus Books; 2009.
[126] Boylston A. The origins of inoculation. J R Soc Med 2012;105(7):309
13.
[127] Plotkin S. History of vaccine development. New York: Springer; 2011. p. 39.
[128] Cheng Y, Wong R, Soo YO, Wong WS, Lee CK, Ng MH, et al. Use of convalescent plasma therapy in SARS patients in Hong Kong. Eur J
Clin Microbiol Infect Dis 2005;24:44
6.
[129] Mair-Jenkins J, Saavedra-Campos M, Baillie JK, Cleary P, Khaw FM, Lim WS, et al. The effectiveness of convalescent plasma and hyperim-
mune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-anal-
ysis. J Infect Dis 2015;211:80
90.
[130] Zhou F, Yu T, Du R, Fan G. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective
cohort study. Lancet 2020;395:1054
62.
[131] Chen X, Zhao B, Qu Y, Chen Y, Xiong J, Feng Y, et al. Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with
drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients. Clin Infect Dis 2020. Available from: https://doi.org/10.1093/
cid/ciaa449.
Chapter 10

Immunological mechanisms associated

with clinical features of Ebola virus
disease and its control and prevention
Nayaka Boramuthi Thippeswamy
Department of Postgraduate Studies and Research in Microbiology, Kuvempu University, Shivamogga, India

10.1 Introduction
For invading microorganisms, immune system is the most important barrier for successful infection and multiplication
in the host. Some of the pathogens specifically target certain immune cells to infect and induce an abnormally altered
level of immune response. The pathogens can also block important and pathogen-specific signaling pathways involved
in the development of immune response. Thus pathogens can complete their life cycle in the immune-compromised
host, causing the disease that eventually could lead to death. Ebola virus (EBOV) is one such pathogen that uses a simi-
lar strategy for pathogenesis in humans. This virus was identified in a village near the Ebola River, hence the name
Ebolavirus. EBOV belongs to the family filoviridae and is prevalent in the regions of the west and equatorial Africa.
This infection causes sporadic but devastating outbreaks of severe hemorrhagic fever especially in humans and nonhu-
man primates (NHP). The EBOV genus is composed of five species; each species is represented by a single virus:
Sudan ebolavirus (SUDV), Reston ebolavirus (RESTV), Bundibugyo ebolavirus (BDBV), Tai Forest ebolavirus
(TAFV), and EBOV (previously known as Zaire ebolavirus). The EBOV, SUDV, and BDBV infections are lethal to
humans, mortality rate lies between 40% and 90% depending upon the type of virus species. TAFV can cause symp-
tomatic disease but not severe and its fatality rate is not known as there is only one documented case in humans.
RESTV infection is not associated with disease in humans but lethal to NHP. The survival rate of infected individuals
depends on the host’s ability to mount an early and robust immune response and keeping a low level of viral load.
However, the virus has developed a variety of methods to counter and collapse both innate and adaptive immune
responses to serve its replicative purposes. These viruses spread from person to person through direct contact via broken
skin or mucous membranes, using bodily fluids such as urine, saliva, sweat, feces, vomit, breast milk, and semen. The
fruit bat (Rousettus aegyptiacus) is considered as the natural reservoir for EBOV and these can transmit the virus
through monkeys, apes, and antelopes in the forest. The EVD is characterized by the development of symptoms that
includes fever, severe fatigue, arthralgia, and hemorrhagic complications resulting in life-threatening conditions such as
shock, multiorgan dysfunction, and death. Young age and pregnancy are considered deleterious prognostic factors asso-
ciated with the early death of infected individuals. Very importantly, the virus persists for a long time in selected body
fluids like semen, amniotic fluid, and breast milk in the survivors of EBOV infection, increasing the risk of disease
transmission. Thus the chronic state of EVD is of great concern in the recovered patients. Keeping this in view, the
EVD epidemiology, virus structure, life cycle, dysregulation of an immune response, disease pathogenesis, clinical
manifestation, therapeutics, and preventive measures have been discussed here in detail.

10.2 Epidemiology
The EBOV outbreak was reported for the first time in 1976 simultaneously in northern Zaire, now the Democratic
Republic of the Congo (DRC), with 318 cases (death rate of 88%) and in Sudan with 284 cases (death rate of 53%) [1].
These two epidemics were caused by different species of the EBOV, Zaire EBOV (ZEBOV) and Sudan EBOV.
Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00001-X
© 2021 Elsevier Inc. All rights reserved. 159
160 Pandemic Outbreaks in the 21st Century

Subsequent outbreaks of EBOV revealed the presence of three more species with varying pathogenicity in humans. In
1989 the Reston Ebola virus was discovered in the United States during an outbreak of viral hemorrhagic fever in cyno-
molgus macaques imported from the Philippines and was found to be nonpathogenic to humans [2]. TAFV was discov-
ered in 1994 and was isolated from an ethnologist who contracted the disease when he was operating a chimpanzee
found dead in the Tai National park; it was nonfatal and remains as the only documented case [3]. Another EBOV
called BDBV was discovered in 2007 in Uganda, where it caused an outbreak by infecting 149 people with a death rate
of 25%.
Among all the species, multiple strains of ZEBOV are responsible for most of the Ebola outbreaks. By the end of
2013, a total of 1383 ZEBOV, 779 SEBOV (SUDV), and 185 BDBV cases were reported in the remote regions of cen-
tral Africa [4]. In December 2013 ZEBOV emerged in Guinea and rapidly extended to Sierra Leone and Liberia, lead-
ing to the largest EBOV epidemic in history. In August 2014 the WHO notified an unrelated ZEBOV outbreak in the
DRC, which resulted in 66 cases with a 74% death rate. As of March 2016, 28,646 positive cases of ZEBOV disease
with 11,323 deaths were reported across the African countries. One more outbreak was recorded on May 8, 2018, in the
northwestern part of the DRC, during which the therapeutic vaccination was used for the first time in the early stage of
the outbreak to control the infection [5]. The WHO declared that the outbreak ended on 24 July 2018. On August 1,
2018, the second outbreak in DRC was declared and confirmed that there is no link between the two outbreaks of DRC.
Although both the outbreaks were caused by the same species of EBOV, they were completely different [6]. In June
2019 the virus entered Uganda through a 5-year-old boy who visited the country for seeking health care but health offi-
cials confirmed the presence of Ebolavirus. From 31 July to 20 August 2019, a total of 216 cases were reported mainly
in the Beni, Mandima, and Butembo provinces of Uganda. During 2018 and 2019, a total of 2927 cases were reported
with a 67% fatality rate. This is the second-largest EVD outbreak compared to the one in West Africa between 2014
and 2016 [7,8]. All these increased numbers of EVD outbreaks since 2000, maybe due to increased contact between
humans and wildlife, high deforestation, as well as climatic changes [9].

10.2.1 Ecology and spreading of Ebola virus

EBOVs are zoonotic pathogens maintained in reservoir species, perhaps bats, with occasional spillover into humans and
other mammals (Fig. 10.1), which may serve as the end, intermediate, or amplifying hosts [10]. Multiple bat species

FIGURE 10.1 Ebola virus transmission. Fruit bats are considered as natural reservoirs of EBOVs and these seem to infect NHP and duikers, which
mostly constitute the spillover event. The virus disseminates from person to person, potentially affecting a large number of people. However, EBOVs
may also spread through the handling. EBOV, Ebola virus; NHP, nonhuman primates.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 161

have been implicated to harbor EBOVs, but the isolation of active virus has not yet been successful [11]. This is rather
uncommon and may be explained by the low level of viral loads, low susceptibility of cells, or inhibitors in bat tissue.
In African countries EVD mostly spreads to humans, living in the forest area due to handling of bushmeat and contact
with infected animals [12]. Human pathogenic EBOVs appear to be epizootic in the regions close to the African
equator.

10.3 Virus structure

Ebola is a filamentous and highly polymorphic enveloped virus containing a 19-kb, negative-strand RNA genome
(Fig. 10.2). The genome has seven sequentially arranged genes from the 30 to 50 end that are, nucleoprotein (NP), virion
protein-35 (VP35), matrix protein VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase also
known as large protein (L), which altogether encoding for 10 proteins [13]. The EBOV genome has overlapping gene
sequences, that is, the stop site of the previous gene sequence overlaps with the start sequence of the subsequent gene
[14]. The genes that encode a single protein include NP, VP24, VP30, VP35, VP40, and L, whereas GP encodes for
four GPs, namely, GP1, GP2, secretory glycoprotein (sGP), and small secretory glycoprotein (ssGP) [15,16]. The ends
of genome contain short, extragenic regions, called leader and trailer sequences, having encapsidation signals as well as
replication and transcription promoters. The leader sequence involved in the initiation of RNA synthesis by viral RNA
polymerase also directs the packaging of the full-length negative-strand RNA genome into nucleocapsids. The trailer
region at the 30 end of the antigenome RNA (1RNA) acts as a promoter for genome replication [17
19].
The GP is the only surface protein on the viral envelope and is important in mediating receptor binding and fusion
[20
22]. The GP gene encodes four different proteins (GP1, GP2, sGP, and ssGP), in which GP1 and GP2 are edited

FIGURE 10.2 EBOV structure and its genomic organization. (A) The core structure comprises of the genomic RNA encapsidated with the viral NP
and linked with the viral transcriptase
replicase complex, which consists of virion proteins (VP30 and VP35) and RNA-dependent RNA polymerase,
which is further associated with VP24. The structure is surrounded by a cell-derived membrane associated with VP40 inside and the glycoprotein
forming spikes on the outside of the viral envelope. (B) The genome of EBOV has approximately 19 kb negative-strand RNA which varies with spe-
cies. RNA contains seven genes represented by colored boxes encoding for a specific protein except GP. Four different GP transcripts (GP1, GP2,
sGP, and ssGP) are produced by the RNA editing process. EBOV, Ebola virus; GP, glycoprotein.
162 Pandemic Outbreaks in the 21st Century

transcripts carried out by the L protein, whereas sGP and ssGP are due to unedited transcripts. Functionally, GP1 binds
to the target cell and GP2 induces fusion with the host cell membrane [23
25]. The sGP and ssGP do not contain the
transmembrane domain hence they are produced only in the secretory form. The ssGP is a truncated form of sGP and
its biological function is not known [26
28]. VP40 is a matrix protein located beneath the viral membrane, where it
maintains the structural integrity of viral particles [20,29]. Expression of VP40 alone can induce the release and forma-
tion of virus-like particles (VLPs) in mammalian cells even in the absence of other VPs revealing its importance in
virus budding [30]. VP24 is a minor matrix protein that plays different roles in nucleocapsid formation, virus release,
and suppression of immune response. VP24 is an antagonist of the IFN response and contributes to the virulence of the
virus, whereas NP, VP35, VP30, and L are associated with the viral RNA genome, forming the nucleocapsid [17,31].
NP is the structural component of the nucleocapsid complex but can also catalyze replication and transcription of the
RNA genome. The active polymerase complex is composed of VP35 (a polymerase cofactor) and the RNA polymerase
L [17,18]. VP30 is a transcriptional activator and a major component of L. VP35 also contributes to innate immune eva-
sion by blocking type I and type II IFN production signaling pathways.

10.4 Life cycle

Host receptors used by the virus to gain entry include TIM, TAM, lectin, and folate receptor proteins present on the sur-
face of DCs, macrophages, and endothelial cells (Fig. 10.3) [32,33]. Once GP binds to its receptor, virions enter the cell
by receptor-mediated endocytosis or macropinocytosis. After the uptake into macropinosomes, particles travel to low
pH compartments of late endosomes and lysosomes where the viral envelope GP is proteolytically cleaved by endoso-
mal cysteine proteases (i.e., cathepsin B and L). This cleavage removes a heavily glycosylated region of GP and
exposes a domain in GP that binds specifically to the endosomal/lysosomal resident EBOV receptor Niemann-Pick C1
protein (NPC1) [34
40]. The experimental evidence suggests that NPC1 binding may be sufficient to trigger the fusion
of viral and cellular membranes [41].
After the fusion, the virus uncoats and releases its genome into the cytoplasm and then transcribes into mRNA using
nucleocapsid-associated viral proteins. The EBOV RNA-dependent RNA polymerase binds to a site within the leader
region of each negative sense genome and slides along the RNA template, transcribing individual genes sequentially in
a 30 to 50 direction [42]. Each gene is comprised of highly conserved transcription start and stop signals with polyadeny-
lation sites marking the termini of the mRNAs [20]. Additionally, EBOV mRNAs are shown to be capped at the 50 end
and polyadenylated at the 30 end [43]. EBOV transcription is dependent on the presence of transcription factor VP30
which is mediated via a complex of VP30, VP35, and the viral polymerase L bound to the NP-coated genome
[18,20,44
48]. The initial transcription and translation of virus genes lead to a buildup of viral proteins, especially NP,
which triggers viral replication [17,18]. Upon phosphorylation, VP30 dissociates from VP35/L complex and is the sig-
nal to switch from transcription to replication [49,50]. During replication, the promoter at the 30 end of the genomic
RNA drives the synthesis of full-length, positive sense antigenomic RNA, which, in turn, serves as a template for the
production of progeny negative-sense genomes [42]. When sufficient virus genomes are replicated, they will undergo
binding assembly with NP, VP24, VP30, and VP35 [18]. During assembly, L associates with the ribonucleoprotein
complex upon interaction with VP35. The nucleocapsids are delivered to sites of viral assembly and are released at the
plasma membrane. The virus release occurs by budding and involves the interaction of VP40 with the components of
the cellular endosomal sorting complex required for the transport pathway. VP40 is sufficient for viral budding, but
budding efficiency is enhanced by other viral proteins, including GP, which reaches the plasma membrane [51].

10.5 Molecular mechanisms of Ebola pathogenesis

10.5.1 Dysregulation of the innate immune response during Ebola infection
The innate immune system constitutes the first line of host defense response to infectious agents which is characterized
by rapid detection of the foreign pathogen and activation of host innate immunity to limit pathogen multiplication and
spread. Antigen-presenting cells such as monocytes, macrophages, and DCs play a significant role in both the stimula-
tion and regulation of innate immune response, as well as subsequent transition to adaptive immunity. Evasion of host
innate immune response is of particular importance to viruses, and many of them have evolved unique mechanisms to
circumvent this line of defense. Studies in animal models and tissue culture suggest that both pathogenesis and inter-
feron antagonism are linked to EBOV proteins, VP35 and VP24 [52
58].
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 163

FIGURE 10.3 The illustration of the EBOV life cycle by the following steps. (1) EBOV binds to the cell surface receptors. (2) Virus gaining entry
into the cell by receptor-mediated endocytosis. (3) Late endosome formation within the vesicle. (4) Acidification of the endocytic vesicle, followed by
fusion of the virus and host membrane releasing the EBOV nucleocapsid into the cytoplasm. (5) The RNA-dependent RNA polymerase transcribes
individual mRNA from the negative-sense genome in a 30 to 50 direction. (6) Each mRNA is capped at the 50 end and a poly-A tail at 30 end, during
posttranscriptional modification. (7) Synthesis of viral proteins after the translation of mRNA transcript. (8) GP and sGP are further modified in the
endoplasmic reticulum and Golgi body. (9) During replication, the promoter at the 30 end of the genomic RNA drives the synthesis of the full-length,
positive-sense, antigenomic RNA, which, in turn, serves as a template for the production of progeny, negative-sense genomes. (10) Nucleocapsid pro-
teins (VP35, L, VP30, and NP) associate with negative-sense genome progeny. (11) When sufficient levels of the negative-sense genomes and viral
proteins are synthesized, they assemble with VP24, VP40, and GP at the plasma membrane. (12) Secretion of sGP into the circulation by exocytosis.
(13) Budding of complete virions from the cell surface. EBOV, Ebola virus; ESCRT, endosomal sorting complexes required for transport; GP, glyco-
protein; TIM-1, T-cell immunoglobulin and mucin domain 1; TAM, Tyro3, Axl, and Mer receptors; VP, virion protein; NP, nucleoprotein.

10.5.2 Subversion of IFN-induced signaling by EBOV

Virulence of EBOV can be attributed to several immunoevasion mechanisms: an early inhibition of innate immunity
initiated by the downregulation of interferon production and inhibition of response to IFN as well as epitope masking
and subversion of the adaptive humoral immunity by secreting a truncated form of the viral GP. A critical component
of innate immunity against viral infection is the IFN response. EBOV has evolved multiple strategies to antagonize the
IFN-α and IFN-β responses in target cells, such as macrophages, monocytes, and DCs, disrupting the normal host innate
immunity function. Ebola VP35 is an important factor for the downregulation of IFN production by the host. Ebola
VP24 inhibits both type I and type II interferon responses in viral-infected cells. This ultimately results in defective DC
maturation, diminished T-cell activation, and proliferation along with apoptosis leading to lymphopenia, a key charac-
teristic feature of EVD.
VP35 plays a prominent role in inhibiting the induction of IFN α, β, and γ through multiple mechanisms
(Fig. 10.4A). Rig-like receptors (RLRs) such as, RIG-I and MDA-5 are innate pattern recognition receptors that detect
foreign cytosolic RNA. RIG-I and MDA-5 recognize 50 -triphosphate of blunt-end RNA and double-stranded RNA
(dsRNA) that may be produced during virus replication. Upon activation, RLRs signal through the adaptor molecule
164 Pandemic Outbreaks in the 21st Century

FIGURE 10.4 Subversion of IFN induction

and response. (A) Interferon (IFN) production
signaling pathway and mechanisms of Ebola
virus evasion. VP35 antagonizes the signaling
pathway that leads to the expression of type I
and type II IFNs. RIG-I and MDA5 get acti-
vated after recognizing the cytoplasmic
double-stranded RNAs (dsRNAs) or RNAs
with 50 triphosphates. Activation of RIG-I is
also facilitated by PACT (PKR activator) pro-
tein. Activated RIG-I and MDA5 signal
through the adaptor molecule IPS-1 activates
the kinases, IKKε and TBK1 by phosphoryla-
tion, which in turn phosphorylate IRF3 and
IRF7. After dimerization, phosphorylated
IRF3 and IRF7 translocate to the nucleus and
promote the expression of type I and type II
IFNs. VP35 binds to dsRNAs and PACT, pre-
venting RIG-I and MDA5 activation. (B) The
IFN response signaling pathway and mecha-
nism of EBOV evasion. Type I and type II
IFNs bind to the extracellular domains of the
heterodimeric type I IFN receptor (IFNAR),
type II IFN receptor (IFNGR) which are com-
posed of two subunits. This activates receptor-
associated tyrosine kinases, Janus kinase 1
(JAK1) and TYK2. JAK1 and TYK2 phos-
phorylate signal transducer and activator of
transcription 1 (STAT1) and STAT2, which
leads to the formation of STAT1
STAT2 het-
erodimers. Similarly, IFNGR activates JAK1
and JAK1 leading to the formation of
STAT1
STAT1 homodimers. Tyrosine-
phosphorylated STAT (pSTAT) dimer is asso-
ciated with karyopherin-α (KPNA) and then
transported to the nucleus, where it binds to
IFN-sensitive response elements (ISREs). The
binding of pSTAT dimers to ISREs induces
the expression of IFN-stimulated genes
(ISGs), which include the genes encoding
antiviral proteins. EBOV VP24 competitively
binds to KPNA and prevents KPNA
STAT
interactions and translocation to the nucleus.
This ultimately inhibits the expression of
interferon signal genes (ISG) involved in the
production of antiviral proteins.

interferon β promoter stimulator protein-1 (IPS-1), which activates the kinases, Tank binding kinase 1 (TBK1), and
I-Kappa-B kinase epsilon (IKKε). The activated TBK1/IKKε phosphorylate interferon regulatory factors IRF3 and IRF7
[59]. After phosphorylation, IRF3/IRF7 dimerize and localize to the nucleus, and activate the transcription and
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 165

FIGURE 10.5 VP35 inhibits PACT-mediated IFN

production by the viral RNA. (A) RIG-I alone or in
presence of PACT gets activated after detecting the
viral RNA, including dsRNA, and triggers a signal
transduction cascade leading to the production of
IFN. (B) VP35 by binding to dsRNA and PACT pre-
vents the interaction with RIG-I and blocks the sig-
naling cascade.

production of IFN-α/β and γ proteins involved in the development of resistance against viral infection (Fig. 10.4A).
IFN-α/β and γ initiate signal in both autocrine and paracrine manner through interaction with its cognate receptor IFN
α/β receptor 1/2 (IFNAR1/2) and IFN γ (IFNGR1/2).
The early experiments have determined that VP35 disrupts the RIG-I pathway by preventing the phosphorylation of
IRF3 and IRF7 [60,61]. VP35 acts as a decoy substrate for IKKε/TBK1 kinases and interact with IKKε/TBK1 to pre-
vent the phosphorylation of IRF3 and IRF7 [62]. The net result of all these interactions leads to the inhibition of induc-
tion of IFN gene promoters (Fig. 10.4B).
In addition to these downstream events in the RIG-I pathway, VP35 directly interacts with ssRNA and dsRNA to
block the interaction between RNA fragments and RLRs such as RIG-I and MDA-5 [63] (Figs. 10.5 and 10.6).
Observations from RNA-bound and -free form structures of VP35 revealed that it can bind both the phosphate backbone
of dsRNA and end-capped RNA in VP35 dimers. Mutations of the basic patch centering on R312 abrogate the dsRNA
binding and structural analysis suggests that R312 mutations disrupt VP35 dimerization [64]. Interestingly, infection of
guinea pigs with recombinant ZEBOV possessing 2 point mutations in VP35 impairs its dsRNA binding activity, result-
ing in the loss of virulence and protected animals against subsequent WT-ZEBOV challenge [53].
The studies have also indicated that VP35 interacts potentially with PACT [65]. Subsequent work showed that VP35
binding to PACT prevents PACT binding to RIG-I and inhibits its activation [66]. Taken together with previous data,
these experiments point to the critical importance of VP35 antagonism of the RIG-I and MDA5 pathways during
EBOV infection.

10.5.3 Degradation of IRF3 and IRF7 by VP35-mediated SUMOylation

SUMOylation (small ubiquitin modification) is a process of degradation of foreign or altered self-proteins, an analo-
gous modification of ubiquitination (Fig. 10.7), which is performed by the enzymes E1, E2, and E3 [67]. During
protein SUMOylation, small ubiquitin-related modifiers (SUMOs) are synthesized as the precursor molecule needs
to be matured by sentrin-specific proteases (SENPs) which cleaves the peptide to expose C-terminal diglycine
motifs in the cytoplasm [68]. The matured SUMO is activated by an ATP-dependent heterodimer of SUMO activat-
ing enzymes 1 and 2 (SAE1, SAE2) [69]. E1 forms a thioester bond between its catalytic cysteine and the SUMO
C-terminal glycine. Activated SUMO is passed on to the catalytic cysteine of conjugating enzyme E2, which is
Ubiquitin Conjugating Enzyme 9 (Ubc9), forming a high-energy thioester bond [70]. The Ubc9 usually acts in
conjunction with ligase enzyme E3 which confers substrate specificity and catalyzes SUMO conjugation to the
166 Pandemic Outbreaks in the 21st Century

FIGURE 10.6 VP35 inhibits MDA5-mediated IFN production

by the viral RNA. (A) MDA5 gets activated after detecting the
viral RNA, including dsRNA, and triggers a signal transduction
cascade leading to the production of IFN. (B) By binding to
dsRNA, VP35 prevents its interaction with MDA5 and stops the
signaling cascade of IFN.

FIGURE 10.7 The mechanism of the SUMO cycle

for the degradation of IRF3 and IRF7. (1) The SUMO
precursor is processed by SUMO-specific protease
SENPs to expose diglycine for maturation. (2) SUMO
is activated by the enzyme E1 (SAE1 1 SAE2) in an
ATP-dependent manner. (3) SUMO is passed on to the
conjugating enzyme E2 (Ubc9). (4) VP35 binds to
ligase enzyme E3 (PIAS1) to select and ligate target
protein, IRF3/7 to SUMO (5) Ubc9, PIAS1, and VP35
are deconjugated from the SUMO-IRF3/7 complex.
(6) Target protein IRF3/7 desumoylated from SUMO
complex by the action of SENPs, where SUMO is
recycled and target protein IRF3/7 is degraded by the
proteasomal activity.

substrate [71]. Finally, the SUMO conjugation process forms an isopeptide bond between the SUMO C-terminus
and ε-amino group of a lysine residue in a specific motif within the target protein [72]. The E3 enzyme is a protein
inhibitor of activated STAT 1 (PIAS1), which enhances SUMO conjugation to target proteins [73]. Removal of
SUMO from the target protein is mediated by SENPs [74]. The separated target protein is subjected to degradation
by the common proteasomal degradation pathway.
During EBOV infection, the viral protein VP35 hijacks the host SUMOylation machinery by interacting with cellu-
lar proteins such as Ubc9 and PIAS1 to degrade IRF3 and IRF7 by the proteasomal degradation pathway. This process
reduces the concentration of IRF3/7 in the cytosol, thereby disconnecting the signaling pathway involved in the produc-
tion of IFNs [75]. Thus VP35-induced SUMOylation of IRF3 and IRF7 leads to a further reduction in the interferon α/β
and γ gene transcription.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 167

10.5.4 VP24 inhibits KPNA-mediated IFN response signaling

EBOV VP24 can block the IFN response signaling pathways by preventing the interaction between phosphorylated
STAT dimers and nuclear transporter protein karyopherin-α (KPNA) (Fig. 10.4B). Secreted interferon binds to type I
and II interferon receptors (IFNAR1/2 and IFNGR1/2), in an autocrine or paracrine manner. Activation of IFNAR1/2
subsequently triggers the TYK2-JAK1 STAT and JAK1-JAK2 STAT signaling cascade, which involves the activation
and autophosphorylation of TYK2-JAK1 and JAK1/2, which in turn phosphorylate STAT1 and STAT2. Upon phos-
phorylation, STAT1/2 dimerizes and translocates to the nucleus with the help of KPNA, where they activate the tran-
scription of the interferon-stimulated genes [76] (Fig. 10.8A). The successful activation and transduction of these
signaling events lead to upregulation of host antiviral genes, thus establishing an antiviral state in the cell. Given the
importance of these pathways for inducing antiviral gene expression in response to interferon, the viruses generally tar-
get this signaling mechanism. For example, the Dengue virus blocks STAT-1 phosphorylation and acts to degrade
STAT-2 via proteasomal degradation pathways [77]. Early experiments have shown that the virus not only blocks the
production of interferon but also inhibits cellular responses that normally result from both interferon α/β (type I) and
interferon γ (type II) signaling. This signaling block is associated with the expression of Ebola VP24 which was later
shown to prevent the nuclear accumulation of phosphorylated STAT1-STAT1/2 dimers (Fig. 10.8B) [78], which partici-
pates in both type I (STAT1-STAT2) and type II (STAT1/STAT1) signal propagation cascades [79,80].
VP35 suppresses IFN production, while VP24 blocks nuclear translocation of phospho-STAT1-STAT1/2, thereby
preventing IFN mediated signaling. Thus Ebola not only diminishes the interferon alarm but also inhibits the alarm
response after it has been heard. This coordinated approach between VP35 and VP24 leads to a highly effective antago-
nism of the innate immune response mediated by interferons.

10.6 Adaptive immune response during EBOV infection

The adaptive immune response is primarily responsible for eliminating the virus from infected hosts as well as prevent-
ing pathogen replication and spread. Therefore enhancing its potency constitutes the primary goal of vaccines and

FIGURE 10.8 Inhibition of KPNA-

mediated IFN response signaling by VP24:
(A) Phosphorylated STAT1-STAT1 or
STAT1-STAT2 dimers enter the nucleus
with the help of karyopherin-α (KPNA) pro-
tein. In the nucleus a STAT1-STAT1/2
complex activates IFN-stimulated response
element (ISRE)-containing promoters. This
leads to the upregulation of IFN stimulated
genes (ISGs). (B) EBOV VP24 competitively
binds to KPNA and blocks the interaction of
phosphorylated STAT1-STAT1 or STAT1-
STAT2 dimers with KPNA that prevents the
translocation of STAT1-STAT1/2 to the
nucleus leading to suppression of ISGs.
168 Pandemic Outbreaks in the 21st Century

treatment options for EVD. Adaptive immune dysregulation involves both humoral and cell-mediated immune
responses (Fig. 10.9). As expected, the aberrant DC differentiation results in ineffective DC-/T-cell synapses that are
unable to induce a correct adaptive immune response. Indeed, EBOV-infected DCs fail to stimulate T-cell proliferation,
suggesting that EBOV suppression of DC function prevents initiation of adaptive immune responses that facilitate

FIGURE 10.9 Complexity of Ebola virus pathogenesis. In the beginning EBOV preferentially infects monocytes, macrophages, and DCs. Infected
macrophages and DCs migrate to regional lymph nodes while producing progeny virions. Infection of DCs impairs their maturation and suppresses
type I and II IFN responses, thereby preventing T cell activation. Infection of monocytes and macrophages leads to the robust expression of inflamma-
tory mediators. Secreted chemokines recruit more monocytes, which act as new targets for viral infection and proliferation. Inflammatory mediators,
reactive oxygen species, and nitric oxide induce apoptosis leading to lymphocyte death. The lack of lymphocytes, such as CD41 T cells inhibits the
ability of the virus to induce the Ab response. Production of sGP seizes any GP-specific Abs produced during infection. Through suppression of intrin-
sic, innate, and adaptive immune responses, systemic distribution of progeny virions and infection of secondary target cells occur in almost all organs.
Eventually, the inflammatory cytokines are responsible for vascular leakage. EBOV systemically disseminates to the liver, spleen, brain, skeletal mus-
cles, kidneys, adrenal glands, gastrointestinal tract and endothelial cells, which contributes to symptoms associated with hemorrhagic fever. EBOV,
Ebola virus.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 169

uncontrolled systemic virus replication [81
83]. On the other hand, the downstream effects of antigen-presenting cell
dysfunction are profound with a marked lack of adaptive immunity noted in fatal cases of EBOV infection. It is well
known that NK cells are crucial in mediating direct protective cytotoxicity and to drive adaptive immune response by
helping DC maturation [84,85]. Thus massive NK cell loss in the peripheral blood may have an impact not only on the
failure of infected cell clearance but also be partially responsible for the imbalanced maturation signals for DC.

10.6.1 Dysregulation of the adaptive immune response

An effective immune response needs the coordinated activities of both humoral and cellular arms. In recovered patients,
robust immune responses, with early and increasing levels of IgM and IgG, were developed during the acute phase of
EBOV infection [86]. This is followed by the clearance of circulating viral replication markers, although fatal infections
are characterized by impaired humoral responses, with the absence of virus-specific IgG and barely detectable IgM
[87]. Interestingly, the humoral immune response seems to be long-lasting, as the survivors of EBOV infection have
been recently shown to present serum-neutralizing activity and GP-specific IgG, after 12 years of infection [88].
Interestingly, several mechanisms have been developed by EBOV to escape humoral immune response and the role of
heavy glycosylation of mucin-like domain of viral GP in shielding the cell-free virus from access to potential virus-
neutralizing antibodies has been described [89]. Furthermore, EBOV can produce a secreted form of GP that can modu-
late or misdirect host immune response [90,91]. In particular, soluble GP promotes immune evasion by serving as an
antibody decoy for GP or by presenting alternative nonneutralizing antibody epitopes [92]. During Ebola infection, the
antibody titer represents the best indication of protection [93,94], however, several evidence suggest a key role of T
cells in mediating a protective immune response [94
96]. The transfer of both serum and splenocytes from Ebola
VLP-vaccinated mice, but not serum or splenocytes alone, conferred protection against lethal-EBOV infection, suggest-
ing that both B and T lymphocytes are required for VLP-mediated protection against EBOV infection [94].
In human fatal cases of EBOV infection, a massive loss of CD41, CD81 T lymphocyte, and NK cells was observed
compared to healthy individuals and survivors. The inflammatory mediators produced after EBOV infection, such as
TNF-α, nitric oxide, and reactive oxygen species, can induce apoptosis [97
99]. In humans the percentage of periph-
eral blood CD41 and CD81 T cells expressing the CD95 apoptotic marker is about 10 times higher than those observed
in healthy individuals [100]. Increased levels of soluble Fas and 41/7 nuclear matrix protein, which is cleaved and solu-
bilized during apoptosis, have been detected in the plasma of patients during the last 5 days of life after infection with
EBOV [94]. Furthermore, although patients who survived EBOV infection showed upregulation of Bcl-2 mRNA in
PBMCs, those who succumbed showed a significant decrease in Bcl-2 mRNA expression during the terminal stage of
infection [94].
The lymphopenia observed during EBOV infection in part explains the lack of EBOV-specific T and B cell
responses. The lack of T cell response is evident by the absence of T cell-derived cytokines (IL-2, IL-3, IL-4, IL-5, IL-
9, and IL-13) in the plasma of fatally infected patients [81,100
102]. Because CD41 T cells are required for B cell iso-
type class switching, the loss of CD41 T cells may explain the lack of Ebola-specific IgM and IgG observed in fatally
infected patients [94]. The T-cell apoptosis is a result of dysregulated DC/T synapse during EBOV infection. It is well
known that T-cell activation needs the coordination of three different signals: (1) TCR recognition of major histocom-
patibility complex (MHC)-peptide; (2) binding of several co-stimulatory molecules between DC and T cells; and (3) a
balanced ensemble of soluble factors in the microenvironment. A well-orchestrated DC/T-cell interaction of all three
signals is necessary to effectively activate CD41 T cells that, in turn, exploit all their help activities, such as the clonal
expansion of specific T-cells, driving CD81 T-cell cytotoxicity, and sustaining antibody-producing B cells. During
EBOV infection, we can speculate that T/DC synapsis is ineffective as it is characterized by TCR/MHC-peptide recog-
nition in a high inflammatory microenvironment but in the absence of co-stimulatory accessory molecules on the DC
surface. This inappropriate interaction induces T-cell apoptosis, thus blocking all T-cell helper functions on CD81-
mediated cytotoxicity and the production of antibodies by B cells. Thus the final result is a marked collapse of the adap-
tive immune response. Notably, in a mice model, the residual T-cell function is observed in the remaining cells despite
their massive loss in numbers [103]. The number of functional T cells that are generated during the late phase of infec-
tion is likely too less to control high viral titers [104].
Studies in the mouse model indicate that although immediate control of EBOV infection may be achieved by CD81
T cells, the support of B and CD41 T cells are important for long-term control and clearance of virus replication [95].
During Ebola infection, lymphoid depletion and necrosis have also been reported in the spleen, thymus, and lymph
nodes of dying patients and experimentally infected NHP. Subversion of the adaptive immune response, coupled with
inactivation of the innate immune system, allows EBOV to disseminate systemically.
170 Pandemic Outbreaks in the 21st Century

10.7 Vascular permeability and coagulation defects

The severity of EBOV infection is a result of robust systemic virus replication and excessive inflammatory responses,
which leads to the clinical manifestations of EVD, including high fever, vascular leakage, and coagulopathy [105].
EBOV can infect many cell types, including macrophages, monocytes, dendritic cells, Kupffer cells in the liver, fibro-
blasts, hepatocytes, cells of adrenal gland tissue, endothelial cells, and epithelial cells but preferentially infects macro-
phages and dendritic cells (Fig. 10.9) [106,107]. EBOV infection begins with the deposition of viral particles on
mucous membranes and skin [14]. After the uptake of viral particles by dendritic cells and macrophages, EBOV replica-
tion potently shuts down early innate immune responses by blocking interferon production and signaling responses
[108,109]. The dissemination occurs through the migration of viral-infected dendritic cells and macrophages to lym-
phoid tissues and the release of the virus into the circulation, leading to infection of fixed macrophages in the liver,
spleen, and other tissues. EBOV induces massive cytokines/chemokines production by infected dendritic cells or mono-
cytes/macrophages [110,111]. Several inflammatory mediators are induced within the first hour of EBOV exposure,
that is, before virus gene expression, suggesting a direct role of the GP present on virion surface in inducing an initial
inflammatory response [110]. The shed GP (resulting from the cleavage of surface GP by the cellular metalloprotease
TACE) can bind and activate the noninfected DCs and macrophages mainly through TLR4 engagement, inducing the
secretion of pro-inflammatory and antiinflammatory cytokines. This activation mechanism of noninfected immune cells
by shed GP could have an important role in the establishment of systemic inflammation during infection, provoking the
excessive cytokine storm that appears to be detrimental to survival after infection. Survivors of EBOV infection showed
an early and short-lived rise in serum cytokines/chemokines, indicative of innate immune response activation, whereas
fatal infection is associated with a dysregulated inflammatory immune response [87].
The pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein 1 (MCP1),
macrophage inflammatory protein 1α (MIP1α), MIP1β, and tumor necrosis factor (TNF), in addition to reactive oxygen
species and nitric oxide are produced by EBOV-infected cells [87,100,112
115]. MIP1α and MCP1 can recruit addi-
tional macrophages and DCs to areas of infection, making more target cells available for viral exploitation and further
amplification of already dysregulated host response [87]. EBOV infection inhibits the maturation of DCs and downregu-
lates the co-stimulatory molecules, CD40, CD80, CD86, and MHC class II molecules leading to poor antigen presenta-
tion to T cells [81
83,116]. Due to inappropriate interaction, infected macrophages induce T-cell apoptosis, thus
blocking all T-cell helper functions on CD81-mediated cytotoxicity and the production of antibodies by B cells. The
final result of all these events is a marked collapse of the adaptive immune response (Fig. 10.9, dotted box). As the dis-
ease progresses, abnormal production of TNF, reactive oxygen species, and nitric oxide have been shown to induce sev-
eral pathological disorders including apoptosis of bystander lymphocytes, tissue damage, and loss of vascular integrity,
which might contribute to virus-induced shock [112]. Subsequent extensive viral replication leads to increased levels of
additional pro-inflammatory cytokines, which further triggers the coagulation cascade.
Hemorrhage and intravascular coagulation are the features of severe EBOV infection which may also lead to the
suppression of immune responses. Hepatocellular necrosis may result in the decreased synthesis of coagulation proteins,
whereas adrenocortical infection and necrosis may negatively affect blood pressure homeostasis, potentially leading to
hemorrhage [106]. The increased vascular permeability and leakage that typically leads to hemorrhagic fever could be
attributed to the release of high levels of TNF from infected macrophages, which further increases the endothelial per-
meability [115]. Similarly, the release of nitric oxide, an important effector molecule in the homeostasis of the cardio-
vascular system, by infected macrophages may result in the loss of vascular smooth muscle tone and hypotension
[112,117]. The major pathogenic events occurring in the endothelial cells seem to be determined by the GP of EBOV.
The GP has been suggested to have a key role in the induction of cytotoxicity and injury in endothelial cells, which is
characterized by cell rounding and detachment associated with downregulating cell-adhesion molecules typical of anoi-
kis, which is a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the sur-
rounding extracellular matrix [118]. It has been shown that VLPs consisting of the EBOV matrix protein VP40 and GP
can activate endothelial cells to induce a decrease in their barrier function. Interestingly, this VLP-induced action is fur-
ther enhanced by TNF-α, which is known to induce a long-lasting decrease in endothelial-cell barrier function and is
hypothesized to have multiple roles in EBOV pathogenesis [119].
The liver is another important target for EBOV, probably having a critical role in the disease pathogenesis and hepa-
tocellular necrosis that have been reported both in patients and in experimental animal models [120
124]. Indeed, the
hemorrhagic events typical of the classic Ebola infection could be related to impaired synthesis of blood coagulation
protein/enzymes owing to the severe hepatocellular necrosis [125]. Studies in NHP have provided direct evidence that
coagulation abnormalities are initiated during the early stage of EBOV infection. A dramatic decrease in plasma levels
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 171

of anticoagulant protein C occurs during early postinfection, approximately after 2 days. This is followed by an increase
in both the levels of tissue plasminogen activator, which is involved in dissolving blood clots and fibrin-degradation
products (D-dimers), 5 days’ postinfection [106].
Besides, EBOV infection of macrophages leads to the upregulation of surface tissue factor (TF) as well as the
release of membrane microparticles containing TF, resulting in the over-activation of the extrinsic pathway of coagula-
tion and the development of disseminated intravascular coagulation [106]. Expression of TF is further upregulated by
pro-inflammatory cytokines, notably IL-6, which are abundant during acute EBOV infection, exacerbating the intravas-
cular coagulation phenotype [126]. The consumption of clotting factors (from disseminated microthrombi), endothelial
dysfunction, and inhibition of platelet function contribute to coagulopathy [14,108,109,120,127,128]. Notably, toward
the terminal stage of infection, after the onset of hemorrhagic abnormalities, EBOV replicates in endothelial cells [107].
Microvascular anomalies, mucosal hemorrhages, hypovolemia, and further fluid losses through vomiting and diarrhea
ultimately lead to tissue hypoperfusion and multiorgan failure eventually causing death (Fig. 10.9) [14,108,127
129].

10.7.1 EBOLA-postinfection manifestation

The 2014
16 ZEBOV outbreak in West Africa resulted in an unprecedented number of survivors (17,000) and
highlighted the complexity of EVD sequelae in clinically recovered patients (Fig. 10.10). In a retrospective study 29
months after the 2007 Bundibugyo outbreak in Uganda, the health status, functional limitations, demographics, blood
chemistry, hematology, and filovirus Ab titers from 49 survivors and 157 seronegative contacts have been evaluated
[130]. The study indicated that, although no differences in blood analysis were observed, the survivors were at a signifi-
cantly greater risk of ocular problems (retro-orbital pain and blurred vision), loss of hearing, difficulty in swallowing
and sleeping, arthralgia, abdominal and back pain, fatigue, impotence, severe headache, memory problem, and confu-
sion [130]. In a study the clinical and laboratory records of surviving patients treated in Port Loko, Sierra Leone, were
assessed during which a higher incidence of arthralgia, ocular symptoms (including uveitis), and auditory problems
were observed. Moreover, a higher ZEBOV viral load leading to the clinical presentation was associated with a higher
incidence of uveitis and other ocular symptoms [131]. Another study of surviving patients showed an increased inci-
dence of anorexia, arthralgia, myalgia, and chest/back pain [132].
Further investigation of recovering survivors revealed that ZEBOV persists in the semen, ocular fluid, cerebrospinal fluid
(CSF), placenta, and amniotic fluid. The genetic sequencing of the ZEBOV strain confirmed that a female patient acquired the

FIGURE 10.10 Postsymptomatic complications of EVD. EVD, Ebola virus disease.

172 Pandemic Outbreaks in the 21st Century

virus via sexual contact from a survivor whose semen tested positive for ZEBOV by quantitative real-time reverse
transcriptase-polymerase chain reaction (qRT-PCR) after 199 days of his recovery [133]. The data further suggest that infectious
virus can persist in the semen for several months after viremia ceases. ZEBOV was also detected in the CSF of a nurse who
developed meningitis after 9 months of recovery from EVD [134]. The ZEBOV also persists in amniotic fluid and placenta
after clearance from the blood in two pregnant women, resulting in the delivery of stillborn fetuses in both cases [135].
Collectively, these observations indicate that ZEBOV can persist in organs that were traditionally considered as immune-
privileged sites (Fig. 10.10). The ability of immune cells to access these sites (anterior chamber of the eye, central nervous sys-
tem, testes, and pregnant uterus) is limited to reduce the risk of irreparable damage to these critical organ systems [136]. The
persistence of ZEBOV in these sites after complete recovery raises many questions regarding the mechanisms and the kinetics
by which this virus gains access, and its ability to persist in these sites. Male survivors and their sexual partners should receive
individual advice, including counseling on safer sex until their seminal fluid is free of viral RNA.

10.8 Diagnosis
Multiple techniques have been established for the laboratory diagnosis of EBOV, including assays for the detection of the
viral genome, viral antigen, and host immune responses [137,138]. The qRT-PCR has emerged as the “Gold-standard” in
the diagnosis of EBOVs. In this method preferably two distinct genome locations are targeted to minimize false-negative
results due to evolving genome mutations. During the acute phase of disease and convalescence, viral RNA can be detected
by Reverse transcriptase-polymerase chain reaction (RT-PCR) in blood and other bodily fluids, such as saliva, tears, sweat,
breast milk, urine, CSF, ocular fluid, amniotic fluid, vaginal fluid, and seminal fluid. Postmortem diagnosis of EVD (people
who did not attend the hospital and died in the community) is best done by RT-PCR on an oral swab [139,140]. The qRT-
PCR assay is expected to be positive in symptomatic patients, with increasing viremia in fatal cases. Even after the negative
results of the assay during the early course of the disease, follow-up testing is warranted in patients who have continuing
symptoms. Accordingly, negative results on at least two sequential tests are required for the discharge from the treatment
center. Besides, simple bedside tests to detect viral antigen have also become available [137,138]. Serology is the method
of choice to diagnose asymptomatic EBOV infections, which are characterized by extremely low viremia due to the devel-
opment of IgG and IgM after about 3 weeks of infection [141,142].

10.9 Vaccines
The development of the EBOV vaccine was initiated during the 1970s using inactivated viral preparations which were
followed during the 1980s and 1990s by subunit and DNA vaccine approaches [143,144]. The high lethality of the
2014 Ebola epidemic prompted an acceleration in the development of different forms of vaccines that include both non-
replicative and replicative vector-based vaccines. The former group uses nonreplicative vectors that code for GP or
another viral antigen with deletions in genes crucial for viral replication. These vaccines need high doses of administra-
tion to develop a significant immune response. The past two decades have seen intensified use of vectored vaccines and
combined approaches. Except EBOV DNA and adenovirus-based vaccines, none of these vaccine candidates have made
it past the preclinical stage at the time of the West African Ebola epidemic [143,144]. Among the various vaccine strat-
egies, the only two vaccine candidates that have advanced further to clinical trials are rAd and rVSV vectors expressing
GP (Table 10.1). Recombinant adenovirus serotype 5 expressing the ZEBOV GP gene (rAd5-EBOV-GP) is a
replication-deficient vector and the immunization of cynomolgus macaques with a single dose of rAd5-EBOV-GP parti-
cles resulted in the production of ZEBOVGP-specific Abs and CD81 T cells that provided 100% protection against
ZEBOV challenge 21 days after vaccination [145]. One of the efficient live-attenuated vectored vaccine based on
recombinant vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein [rVSV-ZEBOV-GP (ERVEBO,
Merck)] has been successfully tested for its efficacy in a randomized trial in Guinea during the West African epidemic
[146,147]. The vaccine, which was approved by the European Medicines Agency and the United States Food and Drug
Administration, has been widely administered during the EBOV outbreak in the DRC. This vaccine has promising pre-
liminary results of 97.5% efficacy with an onset of illness more than 10 days after vaccination [7,8].
Because rVSV-ZEBOV-GP is a live-attenuated, replicating virus, several studies have investigated its safety in
detail. No adverse events have been observed in 80 NHPs immunized with rVSV-MARV-GP or rVSV-ZEBOV-GP
[148,149]. Moreover, no evidence of disease was observed in immunocompromised animal models (NOD-SCID mice
and SHIV rhesus macaques), further confirming the safety of this vaccine [148,150]. Vaccination of cynomolgus maca-
ques with a mixture containing rVSV-MARV-GP, rVSVZEBOV-GP, and rVSV-SEBOV-GP resulted in protection after
infection with MARV, ZEBOV, SEBOV, and CIEBOV, indicating that a multivalent rVSV vaccine is highly
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 173

TABLE 10.1 EVD candidate vaccines in phase I
III clinical trials.

Vaccine Antigen’s Immune responses Clinical trial Associated

origin phase complications
Ad5-EBOV-GP EBOV-1995 High EBOV specific CD8 1 response 1 Headache
rAD5-EBOV-GP EBOV-2014 CD8 1 and EBOV-GP specific Abs 1 Injection site pain
rVSV-EBOV-GP EBOV Kikwit- Strong innate and adaptive responses 2 and 3 Injection site pain
1995
Ad26-ZEBOV 1 MVA- EBOV-1967 90% EBOV-GP specific IgG Abs and 2, 3, and 4 Injection site pain
BN Filo 55% T cells
ChAd3-EBO EBOV-1967 Poor EBOV specific T & B cell 1 Fatigue/malaise
responses

Note: EVD, Ebola virus disease.

efficacious [151]. The results from a cluster-randomized ring phase III trial, in which 7651 people were vaccinated with
rVSV-ZEBOV-GP, showed the vaccine to be 100% efficacious [152]. Importantly, as described for macaques, this vac-
cine has the potential to be a successful postexposure treatment option in humans. In March 2009 a single dose of
rVSV-ZEBOV-GP was administered to a virologist 48 h after EBOV needle-stick injury, the subject developed an acute
fever and rVSV viremia but not EVD [153]. Similarly, during the 2014 outbreak, a physician who sustained a needle-
stick injury while working in an Ebola treatment unit in Sierra Leone was given rVSV-ZEBOV-GP as an emergency
postexposure vaccination, 43 h later. The subject developed a self-limited febrile syndrome, in addition to ZEBOV-GP-
specific adaptive immune responses following vaccination, and recovered [154]. The efficacy of rVSV-ZEBOV was
evaluated in a phase III trial in Guinea using a protocol of ring vaccination, the principle of which relies on the vaccina-
tion of both confirmed EVD patient’s direct contacts and of those contact’s direct contacts [146].
The chimpanzee adenovirus serotype 3
vectored vaccine (ChAd3-EBO-Z), administered as a single shot, was found
to be safe and immunogenic in phase I trials [155]. The booster dose with a modified vaccinia Ankara virus (MVA)-
based vaccine enhanced long-term immunity. The ChAd3-EBO-Z and rVSV-ZEBOV vaccines were assessed in parallel
in a phase III trial to assess safety and immunogenicity. The data obtained supported the safety and immunogenicity
(up to 12 months) of both the vaccines [147].
A human adenovirus-vectored vaccine (Ad26.ZEBOV) boosted with the MVA vaccine was also found to be safe
and immunogenic in phase I trials [156]. Many other vaccines are under development and likely to undergo a further
clinical evaluation. It is to be expected that several licensed vaccines will become available over the next decade, with
many advantages and limitations concerning their safety profiles, the duration of immunity, monovalent versus polyva-
lent, and logistical issues such as storage conditions, cost, and availability. Therefore a wide range of options will be
available to be used in different contexts including ring vaccination, vaccination of healthcare professionals during an
outbreak, and vaccination in high-risk areas before an outbreak [157,158].
In 2000 the vaccination strategy with adenoviral vectors demonstrated cellular and humoral immunity in
Cynomolgus macaques [159,160]. The phase I trial of the recombinant adenovirus serotype 5 vaccine (rAd5) coding for
EBOV GP demonstrated the safety and immunogenicity [161]. After the 2014 outbreak, researchers developed a vac-
cine based on Ad5 vectors (Ad5-EBOV) with the new epidemic strain isolated from the Guinea outbreak. A phase I trial
in China demonstrated its safety, tolerability, and immunogenicity [162]. In 2015 a phase II clinical trial of experimen-
tal Ad5-EBOV was initiated in Sierra Leone. Preliminary results showed that rVSV-EBOV is the first vaccine to confer
a high level of protection. The United States Food and Drug Administration (FDA) has approved the Ebola vaccine
rVSV-ZEBOV on 19 December 2019. Currently, it is the only vaccine approved by the FDA for the prevention of
EBOV disease. The description of some important vaccine candidates is listed in the Table 10.1.

10.10 Therapeutics
Strategies for developing experimental, postexposure treatments against EBOV focuses on: (1) inhibiting viral replication or
translation by nucleotide analogs (remdesivir), and (2) limiting viremia and virus spread by mAb cocktails. Several of these
174 Pandemic Outbreaks in the 21st Century

TABLE 10.2 EVD therapeutics are in clinical trials.

Agent Agent design and biology Clinical Result Notes

name trial phase
ZMapp Cocktail of three monoclonal anti-EBOV- 2/3 84/169 patients (49.7%) It served as a control
GP1,2 chimeric antibodies (derived from died overallTime to first group comparator for
Nicotiana spp. Tobacco plants) negative result: 27 days other developing
therapeutic agents
mAb114 Monoclonal anti-EBOV-GP1,2 IgG1 1 61/174 patients (35.1%) Superior efficacy
antibody (derived from human survivors died overall. Time to
from 1995 Kikwit outbreak) first negative result: 16
days
REGN-EB3 Cocktail of three fully human 1 52/155 patients (33.5%) Superior efficacy
monoclonal anti-EBOV-GP1,2 IgG1 died overall. Time to
antibodies (derived from VelocImmune first negative result: 15
humanized mice) days
Remdesivir Analog, inhibits viral polymerase
93/175 patients (53.1%) Equivalent efficacy
(GS-5734) died overall. Time to (requires monitoring of
first negative result: 28 AST/ALT)
days

Note: EVD, Ebola virus disease.

postexposure therapeutic candidates are currently in clinical trials. Most investigational therapies for EVD are aimed at rapidly
reducing viral replication to limit the inflammatory storm triggered by viral expansion by allowing effective innate and adaptive
immune responses to clear the infection. Some of these investigational treatments were tested during the 2013
16 Ebola out-
breaks in West Africa, but these clinical trials did not show unequivocal efficacy of any treatment. Nevertheless, promising
experimental interventions were identified and the rapid implementation of these trials emerged as a key component of the
response to the global outbreak [163].
In September 2014 WHO inventoried a list of potential drug candidates developed or repurposed to EVD with
shown antiviral efficacy in vitro or animal models (Table 10.2). These therapeutics are nucleoside and nucleotide ana-
logs [164,165] nucleic acid-based drugs and immunotherapeutics [166
169].
Therapeutics with encouraging preliminary efficacy or safety profiles require further investigation to determine effi-
cacy in humans (e.g., remdesivir) and treatments targeting distinct pathways in the viral lifecycle [164,166,170].
Besides, effective drugs that pass the blood
brain barrier are needed for the management of clinical recurrence of EVD
and to penetrate immunologically and anatomically preserved sites and reservoirs in survivors beyond the acute phase
of the disease. To date, no medical therapeutics have been clinically proven to be successful (effective in decreasing
mortality) in the specific treatment of EBOV infections. The promising data on investigational therapeutics exist for
small molecules with direct antiviral activity [164,170] and candidate monoclonal antibodies (mAbs), some of which
act by preventing the binding of the GP of the EBOV (EBOV-GP) to its cellular receptor [166,171,172].
Notably, the nucleoside analog remdesivir administered by the intravenous route was found to be highly effective
against EVD in NHP, with a good safety profile in phase I studies [170]. It is being evaluated to block persistent EBOV
shedding in the semen of male survivors of EVD. ZMapp, a combination of three humanized recombinant mAbs has
shown to have anti-EBOV activity in vitro and NHP. ZMapp efficacy against EVD assessed in a randomized, controlled
trial could not be determined because the trial did not reach the target sample size as the incidence of Ebola infection
decreased in the 2013
16 outbreak in West Africa [166]. Since then, results from a co-formulated cocktail of three
human mAbs (REGN3470
3471
3479) targeting three nonoverlapping epitopes of EBOV look promising, justifying
further clinical development as single-dose therapy for EVD [171]. Other approaches offer protection with a single
mAb, potentially simplifying manufacturing processes and reducing costs (mAb114), [172] and could also provide
cross-species protection of relevance against other EBOVs [173]. During the outbreak in the North Kivu province of
the DRC in August 2018, mAb114, REGN3470
3471
3479, ZMapp, and remdesivir are the investigational treatments
used under an expanded access protocol. There are currently two drugs approved by the FDA to treat EVD caused by
the EBOV in adults and children. Inmazeb external icon is a combination of three mAbs approved during October
2020. Ebangaexternal, a single mAb was approved in December 2020.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 175

10.11 Prevention and control of EVD

Disease control is focused on preventing the transmission and spread of the virus through a multidisciplinary approach
that includes factors such as early case identification, rapid isolation of suspected infected patients, adequate clinical
management, safe burial practices, and health education [174]. Cases of EBOV disease should be identified early using
robust surveillance systems in high-risk areas. Each area must have an access to specialized laboratories for rapid detec-
tion of the virus and specialized centers for the isolation of infected cases. Ebola Treatment Centers (etc.) were estab-
lished in Guinea, Liberia, and Sierra Leone during the 2013
16 outbreaks. The ETCs were divided into two
independent areas: (1) High-risk areas that shelter infected patients, contaminated materials, and dead bodies. (2) Low-
risk areas with offices, laboratory facilities, storage areas, and nursing stations. These centers offer high-quality care for
affected communities through visits to family members to inform them about medical activities and preventive mea-
sures [174]. In the DRC, individual treatment units were developed that facilitated adaptability and access for medical
personnel in cases where the patient did not go to the ETCs. These isolation units are portable, individual rooms with a
BSL4 facility for containment and are located near the patient’s home. The walls are transparent to allow direct commu-
nication with the family and facilitate patient monitoring by medical staff [175]. The contact and monitoring of families
and neighbors of infected patients is an important tool for the control of outbreaks. In Liberia during 2014
15 contact
tracking allowed the detection of 3.6% of new cases [176]. Besides, traditional burial ceremonies with safety precau-
tions contributed to the effective control of the disease [177]. Health campaigns and educational activities help the com-
munity to recognize EVD cases and act appropriately. In addition, these activities overcome cultural beliefs and
practices that challenge disease control. It is highly recommended that traditional healers, religious leaders, or people
exposed to the virus participate in these activities. Humans acquire the infection through direct or indirect contact with
body fluids of infected reservoir species, infected or deceased people, or surfaces and materials contaminated with these
fluids. To prevent transmission, the Pan American Health Organization/WHO develop programs that include protocols
for environmental cleaning and disinfection, hand hygiene, use of disinfectants for surface and equipment decontamina-
tion, use of personal protective elements for health professionals and caregivers, and safe handling of potentially con-
taminated materials or biomedical wastes [178]. Other recommendations for the prevention and integral control of
infection include safe processing of laboratory samples and setting up a committee to supervise activities in hospitals
and communities. Effective communication strategies to combat Ebola at local and global levels should include tradi-
tional and innovative tools such as newspapers, radio, television, and social networks. Traditional healers and religious
leaders should have a central role in vaccination programs, not just for their safety but also to set an example within
their communities, and should receive health education to enhance their potential to recognize cases of EVD and react
appropriately [179].

10.12 Summary
G EVD caused by Filoviridae family virus which is a negative-strand RNA virus discovered in 1976. The infection
causes severe hemorrhagic fever with a mortality rate of up to 90%.
G The genome has seven sequentially arranged genes encoding 10 different proteins wherein, GP gene encodes four
proteins and the other genes encode only one protein each.
G There were two major outbreaks in West Africa between 2014
16 and 2018
19 affected 28,000 and 3000 people,
respectively.
G EVD is mainly transmitted from person to person via direct skin or mucous membrane contact the blood or bodily
fluids of acutely ill patients. Infectious virus is also present in urine, vaginal fluid, semen, saliva, and breast milk. It
is recommended that the men and women, who have recovered from Ebola abstain from sex for as long as the
EBOV is detectable in the semen or vaginal fluid.
G During the initiation of infection, the EBOV preferably infects macrophages, dendritic cells followed by infecting
almost all the body cells. EBOV induces massive cytokines/chemokines production by infected dendritic cells or
monocytes/macrophages.
G The symptoms of EVD may appear from 2 to 21 days after exposure to the virus. Symptoms usually include fever,
headache, joint pain, muscle aches, weakness, diarrhea, vomiting, stomach pain, lack of appetite, internal and exter-
nal bleeding.
G EBOV evades innate immunity using the proteins VP35 and VP24 particularly blocking type I and II IFN production
and response signaling pathways which facilitate rapid multiplication of the virus.
176 Pandemic Outbreaks in the 21st Century

G EBOV dysregulates the adaptive immunity by producing a truncated form of GP and killing lymphocytes by cyto-
kine storm leading to lymphopenia.
G In human fatal cases of EBOV infection, a massive loss of CD41, CD81 T lymphocyte, and NK cells was observed
compared to healthy individuals and survivors. The inflammatory mediators produced after EBOV infection, such as
TNF-α, nitric oxide, and reactive oxygen species, induce apoptosis.
G The severity of EBOV infection results from the robust systemic virus replication and excessive inflammatory
responses, which leads to the clinical manifestations of EVD, including high fever, vascular leakage, and
coagulopathy.
G As the disease progresses, abnormal production of TNF, reactive oxygen species, and nitric oxide has been shown to
induce several pathological complications including apoptosis of bystander lymphocytes, tissue damage, and loss of
vascular integrity, which might contribute to virus-induced shock. Subsequent extensive viral replication leads to
increased levels of additional pro-inflammatory cytokines, which further triggers the coagulation cascade.
G Microvascular anomalies, mucosal hemorrhages, hypovolemia, and further fluid losses through vomiting and diar-
rhea ultimately lead to tissue hypoperfusion and multiorgan failure eventually causing death.
G RT-PCR is one of the most commonly used diagnostic methods because of its ability to detect even low levels of
the virus.
G The United States Food and Drug Administration (FDA) has approved the Ebola vaccine rVSV-ZEBOV on 19
December 2019. This is the first FDA-approved vaccine for Ebola.
G There are currently two drugs approved by the FDA to treat EVD caused by the EBOV in adults and children.
Inmazeb external icon is a combination of three mAbs approved during October 2020. Ebangaexternal, a single
mAb was approved in December 2020.

Acknowledgments
The author is highly thankful to his PhD mentor Prof. Rajeswara N. Achur, for his critical reading and suggestions. The author acknowledges
the efforts of his first PhD student Dr. K. N. Santhosh in the preparation of this chapter. The help rendered by his PhD student Mr. Sayad
Hafeez in the preparation of images is also greatly appreciated. The author is also thankful to Mr. Sandesh K. Gowda for his endless moral
support. The author is thankful to the Kuvempu University for providing the opportunity to write this book chapter.

References
[1] Johnson KM, Lange JV, Webb PA, Murphy FA. Isolation and partial characterisation of a new virus causing acute haemorrhagic fever in Zaire.
Lancet (London, Engl) 1977;1(8011):569
71. Available from: https://doi.org/10.1016/s0140-6736(77)92000-1.
[2] Jahrling PB, Geisbert TW, Dalgard DW, Johnson ED, Ksiazek TG, Hall WC, et al. Preliminary report: isolation of Ebola virus from monkeys
imported to USA. Lancet (London, Engl) 1990;335(8688):502
5. Available from: https://doi.org/10.1016/0140-6736(90)90737-p.
[3] Formenty P, Hatz C, Le Guenno B, Stoll A, Rogenmoser P, Widmer A. Human infection due to Ebola virus, subtype Côte d’Ivoire: clinical and
biologic presentation. J Infect Dis 1999;179(Suppl 1):S48
53. Available from: https://doi.org/10.1086/514285.
[4] Centers for Disease Control and Prevention (CDC). Outbreaks chronology: Ebola virus disease; 2014. Available from: http://www.cdc.gov/vhf/
ebola/outbreaks/history/chronology.html. [Accessed 30 November 2014].
[5] Cohen J. Ebola outbreak continues despite powerful vaccine. Science (N York, NY) 2019;364(6437):223. Available from: https://doi.org/
10.1126/science.364.6437.223.
[6] Languon S, Quaye O. Filovirus disease outbreaks: a chronological overview. Virol Res Treat 2019;10. Available from: https://doi.org/10.1177/
1178122X19849927 1178122X19849927.
[7] World Health Organization. Ebola situation reports. Ebola virus disease—Democratic Republic of the Congo; 2019a. Available from: https://
www.who.int/csr/don/18-july-2019-ebola-drc/en/.
[8] World Health Organization. Preliminary results on the efficacy of rVSV-ZEBOV-GP Ebola vaccine using the ring vaccination strategy in the
control of an Ebola outbreak in the Democratic Republic of the Congo: an example of integration of research into epidemic response. Geneva;
2019b. Available from: https://www.who.int/csr/resources/publications/ebola/ebola-ring-vaccination-results-12-april-2019.pdf.
[9] Patz JA, Olson SH. Climate change and health: global to local influences on disease risk. Ann Trop Med Parasitol 2006;100(5
6):535
49.
Available from: https://doi.org/10.1179/136485906X97426.
[10] Munster VJ, Bausch DG, de Wit E, Fischer R, Kobinger G, Muñoz-Fontela C, et al. Outbreaks in a rapidly changing Central Africa—lessons
from Ebola. N Engl J Med 2018;379(13):1198
201. Available from: https://doi.org/10.1056/NEJMp1807691.
[11] Olival KJ, Hayman DT. Filoviruses in bats: current knowledge and future directions. Viruses 2014;6(4):1759
88. Available from: https://doi.
org/10.3390/v6041759.
[12] Frick WF, Kingston T, Flanders J. A review of the major threats and challenges to global bat conservation. Ann N Y Acad Sci 2020;1469
(1):5
25. Available from: https://doi.org/10.1111/nyas.14045.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 177

[13] Kiley MP, Bowen ET, Eddy GA, Isaäcson M, Johnson KM, McCormick JB, et al. Filoviridae: a taxonomic home for Marburg and Ebola
viruses. Intervirology 1982;18(1
2):24
32. Available from: https://doi.org/10.1159/000149300.
[14] Baseler L, Chertow DS, Johnson KM, Feldmann H, Morens DM. The pathogenesis of Ebola virus disease. Annu Rev Pathol 2017;12:387
418.
Available from: https://doi.org/10.1146/annurev-pathol-052016-100506.
[15] Volchkov VE, Volchkova VA, Chepurnov AA, Blinov VM, Dolnik O, Netesov SV, et al. Characterization of the L gene and 5’ trailer region of
Ebola virus. J Gen Virol 1999;80(Pt 2):355
62. Available from: https://doi.org/10.1099/0022-1317-80-2-355.
[16] Yu DS, Weng TH, Wu XX, Wang F, Lu XY, Wu HB, et al. The lifecycle of the Ebola virus in host cells. Oncotarget 2017;8(33):55750
9.
Available from: https://doi.org/10.18632/oncotarget.18498.
[17] Mühlberger E, Lötfering B, Klenk HD, Becker S. Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to
mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. J Virol 1998;72(11):8756
64. Available from:
https://doi.org/10.1128/JVI.72.11.8756-8764.1998.
[18] Mühlberger E, Weik M, Volchkov VE, Klenk HD, Becker S. Comparison of the transcription and replication strategies of marburg virus and Ebola
virus by using artificial replication systems. J Virol 1999;73(3):2333
42. Available from: https://doi.org/10.1128/JVI.73.3.2333-2342.1999.
[19] Crary SM, Towner JS, Honig JE, Shoemaker TR, Nichol ST. Analysis of the role of predicted RNA secondary structures in Ebola virus replica-
tion. Virology 2003;306(2):210
18. Available from: https://doi.org/10.1016/s0042-6822(02)00014-4.
[20] Sanchez A, Kiley MP, Holloway BP, Auperin DD. Sequence analysis of the Ebola virus genome: organization, genetic elements, and compari-
son with the genome of Marburg virus. Virus Res 1993;29(3):215
40. Available from: https://doi.org/10.1016/0168-1702(93)90063-s.
[21] Will C, Mühlberger E, Linder D, Slenczka W, Klenk HD, Feldmann H. Marburg virus gene 4 encodes the virion membrane protein, a type I
transmembrane glycoprotein. J Virol 1993;67(3):1203
10. Available from: https://doi.org/10.1128/JVI.67.3.1203-1210.1993.
[22] Kawaoka Y. How Ebola virus infects cells. N Engl J Med 2005;352(25):2645
6. Available from: https://doi.org/10.1056/NEJMcibr051754.
[23] Majid MU, Tahir MS, Ali Q, Rao AQ, Rashid B, Ali A, et al. Nature and history of Ebola virus: an overview. Arch Neurosci 2016;3(3):
e35027. Available from: https://doi.org/10.5812/archneurosci.35027.
[24] Bukreyev AA, Chandran K, Dolnik O, Dye JM, Ebihara H, Leroy EM, et al. Discussions and decisions of the 2012
2014 International
Committee on Taxonomy of Viruses (ICTV) Filoviridae Study Group, January 2012
June 2013. Arch Virol 2014;159(4):821
30. Available
from: https://doi.org/10.1007/s00705-013-1846-9.
[25] Kuhn JH, Andersen KG, Bào Y, Bavari S, Becker S, Bennett RS, et al. Filovirus RefSeq entries: evaluation and selection of filovirus type var-
iants, type sequences, and names. Viruses 2014;6(9):3663
82. Available from: https://doi.org/10.3390/v6093663.
[26] Mehedi M, Falzarano D, Seebach J, Hu X, Carpenter MS, Schnittler HJ, et al. A new Ebola virus nonstructural glycoprotein expressed through
RNA editing. J Virol 2011;85(11):5406
14. Available from: https://doi.org/10.1128/JVI.02190-10.
[27] Singh RK, Dhama K, Malik YS, Ramakrishnan MA, Karthik K, Khandia R, et al. Ebola virus—epidemiology, diagnosis, and control: threat to
humans, lessons learnt, and preparedness plans—an update on its 40 year’s journey. Vet Q 2017;37(1):98
135. Available from: https://doi.org/
10.1080/01652176.2017.1309474.
[28] Cantoni D, Rossman JS. Ebolaviruses: new roles for old proteins. PLoS Negl Trop Dis 2018;12(5):e0006349. Available from: https://doi.org/
10.1371/journal.pntd.0006349.
[29] Feldmann H, Mühlberger E, Randolf A, Will C, Kiley MP, Sanchez A, et al. Marburg virus, a filovirus: messenger RNAs, gene order, and regu-
latory elements of the replication cycle. Virus Res 1992;24(1):1
19. Available from: https://doi.org/10.1016/0168-1702(92)90027-7.
[30] Noda T, Sagara H, Suzuki E, Takada A, Kida H, Kawaoka Y. Ebola virus VP40 drives the formation of virus-like filamentous particles along
with GP. J Virol 2002;76(10):4855
65. Available from: https://doi.org/10.1128/jvi.76.10.4855-4865.2002.
[31] Dolnik O, Kolesnikova L, Becker S. Filoviruses: interactions with the host cell. Cell Mol Life Sci 2008;65(5):756
76. Available from: https://
doi.org/10.1007/s00018-007-7406-2.
[32] Chan SY, Empig CJ, Welte FJ, Speck RF, Schmaljohn A, Kreisberg JF, et al. Folate receptor-alpha is a cofactor for cellular entry by Marburg
and Ebola viruses. Cell 2001;106(1):117
26. Available from: https://doi.org/10.1016/s0092-8674(01)00418-4.
[33] Knipe DM, Howley PM, Knipe David M, Howley Peter M, editors. Fields virology. Philadelphia: Wolters Kluwer/Lippincott Williams &
Wilkins Health; 2013.
[34] Misasi J, Chandran K, Yang JY, Considine B, Filone CM, Côté M, et al. Filoviruses require endosomal cysteine proteases for entry but exhibit
distinct protease preferences. J Virol 2012;86(6):3284
92. Available from: https://doi.org/10.1128/JVI.06346-11.
[35] Schornberg K, Matsuyama S, Kabsch K, Delos S, Bouton A, White J. Role of endosomal cathepsins in entry mediated by the Ebola virus glyco-
protein. J Virol 2006;80(8):4174
8. Available from: https://doi.org/10.1128/JVI.80.8.4174-4178.2006.
[36] Carette JE, Raaben M, Wong AC, Herbert AS, Obernosterer G, Mulherkar N, et al. Ebola virus entry requires the cholesterol transporter
Niemann-Pick C1. Nature 2011;477(7364):340
3. Available from: https://doi.org/10.1038/nature10348.
[37] Chandran K, Sullivan NJ, Felbor U, Whelan SP, Cunningham JM. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infec-
tion. Science (N York, NY) 2005;308(5728):1643
5. Available from: https://doi.org/10.1126/science.1110656.
[38] Côté M, Misasi J, Ren T, Bruchez A, Lee K, Filone CM, et al. Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus
infection. Nature 2011;477(7364):344
8. Available from: https://doi.org/10.1038/nature10380.
[39] Dube D, Brecher MB, Delos SE, Rose SC, Park EW, Schornberg KL, et al. The primed ebolavirus glycoprotein (19-kilodalton GP1,2):
sequence and residues critical for host cell binding. J Virol 2009;83(7):2883
91. Available from: https://doi.org/10.1128/JVI.01956-08.
[40] Hood CL, Abraham J, Boyington JC, Leung K, Kwong PD, Nabel GJ. Biochemical and structural characterization of cathepsin L-processed Ebola virus
glycoprotein: implications for viral entry and immunogenicity. J Virol 2010;84(6):2972
82. Available from: https://doi.org/10.1128/JVI.02151-09.
178 Pandemic Outbreaks in the 21st Century

[41] Miller EH, Obernosterer G, Raaben M, Herbert AS, Deffieu MS, Krishnan A, et al. Ebola virus entry requires the host-programmed recognition
of an intracellular receptor. EMBO J 2012;31(8):1947
60. Available from: https://doi.org/10.1038/emboj.2012.53.
[42] Ball LA, Wertz GW. VSV RNA synthesis: how can you be positive? Cell 1981;26(2 Pt 2):143
4. Available from: https://doi.org/10.1016/
0092-8674(81)90297-x.
[43] Weik M, Modrof J, Klenk HD, Becker S, Mühlberger E. Ebola virus VP30-mediated transcription is regulated by RNA secondary structure for-
mation. J Virol 2002;76(17):8532
9. Available from: https://doi.org/10.1128/jvi.76.17.8532-8539.2002.
[44] Modrof J, Becker S, Mühlberger E. Ebola virus transcription activator VP30 is a zinc-binding protein. J Virol 2003;77(5):3334
8. Available
from: https://doi.org/10.1128/jvi.77.5.3334-3338.2003.
[45] Sanchez A, Kiley MP. Identification and analysis of Ebola virus messenger RNA. Virology 1987;157(2):414
20. Available from: https://doi.
org/10.1016/0042-6822(87)90283-2.
[46] Hartlieb B, Modrof J, Mühlberger E, Klenk HD, Becker S. Oligomerization of Ebola virus VP30 is essential for viral transcription and can be
inhibited by a synthetic peptide. J Biol Chem 2003;278(43):41830
6. Available from: https://doi.org/10.1074/jbc.M307036200.
[47] Hartlieb B, Muziol T, Weissenhorn W, Becker S. Crystal structure of the C-terminal domain of Ebola virus VP30 reveals a role in transcription
and nucleocapsid association. Proc Natl Acad Sci U S A 2007;104(2):624
9. Available from: https://doi.org/10.1073/pnas.0606730104.
[48] Bharat TA, Noda T, Riches JD, Kraehling V, Kolesnikova L, Becker S, et al. Structural dissection of Ebola virus and its assembly determinants
using cryo-electron tomography. Proc Natl Acad Sci U S A 2012;109(11):4275
80. Available from: https://doi.org/10.1073/pnas.1120453109.
[49] Biedenkopf N, Hartlieb B, Hoenen T, Becker S. Phosphorylation of Ebola virus VP30 influences the composition of the viral nucleocapsid complex:
impact on viral transcription and replication. J Biol Chem 2013;288(16):11165
74. Available from: https://doi.org/10.1074/jbc.M113.461285.
[50] Martinez MJ, Volchkova VA, Raoul H, Alazard-Dany N, Reynard O, Volchkov VE. Role of VP30 phosphorylation in the Ebola virus replica-
tion cycle. J Infect Dis 2011;204(Suppl 3):S934
40. Available from: https://doi.org/10.1093/infdis/jir320.
[51] Stahelin RV. Membrane binding and bending in Ebola VP40 assembly and egress. Front Microbiol 2014;5:300. Available from: https://doi.org/
10.3389/fmicb.2014.00300.
[52] Mateo M, Carbonnelle C, Reynard O, Kolesnikova L, Nemirov K, Page A, et al. VP24 is a molecular determinant of Ebola virus virulence in
guinea pigs. J Infect Dis 2011;204(Suppl 3):S1011
20. Available from: https://doi.org/10.1093/infdis/jir338.
[53] Prins KC, Delpeut S, Leung DW, Reynard O, Volchkova VA, Reid SP, et al. Mutations abrogating VP35 interaction with double-stranded RNA
render Ebola virus avirulent in guinea pigs. J Virol 2010;84(6):3004
15. Available from: https://doi.org/10.1128/JVI.02459-09.
[54] Reid SP, Valmas C, Martinez O, Sanchez FM, Basler CF. Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin
alpha proteins with activated STAT1. J Virology 2007;81(24):13469
77. Available from: https://doi.org/10.1128/JVI.01097-07.
[55] Cilloniz C, Ebihara H, Ni C, Neumann G, Korth MJ, Kelly SM, et al. Functional genomics reveals the induction of inflammatory response and
metalloproteinase gene expression during lethal Ebola virus infection. J Virol 2011;85(17):9060
8. Available from: https://doi.org/10.1128/
JVI.00659-11.
[56] Ebihara H, Takada A, Kobasa D, Jones S, Neumann G, Theriault S, et al. Molecular determinants of Ebola virus virulence in mice. PLoS
Pathog 2006;2(7):e73. Available from: https://doi.org/10.1371/journal.ppat.0020073.
[57] Hartman AL, Bird BH, Towner JS, Antoniadou ZA, Zaki SR, Nichol ST. Inhibition of IRF-3 activation by VP35 is critical for the high level of
virulence of ebola virus. J Virol 2008;82(6):2699
704. Available from: https://doi.org/10.1128/JVI.02344-07.
[58] Hartman AL, Ling L, Nichol ST, Hibberd ML. Whole-genome expression profiling reveals that inhibition of host innate immune response path-
ways by Ebola virus can be reversed by a single amino acid change in the VP35 protein. J virology 2008;82(11):5348
58. Available from:
https://doi.org/10.1128/JVI.00215-08.
[59] Yoneyama M, Fujita T. RNA recognition and signal transduction by RIG-I-like receptors. Immunol Rev 2009;227(1):54
65. Available from:
https://doi.org/10.1111/j.1600-065X.2008.00727.x.
[60] Basler CF, Wang X, Mühlberger E, Volchkov V, Paragas J, Klenk HD, et al. The Ebola virus VP35 protein functions as a type I IFN antagonist.
Proc Natl Acad Sci U S A 2000;97(22):12289
94. Available from: https://doi.org/10.1073/pnas.220398297.
[61] Basler CF, Mikulasova A, Martinez-Sobrido L, Paragas J, Mühlberger E, Bray M, et al. The Ebola virus VP35 protein inhibits activation of
interferon regulatory factor 3. J Virol 2003;77(14):7945
56. Available from: https://doi.org/10.1128/jvi.77.14.7945-7956.2003.
[62] Prins KC, Cárdenas WB, Basler CF. Ebola virus protein VP35 impairs the function of interferon regulatory factor-activating kinases
IKKepsilon and TBK-1. J Virol 2009;83(7):3069
77. Available from: https://doi.org/10.1128/JVI.01875-08.
[63] Cárdenas WB, Loo YM, Gale Jr M, Hartman AL, Kimberlin CR, Martı́nez-Sobrido L, et al. Ebola virus VP35 protein binds double-stranded
RNA and inhibits alpha/beta interferon production induced by RIG-I signaling. J Virol 2006;80(11):5168
78. Available from: https://doi.org/
10.1128/JVI.02199-05.
[64] Kimberlin CR, Bornholdt ZA, Li S, Woods Jr VL, MacRae IJ, Saphire EO. Ebolavirus VP35 uses a bimodal strategy to bind dsRNA for innate
immune suppression. Proc Natl Acad Sci U S A 2010;107(1):314
19. Available from: https://doi.org/10.1073/pnas.0910547107.
[65] Fabozzi G, Nabel CS, Dolan MA, Sullivan NJ. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the
mammalian RNA interference pathway. J Virol 2011;85(6):2512
23. Available from: https://doi.org/10.1128/JVI.01160-10.
[66] Luthra P, Ramanan P, Mire CE, Weisend C, Tsuda Y, Yen B, et al. Mutual antagonism between the Ebola virus VP35 protein and the RIG-I
activator PACT determines infection outcome. Cell Host Microbe 2013;14(1):74
84. Available from: https://doi.org/10.1016/j.
chom.2013.06.010.
[67] Gill G. SUMO and ubiquitin in the nucleus: different functions, similar mechanisms. Genes Dev 2004;18(17):2046
59. Available from: https://
doi.org/10.1101/gad.1214604.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 179

[68] Yeh ET, Gong L, Kamitani T. Ubiquitin-like proteins: new wines in new bottles. Gene 2000;248(1
2):1
14. Available from: https://doi.org/
10.1016/s0378-1119(00)00139-6.
[69] Desterro JM, Rodriguez MS, Kemp GD, Hay RT. Identification of the enzyme required for activation of the small ubiquitin-like protein
SUMO-1. J Biol Chem 1999;274(15):10618
24. Available from: https://doi.org/10.1074/jbc.274.15.10618.
[70] Desterro JM, Thomson J, Hay RT. Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett 1997;417(3):297
300. Available from: https://doi.
org/10.1016/s0014-5793(97)01305-7.
[71] Sarge KD, Park-Sarge OK. Sumoylation and human disease pathogenesis. Trends Biochem Sci 2009;34(4):200
5. Available from: https://doi.
org/10.1016/j.tibs.2009.01.004.
[72] Mukhopadhyay D, Dasso M. Modification in reverse: the SUMO proteases. Trends Biochemical Sci 2007;32(6):286
95. Available from:
https://doi.org/10.1016/j.tibs.2007.05.002.
[73] Martin S, Wilkinson KA, Nishimune A, Henley JM. Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction.
Nat Rev Neurosci 2007;8(12):948
59. Available from: https://doi.org/10.1038/nrn2276.
[74] Takahashi Y, Kahyo T, Toh-E A, Yasuda H, Kikuchi Y. Yeast Ull1/Siz1 is a novel SUMO1/Smt3 ligase for septin components and functions
as an adaptor between conjugating enzyme and substrates. J Biol Chem 2001;276(52):48973
7. Available from: https://doi.org/10.1074/jbc.
M109295200.
[75] Chang TH, Kubota T, Matsuoka M, Jones S, Bradfute SB, Bray M, et al. Ebola Zaire virus blocks type I interferon production by exploiting the
host SUMO modification machinery. PLoS Pathog 2009;5(6):e1000493. Available from: https://doi.org/10.1371/journal.ppat.1000493.
[76] Katze MG, He Y, Gale Jr M. Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2002;2(9):675
87. Available from: https://doi.
org/10.1038/nri888.
[77] Green AM, Beatty PR, Hadjilaou A, Harris E. Innate immunity to dengue virus infection and subversion of antiviral responses. J Mol Biol
2014;426(6):1148
60. Available from: https://doi.org/10.1016/j.jmb.2013.11.023.
[78] Reid SP, Leung LW, Hartman AL, Martinez O, Shaw ML, Carbonnelle C, et al. Ebola virus VP24 binds karyopherin alpha1 and blocks STAT1
nuclear accumulation. J Virol 2006;80(11):5156
67. Available from: https://doi.org/10.1128/JVI.02349-05.
[79] Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 2005;5(5):375
86. Available from: https://
doi.org/10.1038/nri1604.
[80] Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev Immunol 2014;14(1):36
49. Available from: https://doi.org/
10.1038/nri3581.
[81] Bosio CM, Aman MJ, Grogan C, Hogan R, Ruthel G, Negley D, et al. Ebola and Marburg viruses replicate in monocyte-derived dendritic cells with-
out inducing the production of cytokines and full maturation. J Infect Dis 2003;188(11):1630
8. Available from: https://doi.org/10.1086/379199.
[82] Mahanty S, Gupta M, Paragas J, Bray M, Ahmed R, Rollin PE. Protection from lethal infection is determined by innate immune responses in a
mouse model of Ebola virus infection. Virology 2003;312(2):415
24. Available from: https://doi.org/10.1016/s0042-6822(03)00233-2.
[83] Mahanty S, Hutchinson K, Agarwal S, McRae M, Rollin PE, Pulendran B. Cutting edge: impairment of dendritic cells and adaptive immunity
by Ebola and Lassa viruses. J Immunol (Baltimore, MD: 1950) 2003;170(6):2797
801. Available from: https://doi.org/10.4049/jimmunol.
170.6.2797.
[84] Marcenaro E, Carlomagno S, Pesce S, Moretta A, Sivori S. NK/DC crosstalk in anti-viral response. Adv Exp Med Biol 2012;946:295
308.
Available from: https://doi.org/10.1007/978-1-4614-0106-3_17.
[85] Wehner R, Dietze K, Bachmann M, Schmitz M. The bidirectional crosstalk between human dendritic cells and natural killer cells. J Innate
Immun 2011;3(3):258
63. Available from: https://doi.org/10.1159/000323923.
[86] McElroy AK, Akondy RS, Davis CW, Ellebedy AH, Mehta AK, Kraft CS, et al. Human Ebola virus infection results in substantial immune
activation. Proc Natl Acad Sci U S A 2015;112(15):4719
24. Available from: https://doi.org/10.1073/pnas.1502619112.
[87] Baize S, Leroy EM, Georges AJ, Georges-Courbot MC, Capron M, Bedjabaga I, et al. Inflammatory responses in Ebola virus-infected patients.
Clin Exp Immunol 2002;128(1):163
8. Available from: https://doi.org/10.1046/j.1365-2249.2002.01800.x.
[88] Sobarzo A, Ochayon DE, Lutwama JJ, Balinandi S, Guttman O, Marks RS, et al. Persistent immune responses after Ebola virus infection. N
Engl J Med 2013;369(5):492
3. Available from: https://doi.org/10.1056/NEJMc1300266.
[89] Cook JD, Lee JE. The secret life of viral entry glycoproteins: moonlighting in immune evasion. PLoS Pathog 2013;9(5):e1003258. Available
from: https://doi.org/10.1371/journal.ppat.1003258.
[90] Yang Z, Delgado R, Xu L, Todd RF, Nabel EG, Sanchez A, et al. Distinct cellular interactions of secreted and transmembrane Ebola virus gly-
coproteins. Science (New York, NY) 1998;279(5353):1034
7. Available from: https://doi.org/10.1126/science.279.5353.1034.
[91] Kindzelskii AL, Yang Z, Nabel GJ, Todd 3rd RF, Petty HR. Ebola virus secretory glycoprotein (sGP) diminishes Fc gamma RIIIB-to-CR3
proximity on neutrophils. J Immunol (Baltimore, MS: 1950) 2000;164(2):953
8. Available from: https://doi.org/10.4049/jimmunol.
164.2.953.
[92] Mohan GS, Li W, Ye L, Compans RW, Yang C. Antigenic subversion: a novel mechanism of host immune evasion by Ebola virus. PLoS
Pathog 2012;8(12):e1003065. Available from: https://doi.org/10.1371/journal.ppat.1003065.
[93] Sullivan NJ, Martin JE, Graham BS, Nabel GJ. Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by
the animal rule. Nat Rev Microbiol 2009;7(5):393
400. Available from: https://doi.org/10.1038/nrmicro2129.
[94] Baize S, Leroy EM, Georges-Courbot MC, Capron M, Lansoud-Soukate J, Debré P, et al. Defective humoral responses and extensive intravas-
cular apoptosis are associated with fatal outcome in Ebola virus-infected patients. Nat Med 1999;5(4):423
6. Available from: https://doi.org/
10.1038/7422.
180 Pandemic Outbreaks in the 21st Century

[95] Warfield KL, Olinger G, Deal EM, Swenson DL, Bailey M, Negley DL, et al. Induction of humoral and CD8 1 T cell responses are required
for protection against lethal Ebola virus infection. J Immunol (Baltimore, MD: 1950) 2005;175(2):1184
91. Available from: https://doi.org/
10.4049/jimmunol.175.2.1184.
[96] Warfield KL, Olinger GG. Protective role of cytotoxic T lymphocytes in filovirus hemorrhagic fever. J Biomed Biotechnol 2011;2011:984241.
Available from: https://doi.org/10.1155/2011/984241.
[97] Rath PC, Aggarwal BB. TNF-induced signaling in apoptosis. J Clin Immunol 1999;19(6):350
64. Available from: https://doi.org/10.1023/
a:1020546615229.
[98] Simon HU, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis Int J Program Cell Death
2000;5(5):415
18. Available from: https://doi.org/10.1023/a:1009616228304.
[99] Snyder CM, Shroff EH, Liu J, Chandel NS. Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members. PLoS One
2009;4(9):e7059. Available from: https://doi.org/10.1371/journal.pone.0007059.
[100] Wauquier N, Becquart P, Padilla C, Baize S, Leroy EM. Human fatal zaire ebola virus infection is associated with an aberrant innate immu-
nity and with massive lymphocyte apoptosis. PLoS Negl Trop Dis 2010;4(10):e837. Available from: https://doi.org/10.1371/journal.
pntd.0000837.
[101] Bah EI, Lamah MC, Fletcher T, Jacob ST, Brett-Major DM, Sall AA, et al. Clinical presentation of patients with Ebola virus disease in
Conakry, Guinea. N Engl J Med 2015;372(1):40
7. Available from: https://doi.org/10.1056/NEJMoa1411249.
[102] Leroy EM, Baize S, Debre P, Lansoud-Soukate J, Mavoungou E. Early immune responses accompanying human asymptomatic Ebola infec-
tions. Clin Exp Immunol 2001;124(3):453
60. Available from: https://doi.org/10.1046/j.1365-2249.2001.01517.x.
[103] Bradfute SB, Braun DR, Shamblin JD, Geisbert JB, Paragas J, Garrison A, et al. Lymphocyte death in a mouse model of Ebola virus infection.
J Infect Dis, 196 2007;Suppl 2:S296
304. Available from: https://doi.org/10.1086/520602.
[104] Bradfute SB, Warfield KL, Bavari S. Functional CD8 1 T cell responses in lethal Ebola virus infection. J Immunol (Baltimore, MD: 1950)
2008;180(6):4058
66. Available from: https://doi.org/10.4049/jimmunol.180.6.4058.
[105] Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet (London, Engl) 2011;377(9768):849
62. Available from: https://doi.org/
10.1016/S0140-6736(10)60667-8.
[106] Geisbert TW, Young HA, Jahrling PB, Davis KJ, Kagan E, Hensley LE. Mechanisms underlying coagulation abnormalities in ebola hemor-
rhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event. J Infect Dis 2003;188(11):1618
29. Available
from: https://doi.org/10.1086/379724.
[107] Geisbert TW, Young HA, Jahrling PB, Davis KJ, Larsen T, Kagan E, et al. Pathogenesis of Ebola hemorrhagic fever in primate models: evi-
dence that hemorrhage is not a direct effect of virus-induced cytolysis of endothelial cells. Am J Pathol 2003;163(6):2371
82. Available
from: https://doi.org/10.1016/S0002-9440(10)63592-4.
[108] Muñoz-Fontela C, McElroy AK. Ebola virus disease in humans: pathophysiology and immunity. Curr Top Microbiol Immunol
2017;411:141
69. Available from: https://doi.org/10.1007/82_2017_11.
[109] Prescott JB, Marzi A, Safronetz D, Robertson SJ, Feldmann H, Best SM. Immunobiology of Ebola and Lassa virus infections. Nat Rev
Immunol 2017;17(3):195
207. Available from: https://doi.org/10.1038/nri.2016.138.
[110] Wahl-Jensen V, Kurz S, Feldmann F, Buehler LK, Kindrachuk J, DeFilippis V, et al. Ebola virion attachment and entry into human macro-
phages profoundly effects early cellular gene expression. PLoS Negl Trop Dis 2011;5(10):e1359. Available from: https://doi.org/10.1371/jour-
nal.pntd.0001359.
[111] Gupta M, Mahanty S, Ahmed R, Rollin PE. Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with
ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro. Virology 2001;284(1):20
5. Available from:
https://doi.org/10.1006/viro.2001.0836.
[112] Sanchez A, Lukwiya M, Bausch D, Mahanty S, Sanchez AJ, Wagoner KD, et al. Analysis of human peripheral blood samples from fatal and
nonfatal cases of Ebola (Sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels. J Virol 2004;78(19):10370
7.
Available from: https://doi.org/10.1128/JVI.78.19.10370-10377.2004.
[113] Villinger F, Rollin PE, Brar SS, Chikkala NF, Winter J, Sundstrom JB, et al. Markedly elevated levels of interferon (IFN)-gamma, IFN-alpha,
interleukin (IL)-2, IL-10, and tumor necrosis factor-alpha associated with fatal Ebola virus infection. J Infect Dis 1999;179(Suppl 1):S188
91.
Available from: https://doi.org/10.1086/514283.
[114] Ebihara H, Rockx B, Marzi A, Feldmann F, Haddock E, Brining D, et al. Host response dynamics following lethal infection of rhesus maca-
ques with Zaire ebolavirus. J Infect Dis 2011;204(Suppl 3):S991
9. Available from: https://doi.org/10.1093/infdis/jir336.
[115] Hensley LE, Young HA, Jahrling PB, Geisbert TW. Proinflammatory response during Ebola virus infection of primate models: possible
involvement of the tumor necrosis factor receptor superfamily. Immunol Lett 2002;80(3):169
79. Available from: https://doi.org/10.1016/
s0165-2478(01)00327-3.
[116] Lubaki NM, Ilinykh P, Pietzsch C, Tigabu B, Freiberg AN, Koup RA, et al. The lack of maturation of Ebola virus-infected dendritic cells
results from the cooperative effect of at least two viral domains. J Virol 2013;87(13):7471
85. Available from: https://doi.org/10.1128/
JVI.03316-12.
[117] Kang ES, Acchiardo SR, Wang YB, Tevlin MT, Hughes T, Cardoso S. Hypotension during hemodialysis: role for nitric oxide. Am J Med Sci
1997;313(3):138
46. Available from: https://doi.org/10.1097/00000441-199703000-00003.
[118] Ray RB, Basu A, Steele R, Beyene A, McHowat J, Meyer K, et al. Ebola virus glycoprotein-mediated anoikis of primary human cardiac
microvascular endothelial cells. Virology 2004;321(2):181
8. Available from: https://doi.org/10.1016/j.virol.2003.12.014.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 181

[119] Wahl-Jensen VM, Afanasieva TA, Seebach J, Ströher U, Feldmann H, Schnittler HJ. Effects of Ebola virus glycoproteins on endothelial cell
activation and barrier function. J Virol 2005;79(16):10442
50. Available from: https://doi.org/10.1128/JVI.79.16.10442-10450.2005.
[120] Martines RB, Ng DL, Greer PW, Rollin PE, Zaki SR. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses.
J Pathol 2015;235(2):153
74. Available from: https://doi.org/10.1002/path.4456.
[121] Ryabchikova EI, Kolesnikova LV, Luchko SV. An analysis of features of pathogenesis in two animal models of Ebola virus infection. J Infect
Dis, 179 1999;Suppl 1:S199
202. Available from: https://doi.org/10.1086/514293.
[122] Fisher-Hoch SP, Brammer TL, Trappier SG, Hutwagner LC, Farrar BB, Ruo SL, et al. Pathogenic potential of filoviruses: role of geographic
origin of primate host and virus strain. J Infect Dis 1992;166(4):753
63. Available from: https://doi.org/10.1093/infdis/166.4.753.
[123] Johnson E, Jaax N, White J, Jahrling P. Lethal experimental infections of rhesus monkeys by aerosolized Ebola virus. Int J Exp Pathol
1995;76(4):227
36.
[124] Kindrachuk J, Wahl-Jensen V, Safronetz D, Trost B, Hoenen T, Arsenault R, et al. Ebola virus modulates transforming growth factor β signal-
ing and cellular markers of mesenchyme-like transition in hepatocytes. J Virol 2014;88(17):9877
92. Available from: https://doi.org/10.1128/
JVI.01410-14.
[125] Paessler S, Walker DH. Pathogenesis of the viral hemorrhagic fevers. Annu Rev Pathol 2013;8:411
40. Available from: https://doi.org/
10.1146/annurev-pathol-020712-164041.
[126] Neumann FJ, Ott I, Marx N, Luther T, Kenngott S, Gawaz M, et al. Effect of human recombinant interleukin-6 and interleukin-8 on monocyte
procoagulant activity. Arterioscler Thromb Vasc Biol 1997;17(12):3399
405. Available from: https://doi.org/10.1161/01.atv.17.12.3399.
[127] McElroy A. Understanding bleeding in ebola virus disease. Clin Adv Hematol Oncol 2015;13(1):29
31.
[128] Falasca L, Agrati C, Petrosillo N, Di Caro A, Capobianchi MR, Ippolito G, et al. Molecular mechanisms of Ebola virus pathogenesis: focus on
cell death. Cell Death Differ 2015;22(8):1250
9. Available from: https://doi.org/10.1038/cdd.2015.67.
[129] Leligdowicz A, Fischer 2nd WA, Uyeki TM, Fletcher TE, Adhikari NK, Portella G, et al. Ebola virus disease and critical illness. Crit care
(London, Engl) 2016;20(1):217. Available from: https://doi.org/10.1186/s13054-016-1325-2.
[130] Clark DV, Kibuuka H, Millard M, Wakabi S, Lukwago L, Taylor A, et al. Long-term sequelae after Ebola virus disease in Bundibugyo,
Uganda: a retrospective cohort study. Lancet Infect Dis 2015;15(8):905
12. Available from: https://doi.org/10.1016/S1473-3099(15)70152-0.
[131] Mattia JG, Vandy MJ, Chang JC, Platt DE, Dierberg K, Bausch DG, et al. Early clinical sequelae of Ebola virus disease in Sierra Leone: a
cross-sectional study. Lancet Infect Dis 2016;16(3):331
8. Available from: https://doi.org/10.1016/S1473-3099(15)00489-2.
[132] Qureshi AI, Chughtai M, Loua TO, Pe Kolie J, Camara HF, Ishfaq MF, et al. Study of Ebola virus disease survivors in Guinea. Clin Infect Dis
2015;61(7):1035
42. Available from: https://doi.org/10.1093/cid/civ453.
[133] Mate SE, Kugelman JR, Nyenswah TG, Ladner JT, Wiley MR, Cordier-Lassalle T, et al. Molecular evidence of sexual transmission of Ebola
virus. N Engl J Med 2015;373(25):2448
54. Available from: https://doi.org/10.1056/NEJMoa1509773.
[134] Fink S. Ebola survivor from Scotland is critically ill. New York Times. October 14, 2015. [Accessed February 8, 2016].
[135] Baggi FM, Taybi A, Kurth A, Van Herp M, Di Caro A, Wölfel R, et al. Management of pregnant women infected with Ebola virus in a treatment
centre in Guinea, June 2014. Eurosurveillance 2014;19(49):20983. Available from: https://doi.org/10.2807/1560-7917.es2014.19.49.20983.
[136] Kaplan HJ, Niederkorn JY. Regional immunity and immune privilege. Chem Immunol Allergy 2007;92:11
26. Available from: https://doi.
org/10.1159/000099237.
[137] Shorten RJ, Brown CS, Jacobs M, Rattenbury S, Simpson AJ, Mepham S. Diagnostics in Ebola virus disease in resource-rich and resource-
limited settings. PLoS Negl Trop Dis 2016;10(10):e0004948. Available from: https://doi.org/10.1371/journal.pntd.0004948.
[138] Coarsey CT, Esiobu N, Narayanan R, Pavlovic M, Shafiee H, Asghar W. Strategies in Ebola virus disease (EVD) diagnostics at the point of
care. Crit Rev Microbiol 2017;43(6):779
98. Available from: https://doi.org/10.1080/1040841X.2017.1313814.
[139] Erickson BR, Sealy TK, Flietstra T, Morgan L, Kargbo B, Matt-Lebby VE, et al. Ebola virus disease diagnostics, Sierra Leone: analysis of
real-time reverse transcription-polymerase chain reaction values for clinical blood and oral swab specimens. J Infect Dis 2016;214(Suppl 3):
S258
62. Available from: https://doi.org/10.1093/infdis/jiw296.
[140] Kerber R, Krumkamp R, Diallo B, Jaeger A, Rudolf M, Lanini S, et al. Analysis of diagnostic findings from the European mobile laboratory in
Guéckédou, Guinea, March 2014 Through March 2015. J Infect Dis 2016;214(suppl 3):S250
7. Available from: https://doi.org/10.1093/infdis/jiw269.
[141] Glynn JR, Bower H, Johnson S, Houlihan CF, Montesano C, Scott JT, et al. Asymptomatic infection and unrecognised Ebola virus disease in
Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus. Lancet Infect
Dis 2017;17(6):645
53. Available from: https://doi.org/10.1016/S1473-3099(17)30111-1.
[142] Leroy EM, Baize S, Volchkov VE, Fisher-Hoch SP, Georges-Courbot MC, Lansoud-Soukate J, et al. Human asymptomatic Ebola infection
and strong inflammatory response. Lancet (London, Engl) 2000;355(9222):2210
15. Available from: https://doi.org/10.1016/s0140-6736(00)
02405-3.
[143] Feldmann H, Feldmann F, Marzi A. Ebola: lessons on vaccine development. Annu Rev Microbiol 2018;72:423
46. Available from: https://
doi.org/10.1146/annurev-micro-090817-062414.
[144] Gross L, Lhomme E, Pasin C, Richert L, Thiebaut R. Ebola vaccine development: systematic review of pre-clinical and clinical studies, and
meta-analysis of determinants of antibody response variability after vaccination. Int J Infect Dis 2018;74:83
96. Available from: https://doi.
org/10.1016/j.ijid.2018.06.022.
[145] Sullivan NJ, Geisbert TW, Geisbert JB, Shedlock DJ, Xu L, Lamoreaux L, et al. Immune protection of nonhuman primates against Ebola virus
with single low-dose adenovirus vectors encoding modified GPs. PLoS Med 2006;3(6):e177. Available from: https://doi.org/10.1371/journal.
pmed.0030177.
182 Pandemic Outbreaks in the 21st Century

[146] Henao-Restrepo AM, Camacho A, Longini IM, Watson CH, Edmunds WJ, Egger M, et al. Efficacy and effectiveness of an rVSV-vectored
vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça
Suffit!). Lancet (London, Engl) 2017;389(10068):505
18. Available from: https://doi.org/10.1016/S0140-6736(16)32621-6.
[147] Kennedy SB, Bolay F, Kieh M, Grandits G, Badio M, Ballou R, et al. Phase 2 placebo-controlled trial of two vaccines to prevent Ebola in
Liberia . . . PREVAIL I Study GroupN Engl J Med 2017;377(15):1438
47. Available from: https://doi.org/10.1056/NEJMoa1614067.
[148] Geisbert TW, Daddario-Dicaprio KM, Lewis MG, Geisbert JB, Grolla A, Leung A, et al. Vesicular stomatitis virus-based ebola vaccine is
well-tolerated and protects immunocompromised nonhuman primates. PLoS Pathog 2008;4(11):e1000225. Available from: https://doi.org/
10.1371/journal.ppat.1000225.
[149] Jones SM, Feldmann H, Ströher U, Geisbert JB, Fernando L, Grolla A, et al. Live attenuated recombinant vaccine protects nonhuman primates
against Ebola and Marburg viruses. Nat Med 2005;11(7):786
90. Available from: https://doi.org/10.1038/nm1258.
[150] Jones SM, Stroher U, Fernando L, Qiu X, Alimonti J, Melito P, et al. Assessment of a vesicular stomatitis virus-based vaccine by use of the
mouse model of Ebola virus hemorrhagic fever. J Infect Dis 2007;196(Suppl 2):S404
12. Available from: https://doi.org/10.1086/520591.
[151] Geisbert TW, Geisbert JB, Leung A, Daddario-DiCaprio KM, Hensley LE, Grolla A, et al. Single-injection vaccine protects nonhuman pri-
mates against infection with marburg virus and three species of ebola virus. J Virol 2009;83(14):7296
304. Available from: https://doi.org/
10.1128/JVI.00561-09.
[152] Henao-Restrepo AM, Longini IM, Egger M, Dean NE, Edmunds WJ, Camacho A, et al. Efficacy and effectiveness of an rVSV-vectored vac-
cine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet 2015;386
(9996):857
66. Available from: https://doi.org/10.1016/S0140-6736(15)61117-5.
[153] Günther S, Feldmann H, Geisbert TW, Hensley LE, Rollin PE, Nichol ST, et al. Management of accidental exposure to Ebola virus in the bio-
safety level 4 laboratory, Hamburg, Germany. J Infect Dis 2011;204(Suppl 3):S785
90. Available from: https://doi.org/10.1093/infdis/jir298.
[154] Lai L, Davey R, Beck A, Xu Y, Suffredini AF, Palmore T, et al. Emergency postexposure vaccination with vesicular stomatitis virus-vectored
Ebola vaccine after needlestick. JAMA 2015;313(12):1249
55. Available from: https://doi.org/10.1001/jama.2015.1995.
[155] Tapia MD, Sow SO, Lyke KE, Haidara FC, Diallo F, Doumbia M, et al. Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and United
States adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-
blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis 2016;16(1):31
42. Available
from: https://doi.org/10.1016/S1473-3099(15)00362-X.
[156] Milligan ID, Gibani MM, Sewell R, Clutterbuck EA, Campbell D, Plested E, et al. Safety and immunogenicity of novel adenovirus type 26-
and modified vaccinia ankara-vectored Ebola vaccines: a randomized clinical trial. JAMA 2016;315(15):1610
23. Available from: https://doi.
org/10.1001/jama.2016.4218.
[157] Venkatraman N, Silman D, Folegatti PM, Hill A. Vaccines against Ebola virus. Vaccine 2018;36(36):5454
9. Available from: https://doi.org/
10.1016/j.vaccine.2017.07.054.
[158] Wong G, Mendoza EJ, Plummer FA, Gao GF, Kobinger GP, Qiu X. From bench to almost bedside: the long road to a licensed Ebola virus
vaccine. Expert Opin Biol Ther 2018;18(2):159
73. Available from: https://doi.org/10.1080/14712598.2018.1404572.
[159] Sullivan NJ, Sanchez A, Rollin PE, Yang ZY, Nabel GJ. Development of a preventive vaccine for Ebola virus infection in primates. Nature
2000;408(6812):605
9. Available from: https://doi.org/10.1038/35046108.
[160] Sullivan NJ, Geisbert TW, Geisbert JB, Xu L, Yang ZY, Roederer M, et al. Accelerated vaccination for Ebola virus haemorrhagic fever in
non-human primates. Nature 2003;424(6949):681
4. Available from: https://doi.org/10.1038/nature01876.
[161] Ledgerwood JE, Costner P, Desai N, Holman L, Enama ME, Yamshchikov G, et al. A replication defective recombinant Ad5 vaccine expres-
sing Ebola virus GP is safe and immunogenic in healthy adults & VRC 205 Study TeamVaccine 2010;29(2):304
13. Available from: https://
doi.org/10.1016/j.vaccine.2010.10.037.
[162] Zhu FC, Hou LH, Li JX, Wu SP, Liu P, Zhang GR, et al. Safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based
Ebola vaccine in healthy adults in China: preliminary report of a randomised, double-blind, placebo-controlled, phase 1 trial. Lancet (London,
Engl) 2015;385(9984):2272
9. Available from: https://doi.org/10.1016/S0140-6736(15)60553-0.
[163] Duraffour S, Malvy D, Sissoko D. How to treat Ebola virus infections? a lesson from the field. Curr Opvirol 2017;24:9
15. Available from:
https://doi.org/10.1016/j.coviro.2017.03.003.
[164] Sissoko D, Laouenan C, Folkesson E, M’Lebing AB, Beavogui AH, Baize S, et al. Experimental treatment with Favipiravir for Ebola virus
disease (the JIKI trial): a historically controlled, single-arm proof-of-concept trial in Guinea . . . JIKI Study GroupPLoS Med 2016;13(3):
e1001967. Available from: https://doi.org/10.1371/journal.pmed.1001967.
[165] Dunning J, Kennedy SB, Antierens A, Whitehead J, Ciglenecki I, Carson G, et al. Experimental treatment of Ebola virus disease with
Brincidofovir & RAPIDE-BCV Trial TeamPLoS One 2016;11(9):e0162199. Available from: https://doi.org/10.1371/journal.pone.0162199.
[166] PREVAIL II Writing Group, Multi-National PREVAIL II Study Team, Davey RT, Jr, Dodd L, Proschan MA, Neaton J, et al. A randomized, con-
trolled trial of ZMapp for Ebola virus infection. N Engl J Med 2016;375(15):1448
56. Available from: https://doi.org/10.1056/NEJMoa1604330.
[167] Sahr F, Ansumana R, Massaquoi TA, Idriss BR, Sesay FR, Lamin JM, et al. Evaluation of convalescent whole blood for treating Ebola virus
disease in Freetown, Sierra Leone. J Infect 2017;74(3):302
9. Available from: https://doi.org/10.1016/j.jinf.2016.11.009.
[168] Van Griensven J, Bah EI, Haba N, Delamou A, Camara BS, Olivier KJ, et al. Electrolyte and metabolic disturbances in Ebola patients during
a clinical trial, Guinea, 2015 & Ebola-Tx ConsortiumEmerg Infect Dis 2016;22(12):2120
7. Available from: https://doi.org/10.3201/
eid2212.161136.
 Immunological mechanisms associated with clinical features of Ebola virus disease Chapter | 10 183

[169] Konde MK, Baker DP, Traore FA, Sow MS, Camara A, Barry AA, et al. Interferon β-1a for the treatment of Ebola virus disease: a historically
controlled, single-arm proof-of-concept trial. PLoS One 2017;12(2):e0169255. Available from: https://doi.org/10.1371/journal.pone.0169255.
[170] Warren TK, Jordan R, Lo MK, Ray AS, Mackman RL, Soloveva V, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola
virus in rhesus monkeys. Nature 2016;531(7594):381
5. Available from: https://doi.org/10.1038/nature17180.
[171] Sivapalasingam S, Kamal M, Slim R, Hosain R, Shao W, Stoltz R, et al. Safety, pharmacokinetics, and immunogenicity of a co-formulated
cocktail of three human monoclonal antibodies targeting Ebola virus glycoprotein in healthy adults: a randomised, first-in-human phase 1
study. Lancet Infect Dis 2018;18(8):884
93. Available from: https://doi.org/10.1016/S1473-3099(18)30397-9.
[172] Corti D, Misasi J, Mulangu S, Stanley DA, Kanekiyo M, Wollen S, et al. Protective monotherapy against lethal Ebola virus infection by a
potently neutralizing antibody. Science (New York, NY) 2016;351(6279):1339
42. Available from: https://doi.org/10.1126/science.aad5224.
[173] Mire CE, Geisbert TW. Neutralizing the threat: pan-ebolavirus antibodies close the loop. Trends Mol Med 2017;23(8):669
71. Available
from: https://doi.org/10.1016/j.molmed.2017.06.008.
[174] Gray N, Stringer B, Bark G, Heller Perache A, Jephcott F, Broeder R, et al. ‘When Ebola enters a home, a family, a community’: a qualitative
study of population perspectives on Ebola control measures in rural and urban areas of Sierra Leone. PLoS Negl Trop Dis 2018;12(6):
e0006461. Available from: https://doi.org/10.1371/journal.pntd.0006461.
[175] Malvy D, McElroy AK, de Clerck H, Günther S, van Griensven J. Ebola virus disease. Lancet (London, Engl) 2019;393(10174):936
48.
Available from: https://doi.org/10.1016/S0140-6736(18)33132-5.
[176] Swanson KC, Altare C, Wesseh CS, Nyenswah T, Ahmed T, Eyal N, et al. Contact tracing performance during the Ebola epidemic in Liberia,
2014
2015. PLoS Negl Trop Dis 2018;12(9):e0006762. Available from: https://doi.org/10.1371/journal.pntd.0006762.
[177] Tiffany A, Dalziel BD, Kagume Njenge H, Johnson G, Nugba Ballah R, James D, et al. Estimating the number of secondary Ebola cases
resulting from an unsafe burial and risk factors for transmission during the West Africa Ebola epidemic. PLoS Negl Trop Dis 2017;11(6):
e0005491. Available from: https://doi.org/10.1371/journal.pntd.0005491.
[178] World Health Organization. Interim infection prevention and control guidance for care of patients with suspected or confirmed filovirus hae-
morrhagic fever in health-care settings, with focus on Ebola; 2014. Available from: https://apps.who.int/iris/handle/10665/130596.
[179] Okware SI, Omaswa F, Talisuna A, Amandua J, Amone J, Onek P, et al. Managing Ebola from rural to urban slum settings: experiences from
Uganda. Afr Health Sci 2015;15(1):312
21. Available from: https://doi.org/10.4314/ahs.v15i1.45.
This page intentionally left blank
Chapter 11

SARS-CoV-2—host cell interactions and

pathways: understanding its physiology,
pathology, and targeted drug therapy
Rhea Conchita Gonsalves1, Himavani Pacharla2, Sai Manohar3, Siva Kumar Belliraj4, Ekta Tripathi5, Prashanthi
Karyala5 and Suresh B. Pakala6
1
Department of Biochemistry, Indian Academy Degree College—Autonomous, Bengaluru, India, 2Department of General Medicine, Apollo Hospital,
Hyderabad, India, 3Department of Chemistry, Sri Sathya Sai Institute of Higher Learning, Anantapur, India, 4Grey Scientific Labs, Visakhapatnam,
India, 5Department of Biotechnology, Faculty of Life and Health Science, Ramaiah University of Applied Sciences, Bengaluru, India, 6Biology
Division, Indian Institute of Science Education and Research (IISER) Tirupati, Tirupati, India

11.1 Introduction
Coronaviruses (CoVs) are a group of enveloped and nonsegmented positive-sense RNA viruses belonging to the family
of Coronaviridae of the order Nidovirales [1]. They are classified into four genera: alpha, beta, gamma, and delta. CoVs
can infect a varied range of hosts, from humans to other animals [2]. Human coronaviruses (HCoVs) were initially dis-
covered in the 1960s and currently, there are seven HCoVs that have been identified to date. All HCoVs are believed to
have emerged originally as zoonoses and infect humans by crossing species barriers [3]. HCoV-229E and HCoV-NL63
are α-CoVs and HCoV-OC43, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East
respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 are β-CoVs. Four of the HCoVs infecting humans
result in mild symptoms of cold. However, in recent times, three exceedingly pathogenic HCoVs, SARS-CoV, MERS-
CoV, and SARS-CoV-2, have emerged producing severe acute respiratory diseases with human-to-human transmission
and high morbidity and mortality [4].
In December 2019 World Health Organization (WHO) reported the presence of a cluster of pneumonia cases of
unknown etiology in Wuhan, China [5
7]. A novel coronavirus was subsequently identified as the causative pathogen,
provisionally named 2019 novel coronavirus (2019-nCoV) but later called SARS-CoV-2 [8]. On February 11, 2020,
WHO announced the rapidly spreading coronavirus disease as COVID-19 [9]. As of January 26, 2020, more than 2000
cases of COVID-19 infection were confirmed [10]; most of which included people living in or visiting Wuhan, and
human-to-human transmission was established [11]. WHO declared the COVID-19 outbreak a public health emergency
of international concern on January 30, 2020 [12] and a pandemic on March 11, 2020 [13]. As of December 30, 2020,
there have been more than 80 million confirmed COVID-19 cases, including more than 1,783,619 deaths in over 180
countries globally as reported to WHO [14].
Although investigation into the clinical features of SARS-CoV-2 has progressed at a fast pace, its pathogenesis is still
incompletely understood. The SARS-CoV-2 infection, disease, morbidity, and mortality are complex and unprecedented
with very few insights from prior research in other related infectious diseases. Therefore, the mechanisms behind the
pathogenesis of the SARS-CoV-2 require the utilization of high-throughput technologies. Owing to its emphasis on sys-
tems science, omics technologies are better poised to address the pandemic in ways that offer novel mechanistic insights
into the pathogenesis of life-threatening infectious diseases. In this background, the omics level studies are steadily being
employed to unravel the mysteries of the basic biology of SARS-CoV-2 and its interaction with the host cell and sys-
tems. Many studies have shown profound changes in host transcriptional and immunological changes in both in vitro and
patient studies. Understanding the host response to SARS-CoV-2 infection will finally help in obtaining a comprehensive

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00006-9

© 2021 Elsevier Inc. All rights reserved. 185
186 Pandemic Outbreaks in the 21st Century

picture of disease trajectory, identification of biomarker, and guide the development of therapeutics and vaccines against
this disease.

11.2 COVID-19 disease

11.2.1 History and epidemiology
In the past two decades, we have witnessed thrice the devastating effects of viral outbreaks on human population.
Several virus strains have been recognized and documented by WHO that exhibit human disease. Among these, the
genus CoVs, in the Coronaviridae family, pleomorphic enveloped viruses with single-stranded RNA (1ssRNA) genome
have known to have become the major pathogens of emerging respiratory disease outbreaks [15].
According to the International Committee on Taxonomy of Viruses, coronaviruses are classified under the order
Nidovirales, family Coronaviridae and subfamily Coronavirinae. Based on early serological and later genomic evidence,
Coronavirinae is divided into four genera of CoVs (Fig. 11.1): Alphacoronavirus (alpha-CoV), Betacoronavirus (beta-
CoV), Deltacoronavirus (delta-CoV), and Gammacoronavirus (gamma-CoV) [16,17]. Alphacoronavirus and
Betacoronavirus usually infect mammalians, whereas Gammacoronavirus usually infect birds [18,19].
Strains of CoVs that are human pathogens are referred to as human coronaviruses (HCo-V) [15]. Seven HCoVs
have been reported since the 1960s [15,20,21]. Four of them (OC43, 229E, NL63, and HKU1) cause mild symptoms
similar to those of common cold and gastrointestinal tract infections. The other three, SARS-CoV, MERS-CoV, and
SARS-CoV-2, belong to genus Betacoronavirus and are known to cause more severe symptoms [22
24].
In the month of November 2002, several patients were hospitalized presenting atypical pneumonia-like symptoms in
Guangzhou. Around February 2003, tests revealed positive results for SARS-CoV in the nasopharyngeal aspirates of
these patients and by March 2003, a new CoV was confirmed as the causative agent for SARS and thus was referred to
as SARS-CoV. The SARS epidemic ended in July 2003, with a total of 8098 cases and a mortality rate of 774 deaths
across 29 countries [25,26].
The Middle East respiratory syndrome (MERS) is a highly fatal respiratory tract disease in humans that was first
detected in 2012 in Saudi Arabia later spread to the surrounding countries. By March 2018, the WHO estimated a total
of 2189 human laboratory-confirmed cases from 27 countries have been reported including 782 associated deaths
[26,27]. Dromedary camels (Camelus dromedarius) have been shown to be the natural reservoir and thus it is theorized
that human MERS infection occurred as a spill-over from the infected camels [24
26,28
30,31].
SARS-CoV-2 emerged in Wuhan City, China, causing severe respiratory illness and mortality. In the month of
December 2019, several local health facilities reported clusters of cases presenting with pneumonia of unknown etiol-
ogy [6,7]. Following epidemiological analysis, the first four cases reported were linked to the Huanan Seafood
Wholesale in Wuhan, Hubei Province, China [32
34]. Thus, the Chinese Center for Disease Control and Prevention
(China CDC) dispatched a rapid response team to accompany Hubei provincial and Wuhan city health authorities and

FIGURE 11.1 Classification of coronaviruses. From Rehman

SU, Shafique L, et al. Evolutionary trajectory for the emergence
of novel coronavirus SARS-CoV-2. Pathogens 2020;9(3):240.
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 187

to conduct epidemiologic and etiologic investigations. A novel CoV was identified as the SARS-CoV-2 from the throat
swab sample of an infected patient on January 7, 2020 [35
38]. The spread of the SARS-CoV-2 globally across the
countries later led to a worldwide pandemic. On Wednesday, February 12, 2020, the WHO established the official
name of the new coronavirus from “Wuhan Virus” as “COVID-19” [39]. On March 11, 2020, the WHO announced the
outbreak of the novel coronavirus, or “COVID-19”, a pandemic worldwide [13].
The origin of SARS-CoV-2 remains unclear; however, comparative analysis of its genome with other CoVs shows
that SARS-CoV-2 is not a laboratory construct rather suggests that it has a zoonotic origin that underwent multiple muta-
tions before spillover to humans [25,40
42]. SARS-CoV-2 has been found to be 96% identical at the whole genome
level to the bat SARS-like coronavirus strain Bat CoV RaTG13, making it likely that bats served as reservoir hosts.
With many theories not supportive of direct spillover from bats to humans, further investigation was conducted [41].
Malayan pangolins (Manis javanica) illegally imported into Guangdong province were reported as potential intermediate
hosts as they can be infected by coronaviruses similar to SARS-CoV-2. Samples from the pangolins showed new corona-
virus genomes with 85.5%
92.4% resemblance to SARS-CoV-2. Even more remarkable was the 97.4% amino acid
similarity in the receptor-binding domain (RBD) of coronavirus genomes from pangolins compared to SARS-CoV-2. In
comparison, the Bat CoVRaTG only had 89.2% amino acid similarity in the RBD with SARS-CoV-2 [34,40].
According to the Weekly epidemiological update, the data as received by WHO from national authorities, as of
December 30, 2020, have reported over 5 lakh new cases in the past 24 h globally. As of December 30, 2020, nearly
80 million cases and 1,783,619 deaths have been reported globally. The further acceleration of the incidence of new
cases was most notable in the European Region. A substantial rise in deaths globally was also documented.

11.2.2 Transmission and course of infection

After the original infection of SARS-CoV-2 at the Huanan Seafood market, the virus has been known to be transmitted
by human-to-human interactions [33,43]. SARS-CoV-2 is predominantly transmitted through indirect or direct contact
with mucous membranes in the mouth, eyes, or nose with infectious respiratory droplets or fomites. Droplet transmis-
sion occurs through direct contact when a person is within 1 m of someone with respiratory symptoms including cough-
ing and sneezing [11,44]. Having their mucous membranes, including their mouth, nose, and eyes, exposed to the
droplets puts the individual at risk. Transmission can also occur through indirect contact by way of fomites on surfaces
in the immediate environment around the infected person as it is postulated that SARS-CoV-2 survives for many days
when dried on surfaces and in feces at an alkaline pH [44
46]. Studies have shown that infected people are most conta-
gious on the 10th day of illness as the viral load reaches its peak at that time [16].
Clinical symptoms have been shown to occur most commonly between days 4 and 5 after exposure. Infected persons
present initially with fever, myalgia, malaise, and chills or rigor, and dry cough [47,48]. Shortness of breath, tachypnea,
or pleurisy are prominent only later in the course of the illness while few patients reported gastrointestinal symptoms
such as nausea and diarrhea [49]. The severity of the illness ranges from mild to fatal. A study done by China CDC
showed that the majority of the cases were classified as mild disease and had either mild pneumonia or no pneumonia.
However, the cases classified as severe disease presented dyspnea with increased respiratory rate, low blood oxygen sat-
uration, reduced partial pressure of arterial oxygen to fraction of inspired oxygen ratio, and/or the development of dif-
fuse lung infiltrates. The critical patients showed respiratory failure, shock, and/or multiorgan failure that mostly led to
the death of the patient [5,50].

11.2.3 Pathogenesis
Although the exact mechanism of pathogenesis of SARS-CoV-2 has not been deciphered entirely, models of pathogene-
sis have been postulated based on similar human beta-coronaviruses: SARS-CoV and MERS-CoV due to their similari-
ties with SARS-CoV-2 [51]. A SARS disease model proposed consists of three phases: viral replication, immune
hyperactivity, and pulmonary destruction [52]. SARS-CoV-2 enters through the nasal or oral route and binds to
the epithelial cells followed by diffusion down the respiratory tract. SARS-CoV-2 is hypothesized to infect primary
ciliated cells as observed in SARS-CoV. Further progression of the virus can cause the infection of the alveolar type II
pneumocyte cells and cause cell apoptosis resulting in diffuse alveolar damage, severe scarring, and fibrosis. Type II
pneumocytes produce surfactants that are responsible for maintaining the surface tension in alveolar walls and also the
lung epithelium after injury through epithelial regeneration. Therefore, the resulting apoptosis eventually causes diffuse
alveolar damage and impaired gas exchange, which is hypothesized to lead to acute respiratory distress syndrome
(ARDS) [50
52].
188 Pandemic Outbreaks in the 21st Century

The virus is said to primarily infect epithelial cells within the lung but an abortive infection in the macrophages and
dendritic cells may induce pro-inflammatory cytokines that may contribute to disease. Pro-inflammatory cytokines
released by stimulated macrophages in the alveoli may have a role in the pathogenesis of the virus. This is based on the
cytokine deregulation hypothesis. Also, the activated helper T cells release cytokines and chemokines at high levels,
which leads to the development of cytokine storm syndrome (CSS) [53]. Acute lung injury, including its severe form
ARDS, is a common consequence of CSS. This has been shown to occur in patients with severe SARS-CoV-2 infection
with the development of diffuse lung injury, inflammation, and fluid buildup, which can ultimately lead to death.
In severe cases the SARS-CoV-2 can also attack several organs and organ systems leading to multiple organ damage
[50,54]. CoVs can easily penetrate the brain via the nerve cells and enter the central nervous system and cause damage
to the respiratory center. Neurological manifestation of CoV’s is associated with confusion, loss of the sense of smell
and taste, lethargy, disorientation, decreased consciousness, and acute necrotizing hemorrhagic encephalopathy [55
58].
Several studies have been conducted that associate COVID-19 with cardiac damage. Terminal cases of COVID-19 have
shown severe inflammation in the cardiac muscles. This inflammation also causes rhythm disturbances and muscle dam-
age. The presence of elevated levels of cytokines and inflammatory markers in patients with preexisting heart conditions
are associated with either apoptosis or necrosis of myocardial cells [59,60]. Studies revealed a loss of kidney functions
developed shortly with COVID-19 infection [61].

11.2.4 SARS-CoV-2 association with other comorbidities

SARS-CoV-2 causes disease in individuals of all age groups, nevertheless, it is becoming clear that people aged above
60 years and having comorbidities such as cardiovascular diseases, diabetes, and chronic respiratory disease have a
greater risk of developing disease and death from COVID-19. However, the precise role of different comorbidities in
disease severity is unclear. Across the world, it has been observed that the elderly possessing preexisting medical ail-
ments like asthma, diabetes, heart disease, and asthma are relatively more vulnerable to the coronavirus. Meta-analysis
of published literature showed that the prevalence of chronic comorbid conditions among COVID-19-infected popula-
tion with at least one underlying chronic comorbid condition (37%), hypertension (22%), diabetes (14%), cardiovascular
diseases (13%), respiratory disease (5%), and other chronic diseases (8%) [60
62].
Due to known impact of COVID-19 on the lung, it is natural to have fear for patients with underlying Chronic
obstructive pulmonary disease (COPD). In reports from China on hospitalized patients, the prevalence of COPD
was found to be between 0% and 10% [63]. Patients with hypertension, on therapy with ACE inhibitors or ARBs, are
at higher risk of morbidity and mortality if they become infected with SARS-CoV-2, although this is confounded by
additional factors such as vascular disorders and age [64
66]. Even though respiratory illness is the main clinical mani-
festation of COVID-19, analysis of a large number of patients with COVID-19 revealed that many had preexisting
cardiovascular diseases or develop new-onset cardiac dysfunction during the course of the illness [60,67,68]. Chronic
diseases have many clinical features with complications, including the pro-inflammatory state, and the decrease in
the innate immune response [69]. For example, diabetes mellitus arises in part due to buildup of activated innate
immune cells in metabolic tissues that result in the release of inflammatory mediators, predominantly, IL-1β and TNFα,
which progresses into systemic insulin resistance and β-cell damage [69]. Therefore chronic comorbid conditions of
COVID-19 patients presents main clinical challenges in terms of diagnosis, ill health, the course of treatment, and dis-
ease management, which poorly affects treatment choices and outcomes.

11.3 The molecular biology of SARS-CoV-2

11.3.1 Overview of SARS-CoV-2 replication cycle
The coronavirus replication cycle is divided into several steps: attachment and entry, translation of viral replicase,
genome transcription and replication, translation of structural proteins, and virion assembly and release (Fig. 11.2) [70].

11.3.1.1 Attachment and entry

The first step in a viral life cycle is the binding of the viral particle to the target cell. The SARS-CoV-2 has been shown
to use a surface glycoprotein, called spike (S) protein that recognizes the angiotensin-converting enzyme 2 (ACE2)
receptor on the host cell surface [71]. ACE2 receptors have been found in various organs and cells including the naso-
pharynx, nasal and oral mucosa, small intestine, colon, kidney, liver, vascular endothelium, and epithelial cells of lung
alveoli (mainly type II pneumocytes) [71,72]. The homo-trimeric spike glycoprotein comprises two subunits involved in
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 189

FIGURE 11.2 Replication cycle of human coronaviruses. Coronavirus particles bind to cellular attachment factors and specific spike protein (S)
interactions with the cellular receptors (such as angiotensin-converting enzyme 2, ACE2), together with other host factors (such as the cell surface ser-
ine protease, TMPRSS2), promote viral uptake and fusion at the cellular or endosomal membrane. Following entry, the release and uncoating of the
incoming genomic RNA occurs. The genomic RNA (gRNA) serves as the template for translation of polyproteins pp1a and pp1ab, which are cleaved
to form non-structural proteins (nsps). Nsps induce the rearrangement of cellular membrane to form double-membrane vesicles (DMVs), where the
viral replication transcription complexes (RTCs) are anchored. Full-length gRNA and a nested set of sub-genomic RNA (sgRNA) is synthesized via a
negative-sense intermediate through transcription. These sgRNAs encode viral structural and accessory proteins. Particle assembly occurs in the ER-
Golgi intermediate complex (ERGIC), and mature virions are released in smooth-walled vesicles via the secretory pathway. From V’kovski P, Kratzel
A, Steiner S, et al. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol 2020; 19:155
170. https://doi.org/10.1038/
s41579-020-00468-6.

the attachment and fusion of the virus to the host cell: S1 subunit contains a C-terminal RBD that recognizes the ACE2
receptor and S2 subunit. The interaction of the S1 subunit of the spike protein with the ACE2 leads to the transition of
the S2 subunit from a semistable perfusion state to a stable postfusion state [72,73]. Following the attachment, membrane
fusion occurs by acid-dependent proteolytic cleavage of S protein at two sites within the S2 subunit. These cleavages are
190 Pandemic Outbreaks in the 21st Century

catalyzed by host-encoded proteases: endosomal cysteine protease cathepsin L and trypsin-like serine protease
TMPRSS2 [74]. The first cleavage is important for separating the RBD and fusion domains of the S protein while the
second cleavage exposes fusion peptides [74,75]. Fusion generally occurs within acidified endosomes. The exposed
fusion peptides then insert themselves into the membrane, which is followed by joining of viral and vesicle membrane
ultimately resulting in the release of the viral genome into the cytoplasm [70].

11.3.1.2 Replicase protein expression

The release of the viral genome into the host cell cytoplasm is followed by the translation of the replicase gene from
the virion genomic RNA. The replicase gene consists of two large open reading frames (ORFs) that occupy two-thirds
of the viral genome: rep1a and rep1b that encodes two polyproteins pp1a and pp1ab, respectively. While translation
of pp1a the ribosome unwinds an RNA pseudo-knot structure to translate the rep1a gene until it encounters the stop
codon and terminates translation. In the case of pp1ab, the presence of a slippery sequence (50 -UUUAAAC-30 ) causes
the synthesis of a longer polypeptide. Occasionally when the ribosome encounters the pseudoknot, a pause on the slip-
pery sequence causes an a -1 frameshift and change in the reading frame by moving back one nucleotide before dissol-
ving through the knot. This frameshift causes the ribosome to bypass the stop codon resulting in a longer polyprotein,
pp1ab [22,75].
The autoproteolytic cleavage of pp1a and pp1ab generates 16 nonstructural proteins (nsps) with various functions;
pp1a and pp1ab contain the nsps 1
11 and 1
16, respectively. They are subsequently cleaved into the individual nsps
by viral-encoded proteases. These proteases include papain-like proteases (PLpro), encoded within nsp3, and a serine-
type protease, the main protease, or Mpro, encoded by nsp5 (Table 11.1; Fig. 11.3). The PLpro cleaves the nsp1/2,
nsp2/3, and nsp3/4 boundaries, while the Mpro is responsible for the remaining 11 cleavage events. Many of the nsps
assemble to produce a replicase
transcriptase complex [76,77].

TABLE 11.1 Various nonstructural proteins formed due to the cleavage of polypeptide 1a and 1ab and their functions.
From Fehr A.R., Perlman S., Coronaviruses: an overview of their replication and pathogenesis Springer New York
New York, NY 2015 1-23 [1].

Protein Function
nsp1 Promotes cellular mRNA degradation and blocks host cell translation, results in blocking the innate immune response
nsp2 No known function binds to prohibitin proteins
nsp3 Large, multidomain transmembrane protein activities include:G Ubl1 and Ac domains interact with N proteinG ADRP
activity promotes cytokine expressionG PLPro/deubiquitinase domain cleaves viral polyprotein and blocks host innate
immune responseG Ubl2, NAB, G2M, SUD, Y domains, unknown functions
nsp4 Potential transmembrane scaffold protein, important for proper structure of DMVs
nsp5 Mpro cleaves viral polyprotein
nsp6 Potential transmembrane scaffold protein
nsp7 Forms hexadecameric complex with nsp8 may act as processivity clamp for RNA polymerase
nsp8 Forms hexadecameric complex with nsp7 may act as processivity clamp for RNA polymerase; may act as primase
nsp9 RNA binding protein
nsp10 Cofactor for nsp16 and nsp14 forms heterodimer with both and stimulates ExoN and 2-O-MT activity
nsp12 RdRp
nsp13 RNA helicase, 50 triphosphatase
nsp14 N7 MTase and 30 -50 exoribonuclease, ExoN; N7 MTase adds 50 cap to viral RNAs, ExoN activity is important for
proofreading of viral genome
nsp15 Viral endoribonuclease, NendoU
nsp16 20 -O-MT; shields viral RNA from MDA5 recognition
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 191

FIGURE 11.3 The genome of SARS-CoV-2 showing transcription sites and protein coding domains. Genes coding for structural elements of the
SARS CoV-2 include the Spike (S) protein responsible for attachment of the virion to the host cell and entry; a membrane (M) protein that is responsi-
ble for membrane formation and assembly; an envelope protein E; and a nucleocapsid N. ORF1a and ORF1b form two polypeptides; pp 1 a and pp
1ab that on cleavage produce several nonstructural proteins (nsp 1-16). These non structural proteins are necessary for the production of genomic
RNA, structural proteins and several accessory proteins necessary for the assembly of the daughter virions. From https://viralzone.expasy.org/
resources/nCoV_genome_bis.png.

11.3.1.3 Replication and transcription

SARS-CoV-2 has an enzyme called the RNA-dependent RNA polymerase, which, along with other viral and cellular
proteins, comprises the main replication complex responsible for replicating the viral genome. Using the genomic RNA
as a template, the viral RNA synthesis produces a negative-sense antigenome, which in turn serves as a template for the
synthesis of new genomic RNA. Transcription termination occurs at transcription regulatory sequences (TRS), located
between ORFs that work as templates for the production of subgenomic mRNAs. Shifting of frames during discontinu-
ous transcription at TRSs produces a set of nested negative-sense subgenomic RNAs (
sgRNA). These
sgRNA act as
templates for the synthesis of positive-sense sgRNAs (1sgRNA) [78,79].

11.3.1.4 Translation of structural proteins

The 1 sgRNAs encode for several membrane proteins, N protein, and a variety of accessory proteins. Most of the
sgRNAs are functionally monocistronic. However, some employ other mechanisms to translate additional ORFs.
Transmembrane structural proteins (S, M, and E) and some membrane-associated accessory proteins are translated in the
ER, whereas the N protein is translated by cytosolic free ribosomes, which fuse into the endoplasmic reticulum
Golgi
intermediate compartment (ERGIC). Most coronavirus structural proteins are subjected to posttranslational modifications
that modulate their functions. The membrane proteins are inserted into the rough endoplasmic reticulum (ER) and are
transported to the ERGIC, where the N proteins along with genomic RNA, then, form nucleocapsids [80].

11.3.1.5 Assembly and release

Particle assembly occurs in the ERGIC and is orchestrated by the M protein. M-S and M-N interactions facilitate the
recruitment of structural components to the assembly site. M protein interacts with E protein to form virus-like proteins
192 Pandemic Outbreaks in the 21st Century

(VLPs) suggesting their function to produce the virion envelope. The N protein enhances the formation of VLP, fusion
and encapsulation of the viral genome into the ERGIC. The S protein is incorporated into the virions at this step. The
M protein also binds to the nucleocapsid completing the virion assembly. Following assembly, virions are transported
to the cell surface by smooth-walled vesicles and trafficked via the secretory pathway for release by exocytosis [81,82].

11.3.1.6 Elements of SARS-CoV-2 genome

CoVs are enveloped, positive-stranded RNA viruses with nucleocapsid with the largest known RNA genomes. In CoVs,
the genomic structure is organized in a 1 ssRNA of approximately 30 kb in length with a 50 -cap structure and 30 -poly-A
tail. Approximately, the first two-thirds of the 26
32 kb encodes for a large polyprotein (ORF1a/b) that is proteolyti-
cally cleaved to generate 15 or 16 nonstructural proteins (nsps) (Fig. 11.3). The 30 -end of the genome encodes four struc-
tural proteins—spike (S), membrane (M), envelope (E), and nucleocapsid (N) along with a set of accessory proteins.
The genome has a 50 -UTR, ranging from 210 to 530 nucleotides and a 30 -UTR, ranging from 270 to 500 nucleotides.
There is a frameshift element which is an RNA structural motif that incorporates nucleotides of the overlapping frames
of ORF1a and ORF1b. It consists of two stable hairpins: the first of which contains a loop sequence that forms a pseudo-
knot [82].

11.3.1.7 Viral proteins and their function

11.3.1.7.1 Nonstructural proteins
The replicase gene consists of two large ORFs: rep1a and rep1b. rep1a and rep1b together occupy two-thirds of the viral
genome that gives rise to 16 nsps (Table 11.1; Fig. 11.3). These nsps are not directly incorporated into the new virion
but are necessary for the synthesis and assembly of the genomic and components that will be incorporated into the
virion as well as various accessory proteins [1,77,82].

11.3.1.7.2 Structural proteins

Besides ORF1a and ORF1b, the rest one-third of the viral genome can be divided into another set of ORFs that
encode for several structural and accessory proteins required for the assembly and release of the newly synthesized vir-
ions [1,76].

11.3.1.7.3 Spike protein

The coronavirus spike protein is a type I glycoprotein that forms the peplomers on coronavirus particles. It is a large
N-exo, C-endo transmembrane protein that assembles into trimers to form the distinctive inactive surface spikes. The
larger protein is cleaved into two subunits by a furin-like enzymatic activity during processing in the Golgi and inserted
into the ER via the cleaved, amino-terminal signal peptide (Fig. 11.4). The ectodomain makes up most of the molecule,
with only a small carboxy-terminal segment constituting the transmembrane domain and endodomain. The amino-
terminal S1 subunit, which is believed to form the globular head of the mature protein, contains a RBD. The carboxy-
terminal S2 subunit is believed to form a stalk-like structure anchored in the membrane. It contains two heptad repeat
domains as well as the putative fusion peptide. Viral attachment triggers a conformational change in the spike protein
that promotes the fusion of viral and cellular membranes [53,70,73].

11.3.1.7.4 Nucleocapsid
The N polypeptide is the protein part of the helical nucleocapsid component of the virus. N protein is divided into three
conserved domains, separated by two spacer regions (Fig. 11.5). Domains 1 and 2 occupying the majority of the poly-
peptide are rich in arginine and lysine residues. The short, carboxy-terminal domain 3 has a net negative charge due to
the presence of acidic amino acids. The coronavirus ribonucleoprotein complexes have been shown to be sensitive to
the action of ribonucleases. The N protein is also important for encapsidation of viral RNA and acts as an interferon
(IFN) antagonist [77,83
85].

11.3.1.7.5 Membrane protein

The M polypeptide is a transmembrane protein with a small, amino-terminal domain that faces toward the exterior of
the virion. The ectodomain is followed by three helical transmembrane segments. A large carboxy terminus compro-
mises the major part of the molecule and is situated toward the interior of the virion (Fig. 11.5). It is theorized that lat-
eral interactions between M protein monomers are the driving force for virion envelope formation [82].
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 193

FIGURE 11.4 Structural diagrams of spike glycoproteins of SARS-CoV, MERS-CoV, and SARS-CoV-2. All spike proteins of coronaviruses contain
S1 subunit and S2 subunit, which are cleaved at the S cleavage sites. The S protein has the following domains: FP, fusion peptide; HR, heptad repeat
1 and heptad repeat 2; RBD, receptor-binding domain, contains core binding motif in the external subdomain; and SP, signal peptide. (A) From
Masters PS. The molecular biology of coronaviruses. In: Advances in virus research, vol. 66. Academic Press. p. 193
292. (B) From Liu Z, Xiao X,
Wei X, et al. Composition and divergence of coronavirus spike proteins and host ACE2 receptors predict potential intermediate hosts of SARS-CoV-2.
J Med Virol 2020;92:595
601.

FIGURE 11.5 Structure of the protein domains of the envelope

protein E, Membrane protein M, and nucleocapsid protein N.
From Masters PS. The molecular biology of coronaviruses. In:
Advances in virus research, vol. 66. Academic Press. p. 193
292.

11.3.1.7.6 Envelope protein

The Envelope (E) protein has been proposed to induce membrane curvature in the ERGIC, so that it can act to pinch
off the neck of the viral particle in the final stage of the budding process. It has a transmembrane domain toward the N
terminal (Fig. 11.5) [1,82].
194 Pandemic Outbreaks in the 21st Century

11.4 Identifying SARS-CoV-2 host cell interface, host dependency factors and
cytokine storm: high-throughput and low-throughput approaches
11.4.1 Requirement of host factors for SARS-CoV-2 life cycle
Viruses are obligate intracellular parasites that have limited genomic capacities, therefore, have evolved to hijack host
factors to facilitate their replication. In response to infection, host cells have also developed intricate signaling pathways
to detect and control viruses, although these antiviral networks are further evaded by various viral counter mechanisms.
The host factors are required at every step of viral life cycle including viral entry, replication, assembly, and release
[86]. The sudden emergence of SARS-CoV-2 requires an in-depth understanding of viral
host interactions to identify
critical viral and host factors that regulate the COVID-19 pathology and to develop effective therapeutic strategies
against SARS-CoV-2 infection.
The interactions between the virus and host proteins are essential for viral pathogenesis and host immune response
which ultimately determine the disease outcome. The first essential step in the viral replication cycle is the entry of
virus into the target cell. The binding of the viral surface protein to the cell surface receptor is the major determinant of
host range and cell tropism. The RBD in S1 domain of S protein of SARS-CoV-2 binds to the cell surface receptor and
the transmembrane S2 domain mediates the fusion between the viral envelope and cellular membrane upon conforma-
tional changes. Different coronaviruses utilize different cell surface proteins as receptors. SARS-CoV, SARS-CoV-2,
and NL63 use ACE2, MERS-CoV uses dipeptidyl peptidase-4 (DPP4), while HCoV-OC43 and HCoV-HKU1 use 9-O-
acetylated sialic acid as a receptor.
Besides receptor binding, the proteolytic cleavage of S protein into S1/S2 is mediated by one or more host proteases
such as cell surface serine protease, TMPRSS2, and endosomal cysteine protease cathepsin B/L during viral entry and
membrane fusion. The inhibition of TMPRSS2 efficiently prevents SARS-CoV-2 entry in lung cell lines and primary
lung cells [85]. Moreover, S protein of SARS-CoV-2 has acquired a polybasic cleavage site (RRAR) at the S1
S2
boundary, which is cleaved by another host protease furin. This cleavage results in enhanced infection and contributes
to the increased cell tropism and zoonotic potential of SARS-CoV-2 [85,87]. Such motifs can bind and activate
Neuropilin (NRP-1 and NRP-2) cell surface receptors. Two groups discovered independently that SARS-CoV-2 uses
NRP-1 as an alternative doorway for entry and infection in human cells [88,89]. Interestingly, gene expression analysis
in lung tissue from COVID-19 patients revealed an upregulation of NRP1 and NRP2 receptors. NRP-1 may serve as a
potential target for therapeutic intervention [88].
Upon entry, the viral RNA is released into the host cytoplasm and it utilizes both host and its own machinery for
replication, translation, and assembly. The successful completion of virus life cycle depends on interactions with host
proteins that are repurposed to meet the requirements of the virus. This includes host factors required for virus entry
(host cell receptor and host proteases), viral RNA synthesis and virus assembly (protein associated with ER/Golgi and
vesicular trafficking), and translation of viral mRNAs (host translational initiation factors) [87].
The host cells detect viral RNA using different cytosolic sensors and recruit an innate antiviral response to limit the
spread of the infection. In case of SARS-CoV-2 infection, the viral RNA is recognized by sensors like RIG-I/MDA-5/
TLRs, which begins a signaling cascade, ultimately leading to the production/activation of interferons and nuclear fac-
tor kappa B (NF-kB) resulting in an antiviral state of the infected cells. On the other hand, the viruses have evolved
strategies to evade the host immune responses, which influence the pathogenesis and disease course. The viral proteins
modulate cellular translation in favor of viral mRNAs over cellular mRNAs and decrease the expression of host
immune response proteins. Indeed, SARS-CoV-2 nsp1 protein binds to ribosomes and mediates impairment of transla-
tion [90]. Furthermore, SARS-CoV-2 modulates the transcriptional landscape of infected cells by inducing inflamma-
tory cytokine and chemokine signatures [91].
Advanced technologies, such as transcriptomics, proteomics, metabolomics, clustered regularly interspaced short palin-
dromic repeats (CRISPR), single-cell RNA sequencing, and global single-cell profiling of patient samples, have been valu-
able tools to understand the pathophysiology of virus, host
virus dependencies, and combat SARS-CoV-2 infections
(Fig. 11.6 and Fig. 11.7). Using these high-throughput techniques, hundreds of host proteins and pathways have been iden-
tified that are implicated in SARS-CoV-2 infection. Furthermore, identification and characterization of the critical viral
and host factors that promote SARS-CoV-2 replication will guide the development of efficient and targeted therapeutics.

11.4.2 Analysis of the viral transcriptome of SARS-CoV-2

Next-generation sequencing technologies based on “sequencing-by-synthesis” methods such as MGI and Illumina plat-
forms provide a powerful means to investigate viral transcriptome but are limited by short read length. More recently, a
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 195

FIGURE 11.6 “Omics” technologies are primar-

ily aimed at the study of genes (genomics), mRNA
(transcriptomics), proteins (proteomics), and meta-
bolites (metabolomics) in a specific cell or organ-
ism. Adapted from Wang Z, et al. Chapter 15:
Metabolomics, proteomics, and genomics: an
introduction to a clinician; 2019. p. 159
170.

FIGURE 11.7 CRISPR-Cas9 sys-

tem consists of a guide RNA mole-
cule that guides the Cas9 nuclease
to a gene sequence inducing a site-
specific cleavage. DNA damage is
then repaired by cellular DNA
repair pathways—non-homologous
end-joining repair (NHEJ) or
homology-directed repair (HDR).
The NHEJ pathway is efficient but
error-prone, introducing insertions
and deletions that may affect gene
function.

novel approach called nanopore-based direct RNA sequencing (dRNAseq) was discovered. It enables long-read
sequencing, which can be used for the SARS-CoV-2 transcript analysis, although less accurately. Moreover, RNA mod-
ification information can also be obtained directly by dRNAseq. This technique allows individual RNAs to be
sequenced directly without the amplification bias as in other sequencing methods. In this method, individual strands of
RNA translocate through a nanopore and the electric current is measured to infer the individual bases.
Two independent groups [92,93] mapped the transcriptome and epitranscriptome (RNA base modifications) archi-
tecture of SARS-CoV-2 using dRNAseq. The study showed that the viral transcriptome is highly complex due to dis-
continuous transcription events and identified novel RNA modification sites in viral transcripts [93]. SARS-CoV-2
undergoes frequent recombination events which may allow them to evolve and change their host specificity rapidly.
The evolutionary data generated provided new insights into the biology and evolution of this emerging pathogen [92].
The comprehensive mapping is a prerequisite for the investigation of viral replication mechanism, host
viral interac-
tions, pathogenesis, evolution, molecular diagnostics, and public health responses.
196 Pandemic Outbreaks in the 21st Century

11.4.3 Identification of virus
host proteome and interactome using high-throughput technologies

An in-depth study of SARS-CoV-2 protein
protein interaction (PPI) was performed using tandem mass spectrometry
which identified 332 PPIs between SARS-CoV-2 protein and human proteins that were associated with various com-
plexes and biological processes [94]. Briefly, all the SARS-CoV-2 proteins (26 out of 29 proteins) were expressed in
HEK293T cells and affinity purified using Strep-tag followed by mass spectrometric analysis. The human proteins of the
SARS-CoV-2 human interactome are involved in major cellular processes such as DNA replication, epigenetic and gene
expression regulation, vesicle trafficking, lipid metabolism, and RNA processing. Several SARS-CoV-2 proteins (NSP2,
NSP6, NSP7 NSP8, NSP10, ORF8, M, and NSP13) interact and modify ER and Golgi components for its replication.
Moreover, many innate immune signaling pathway proteins were also targeted by SARS-CoV-2 viral proteins. The viral
proteins, NSP13, NSP15 and ORF9b, modulate the IFN and NF-kB response. They also identified 66 host factors that
were druggable and targeted by various compounds. Some of the compounds screened showed antiviral activity [94].
Another study by the same group conducted a mass spectrometric-based phosphoproteomic analysis of SARS-CoV-
2 in Vero E6 cells which revealed significant rewiring of signaling networks [95]. The phosphorylation profile revealed
large changes in phosphorylation status of host proteins due to altered activities of kinases. SARS-CoV-2 infection acti-
vated p38 MAPK signaling and caused cell cycle arrest. Upregulation of CK2 cytoskeleton-related targets stimulated
a marked induction of filopodial protrusions with budding viral particles which allow cell
cell spread. Cells were
arrested in S/G2 phase which provides benefits for viral replication by ensuring an abundant supply of nucleotides and
other essential host proteins. Eighty-seven drugs and compounds were identified that targeted host kinases and most of
them possessed antiviral efficacy, representing potential COVID-19 therapies [95].
A study by Stukalov et al. utilized the multilevel proteomic approach and investigated the influence of SARS-CoV
and SARS-CoV-2 on transcriptome, proteome, ubiquitinome, and phosphoproteome of a lung-derived human cell line
(A549). This systematic interactome and proteome profiling revealed common and unique characteristics in the patho-
genicity and transmission capabilities of each strain which can be targeted with drugs [96]. Bojkova et al. developed a
SARS-CoV-2 - CaCo-2 cell infection model which was used to analyze the quantitative proteome profiles of the cellu-
lar response after infection with SARS-CoV-2. These analyses revealed that SARS-CoV-2 modulates various host
cell pathways such as translation, splicing, carbon metabolism, protein homeostasis, and nucleic acid metabolism. This
study enabled the identification of host genes that can be targeted by small-molecule inhibitors [97]. Consistent with
these data, shotgun proteomic analysis of the host cell proteome following SARS-CoV-2 infection in Vero cells, identi-
fied RNA modification proteins, such as spliceosome components, and proteins involved in carbon metabolism, further
supporting that splicing is required for replication of SARS-CoV-2, and thus a potential target [98].
Multiomic characterization at the level of transcriptome, proteome, and phosphoproteome was performed in Vero
E6 cells infected with SARS-CoV-2, revealing new and critical aspects of S glycoprotein of SARS-CoV-2 [99]. This
integrated analysis identified an eight aa deletion in the furin cleavage site of S protein that potentially affects protein
cleavage, cell tropism, and infectivity. All factors identified through various studies have extended our knowledge about
the metabolic and immune imbalance induced during the viral infection.
Functional analysis of virus
host interactome was carried out in order to develop a network-based model to predict
new targets for SARS-CoV-2 diagnostics and therapeutics [100]. Based on PPI and gene expression data obtained
through public databases, network analysis was done to describe the interactome of coronavirus S protein and host pro-
teins. The host interactome with S protein mainly highlighted innate immunity pathway components, such as Toll-like
receptors, cytokines, and chemokines. CoVex is an online platform that integrates SARS-CoV-2—host interactions and
drug
target interactions for network-based prediction of drug candidates [101].

11.4.4 Identification of virus
host dependency factors using CRISPR/Cas9 technology

CRISPR is a powerful technique for editing genomes. CRISPR-Cas9 system consists of the enzyme Cas9 which can be
directed to cut any DNA sequence using guide RNA (gRNAs) that binds to complimentary target DNA. Once the DNA
is cut, the error-prone DNA repair machinery of cell (Non-homologous end joining, NHEJ) works to introduce muta-
tions (deletions or insertions) in the genome which could disrupt a gene (Fig. 11.7).
Genome-wide screens using CRISPR/Cas9 technology are powerful methods for identification of host factors
required for infection by different viruses. In the study by Daniloski et al. a genome-scale CRISPR loss-of-function
screen was conducted to identify key host genes required for SARS-CoV-2 in human lung epithelial cells (A549) over-
expressing ACE2 [102]. The GeCKOv2 CRISPR Cas9 library containing gRNAs against 19,050 genes in the human
genome was used. The screen was based on identifying genes whose loss confers resistance to SARS-CoV-2 viral
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 197

infection (better survival). A549 cells transduced with the library were infected with the SARS-CoV-2 virus and postin-
fection, genomic DNA was extracted and gRNA was sequenced from the surviving cells. The 30 out of 200 genes iden-
tified in the screen were validated and were essential for initial attachment and endocytosis, spike protein cleavage and
viral membrane fusion, endosome recycling, ER-Golgi trafficking, and transcriptional modulators. Some of the top-
ranked genes from the screen had direct PPI with different viral proteins. For example, ATP6AP1 and ATP6V1A (subu-
nits of the vacuolar-ATPase proton pump), interact with SARS-CoV-2 nonstructural protein 6 (nsp6) and membrane
(M) protein, respectively [102]. Interestingly, similar genes were identified in CRISPR screens for Zika virus and pan-
demic H1N1 avian influenza (IAV) essential for acidification and endosomal processing. Furthermore, to understand
the mechanisms underlying how individual genes identified in the screen drive infection resistance, they utilized the
Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing (ECCITE-seq) method
which couple CRISPR with a single-cell transcriptomic and proteomic readout. ECCITE-seq experiment resulted in
transcriptomic shifts upon target gene perturbation. Loss of six genes—ATP6AP1, ATP6V1A, CCDC22, NPC1,
PIK3C3, and RAB7 (members of the endosomal entry pathway) had a similar transcriptional signature, that is, upregu-
lation of pathways affecting lipid and cholesterol homeostasis [102].
Zhu et al. also performed an independent genome-scale CRISPR screen for SARS-CoV-2 infection in ACE2 over-
expressing A549 cells but using a different CRISPR library [103]. These two studies have substantial overlap in their
screens. Limitation of these studies was using ACE2 overexpression in the screen. Furthermore, a genome-wide screen
was performed with SARS-CoV-2, SARS-CoV, and MERS-CoV in Vero-E6 cell line (African green monkey cell line).
Vero-E6 cells were chosen based on their susceptibility to infection with both SARS-lineage (SARS-CoV and SARS-
CoV-2) and MERS-CoV viruses and endogenous expression of ACE2 and DPP4 receptors enabling direct comparisons
across all three strains of coronaviruses. The screen was based on a survival assay that confers either resistance (pro-
viral) or sensitization (antiviral) when targeted by gRNAs. The genes identified were involved in diverse biological
processes including chromatin remodeling, histone modification, cellular signaling, and RNA regulation. The genetic
screen identified pro-viral factors such as HMGB1 and the SWI/SNF chromatin remodeling complex as well as host
proteins required for viral entry and the antiviral factors such as IFN-stimulated gene LY6E [104].
A parallel genome-wide CRISPR screen was conducted in cells infected with SARS-CoV-2 and three other common
cold coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E) to compare and contrast virus-specific and pan coro-
navirus host requirements. This approach identified multiple genes and pathways such as glycosaminoglycan biosynthe-
sis, sterol regulatory element-binding protein signaling, glycosylphosphatidylinositol biosynthesis that support infection
by all three coronaviruses [105]. In addition, Transmembrane Protein 41B (TMEM41B) was critical for all corona-
viruses infection, thereby, making it a high-priority and broad-spectrum potential target for drug development. In a fol-
low up study, the authors showed that TMEM41B is required for all flavivirus infection for use in autophagy [106].

11.4.5 Identification of host factors in COVID-19 patient samples

Although the genome and proteome characterization of SARS-CoV-2 provides valuable information regarding the structure
of the virus and host
protein interactions, understanding how the infection alters the metabolic homeostasis in COVID-19
patients will provide better information to develop novel diagnostic and therapeutic strategies. A number of transcriptomic
and proteomic studies on cells, tissues, fluids, or blood samples from COVID-19 patients have provided valuable insights
into the expression of SARS-CoV-2 receptors, coreceptors, immune responses, as well as risk factors for comorbidities.
Transcriptome sequencing of bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) of
COVID-19 patients provided an association between COVID-19 severity and increased levels of certain cytokines, for
example, CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B [107]. This study also found that activation of
apoptosis and the p53 signaling by SARS-CoV-2 infection could be the cause of lymphopenia in the patients.
A comparison of transcriptional signature of COVID-19 patients to that of individuals infected with SARS-CoV or
IAV (Influenza A virus) revealed higher expression of genes involved in metabolic pathways (heme biosynthesis, oxida-
tive phosphorylation, and tryptophan metabolism) in COVID-19 patient samples but no significant change in expression
levels of type I IFN pathway genes. This suggests a critical role of mitochondrial activity during SARS-CoV-2 infec-
tion. Metatranscriptome sequencing and functional analysis of BALF of COVID-19 patients showed distinct patterns
of host innate immune response compared to community-acquired pneumonia patients, and healthy controls [108]. The
expression of pro-inflammatory genes, especially chemokines, was elevated in COVID-19 patient samples suggesting
the causes of hypercytokinemia by SARS-CoV-2 infection.
To understand the molecular basis of SARS-CoV-2 severity and poor outcomes in chronic lung disease patients, an
integrated analysis of the single cell transcriptomes from 605,904 single cells derived from from COVID-19 patients,
198 Pandemic Outbreaks in the 21st Century

healthy and diseased human lungs was performed [109]. Interestingly, the two main host entry factors, ACE2 and
TMPRSS2, showed similar expression profiles in the diseased and control samples. However, the diseased cells were
associated with altered gene expression regulating viral infection and immune response. A single-cell RNA expression
map using 28 sets of genes referred as SCARFs (SARS-CoV-2 and coronavirus-associated receptors and factors) was
generated across a wide range of healthy tissues. Consistent with the clinical data, specific cell types of intestine, pla-
centa, and kidneys appear more susceptible to SARS-CoV-2 infection [110].
Shen et al. performed proteomic and metabolomic profiling of COVID-19 patient sera using stable isotope-labeled
proteomics strategy TMTpro (16plex) and ultraperformance liquid chromatography/tandem mass spectrometry untar-
geted metabolomics approach. The study showed a dysregulation of macrophage, platelet degranulation, complement
system pathways, and massive metabolic suppression [111]. Another study by Li et al. used quantitative proteomics to
investigate the PBMC proteomic profile from COVID-19 patients, revealing an imbalance in neutrophil activation by
nsp8-NKRF interaction and IFN signaling [54]. Accordingly, other studies have also demonstrated changes in neutro-
phil, platelet degranulation, and the immune response within nasopharyngeal and urine samples derived from COVID-
19 patients [112,113]. All these studies are consistent with the association between immune dysfunction and outcome
of disease severity in patients with COVID-19 [114].
All the datasets generated from transcriptomic, proteomic, and CRISPR analysis in cell culture model or COVID-19
patients provide a valuable resource for understanding the molecular mechanisms of host response and guidance for
treatment strategies.

11.4.6 Cytokine storm and COVID-19

Cytokine storm syndromes or hypercytokinemia or CSS is an activation cascade of pro-inflammatory cytokines due to
unregulated host immune response system to different stimuli such as infections, malignancy, rheumatoid disorders,
drugs, and so on [114
116]. The main characteristic feature of CSS is feed forward activation and amplification of
host immune system, resulting in excessive release of wide range of cytokines and chemokines such as IFN-gamma,
TNF-alpha, IL-1, IL-6, IL-8, CXCL10, and CCL2, which contributes to the onset of cytokine storm [117].
Accumulating evidence revealed that CSS is one of the important and deadly obstacle in COVID-19 patients. COVID-
19 patients possess significantly high levels of inflammatory parameters, including C-reactive protein, ferritin, and pro-
inflammatory cytokines, indicating cytokine storm in severe COVID-19 patients [118,119]. In some patients, there were
elevated levels of D-dimer and myocardial dysfunction, which is again partly associated with cytokine storm [120]. In
addition to lung injury, liver, renal injury, and multiorgan failure were also observed due to cytokine storm in COVID-
19 patients [119,121,122]. Furthermore, the number of WBC, neutrophils, procalcitonin, C-reactive protein, and other
inflammatory manifestations are significantly higher in the intensive care unit (ICU) patients compared to non-ICU
patients [123]. Several studies reported that there were higher levels of pro-inflammatory cytokines, especially IL-6 and
lower levels of antiinflammatory cytokines such as IL-10 in severely ill patients than moderately ill patients
[119,124
127]. The transcriptome sequencing analysis of BALF cells of SARS-CoV-2-infected patients revealed
excessive release of chemokines such as CXCL10, CCL2, CCL3, and CCL4 [107]. Furthermore, high level of cytokines
in COVID-19 patients is associated with poor prognosis of the disease [125,128]. Observed ARDS (Acute Respiratory
Distress syndrome) and T-cell overactivation in patients who died with COVID-19, this might be due to an increase in
the number of T-helper 17 cells and high cytotoxic effect of CD8 1 T cells [129]. Also, the innate and adaptive immune
responses induced by the SARS-CoV-2 infection led to uncontrolled inflammatory responses and in turn the cytokine
storm [126]. The resulted cytokine storm brings apoptosis of epithelial cells and endothelial cells, vascular leakage, and
finally result in ARDS and sometimes death [130].
Overall, SARS-CoV-2 infection selectively induces high-level secretion of IL-6 and results in the exhaustion of
lymphocytes, and hence use of tocilizumab, an inhibitor of IL-6 is effective and safe. Also, corticosteroids, cytoki-
ne
adsorption devices, intravenous immunoglobulins, and antimalarial agents could be potentially useful and reliable
approaches to counteract CSS in COVID-19 patients [116]. Targeting the cytokines during the treatment of COVID-19
patients improves the survival rates and reduces the risk of mortality.

11.4.7 Chemical compounds virtual screening

Computer-aided drug discovery (CADD) is a relatively modern platform that has affected manifold, the design of drugs
most notably via rapid screening. As captured in Fig. 11.8, the computational methodologies are today, a key compo-
nent of drug discovery—right from hit identification to lead optimization, employing a holistic rationale to ascertain all
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 199

FIGURE 11.8 The contribution

of in silico methods in the various
stages of a drug design process.

the facets of the drug. The value of CADD is most evident in the backdrop of an emergency/pandemic-like
Coronavirus as there is a race against time and resources to design therapeutics and vaccines to mitigate the damage to
life. As expected, this tool has served as an enormous aid to the current COVID-19 crisis.
The Bioinformatics’ tools of Homology modeling have enabled target design via assessment of the crystallized
protein-ligand complexes for accurate characterization of the binding site. This has in turn increased the efficacy of
high-throughput molecular docking studies for in silico screening. All of these aspects together have helped evolve the
in silico techniques of 3-D QSAR and molecular docking for gaining mechanistic insight into the interaction of small
molecules for potential leads [131
133].
One of the first and most effective strategies has been drug repurposing. While the receptors for docking are avail-
able from the X-ray crystal structures in Protein Data Bank (http://www.rcsb.org), the exhaustive database of chemical
compounds for virtual screening is present in DrugBank (https://go.drugbank.com/) and Zinc Natural Product database
[134]. Some of the early in silico studies probed the repurposing of antivirals [135]. They have been chiefly evaluated
for their binding interactions with SARS-CoV-2: Mpro, SARS-CoV-2: 3CLpro, SARS-CoV-2 papain-like protease
(PLpro), and the viral S protein
human ACE2 interface. While there is an exponentially increasing number of
these studies on a monthly basis, these are but a glimpse of the strategy employed effectively. There have also been in
silico studies to probe the efficacy of antimalarials, antiparasitics, and antibiotics [136]. Today, there are several clinical
trials conducted worldwide by WHO and major pharmaceutical companies on a combination of one or more of these
drugs.
Furthermore, a combination of CADD and high-throughput screening provides new fingerprint molecular descriptors
and recognition algorithms to develop drugs [137]. In recent times machine learning (ML) and artificial intelligence
(AI) have demonstrated the ability to generate novel leads of required chemical and pharmacological properties
[138
140]. AI and ML provide adequate support in identifying drugs with potency against the Coronavirus and help
overcome the probable barriers that exist between repurposed drugs, extensive laboratory/clinical testing, and the final
approval of drug. It makes easy the perusal and analysis of the vast amount of data provided by various health bodies
that are in open access to the public at large [141].
As presented in the Fig. 11.9, we require the Repurposed Drugs’ Database, an Open Drug/Chemical Database as
inputs to the modeling. Consequently, a drug is designed by optimization using rigorous algorithms on these inputs
[142]. The merit of this protocol is affirmed in the vast number of major companies like Benevolent AI in the United
Kingdom to Deargen in Korea [143].
200 Pandemic Outbreaks in the 21st Century

FIGURE 11.9 The drug repurposing using AI. ML, Machine learning; DL, deep learning;
RNN, repetitive neural networks; CNN, convolutional neural networks; DBN, deep belief net-
works. From Mohanty S, AI Rashid H, et al. Application of Artificial Intelligence in COVID-
19 Drug Repurposing. Diabetes Metab Syndrome Clin Res Rev 2020;14(5):1027
1031.

FIGURE 11.10 The representative

mechanism of entry and replication
of the viral particles inside the host
cell. The host proteins that are poten-
tial drug targets are highlighted.

11.5 Host cell factors and viral proteins as target for antiviral agents
11.5.1 Viral life cycle as drug targets
One of the first steps in the process of drug discovery is the identification of the drug target. In this context the direct strat-
egy is to understand the life cycle of coronaviruses, that is, their entry, replication, and proliferation. The different host and
viral enzymes and proteins involved in viral life cycle at different stages, then, become the potential druggable targets.
As presented in Fig. 11.10, the entry of SARS-CoV-2 infection begins via the mediation of the Spike (S) glycopro-
tein present in the virus that interacts with ACE2 receptor of the host [71], and subsequent cleavage of this protein by
the transmembrane serine protease 2 (TMPRSS2) of the host cell. This leads to the fusion of the viral particle into the
host’s cell membrane [144]. Once inside, the viral particle releases the Nucleocapsid (N) protein and the viral genome
load into the cytosol. The ribosomes present in the host cell then synthesize the ORF into polyproteins that eventually
encode 16 nsps, and the remaining ORFs encode structural proteins. At this stage, the proteases play a key role in the
generation of the replication
transcription complex, which eventually generates more viral payload in situ [145].
Therefore, in essence, there are three classified targets of interest, for design of therapeutics to treat/mitigate the
disease. They are as follows: (1) virus-based targets, (2) host-based druggable targets, and (3) host-immune response
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 201

mechanisms. In a snapshot, to give a brief overview, the virus-based targets consist of two kinds, namely, the structural
proteins and the nonstructural proteins. ACE2, TMPRSS2, Furin, Cathepsin L, AAK and GAK1, PIK, and TPC2 are the
seven unique host-based targets. Lastly, as a consequence of the disease, the host immune responses provide a pathway
for probing therapeutic targets. In the following sections, we detail each of these targets.

11.5.2 Virus-based targets

11.5.2.1 Structural proteins
The early determination of the viral structure is one of the key aspects leading to the rapid design of drugs and vaccines
for COVID-19. Fig. 11.11 provides the representation of SARS-CoV-2 and its structural proteins. As presented, there
are various proteins in the structure but two of them are of specific interest as targets, namely, the “N” protein and the
“S” protein.

11.5.2.1.1 Nucleocapsid protein

There are two highly conserved domains in the Nucleocapsid (N) protein—(1) N-terminal RNA binding domain and (2)
C-terminal dimerization domain. In addition, there is a disordered central Ser/Arg-rich linker for primary phosphoryla-
tion [146
148]. This is of particular interest, given that there are successful compounds found to interfere with N pro-
teins of other CoVs, such as, recent discovery of stabilizers of the protein 2 protein interaction of MERS N protein.
Furthermore, this protein is highly immunogenic and considered as a potential vaccine target and development of
COVID-19 diagnostic methods.

11.5.2.1.2 Spike protein

On the other hand, the spike (S) protein consists chiefly of two functional groups, namely, the S1 subunit which is
responsible for host recognition and S2 subunit, which participates in the host 2 guest membrane fusion [73]. This has
been the key target of various groups for designing an effective drug. One of the key aspects in the particular protein is
the up and down conformation of the protein as presented in Fig. 11.12, which renders it active or inactive as a selective
target for the drug. This conformational selectivity as a strategy has enabled the structure-based drug discovery on the
inhibition of the interaction between S-RBD and the host protein ACE2 [149,150].

11.5.2.2 Nonstructural proteins

These proteins are key to the replication and transcription of the virus in the host. The main protease (3CLpro, nsp5)
along with Papain-like protease (PLpro, nsp3) cleave the polyproteins to generate nsps: “nsp2 2 16s” that form the
replication 2 transcription complex [145]. Some of these nsps include RNA-dependent RNA polymerase (RdRp, nsp12)
and helicase (nsp13). Therefore, any of these proteins could become potential targets to stop the replication of the virus.

11.5.2.2.1 3CLpro and PLpro

These proteins are both crucial for virus replication and controlling the host cell response. The importance of these tar-
gets is validated by the growing number of studies that focus on these proteases for drug repurposing. In fact, PLpro is
an interesting target as it not only inhibits viral replication but also dysregulates signaling cascades within the infected
cells, that results in the apoptosis of neighbor uninfected cells [151].

FIGURE 11.11 Schematic representation of SARS-CoV-2 and its structural pro-

teins. From Gil C, Ginex T, et al. COVID-19: drug targets and potential treatments.
J Med Chem 2020;63:21. https://doi.org/10.1021/acs.jmedchem.0c00606.
202 Pandemic Outbreaks in the 21st Century

FIGURE 11.12 Representation of the closed “down” (6VXX), open “up” (6VSB), and the interaction of a drug Setrobuvir against the “up” state S
glycoprotein as captured using PyMOL.

11.5.2.2.2 RNA-dependent RNA polymerase

RdRp mediates the transcription and replication of the RNA genome during infection. Given the lack of a human coun-
terpart, coupled with its necessity for the virus’s life cycle, this target is a great candidate that leads the way for anti-
viral development [152].

11.5.2.2.3 Helicase (nsp13)

These helicases are essential for RNA viral synthesis and are the most conserved proteins across the family of nido-
viruses. These factors make it an interesting target, with several inhibitors already reported in literature [153].

11.5.3 Host-based druggable targets

11.5.3.1 Angiotensin I converting enzyme 2 receptor (ACE2)
The ACE2 receptor is confirmed as the main virus receptor in the host cell. It is a multifunctional Zn-metalloprotease
with an amino-terminal catalytic domain and a carboxy-terminal domain [154]. There are diverse therapeutic strategies
for COVID probed via ACE2 that include attempting to interfere with dynamics of the virus
host ACE2
S-RBD inter-
face [155,156], inhibiting the ACE receptor using Angiotensin blockers [157], and supplementing the system with deliv-
ery of soluble ACE2 [158].

11.5.3.2 Transmembrane serine protease 2

The entry of SARS-CoV-2 in the host cell is facilitated by TMPRSS2 [144]. It has been observed that the viral infection
is decreased by the use of the TMPRSS2 inhibitor Camostat [144]. Furthermore, given that the its expression in lungs is
modulated by estrogens and androgens, the activation of estrogen/inhibition of androgen pathways is an interesting new
target for therapeutic clinical intervention for symptom amelioration in COVID-19 patients [159]. However, as yet,
there is no crystal structure of TMPRSS2 elucidated, and the small molecules are being developed using homology
models of TMPRSS2 developed based on other well-known serine protease structures.

11.5.3.3 Furin
Earlier studies have reported that the inhibition of furin with peptides and small molecules arrests tumor growth, inflam-
mation, and some viral and bacterial infections, indicating its role in the process [160]. However, since furin-like
enzymes are pleiotropic in nature participating in multitudinous cellular processes, there is a major drawback of com-
pounded side effects [161].
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 203

11.5.3.4 Cathepsin L
It was observed that coronavirus needs Cathepsin L, given that its inhibition reduced the entry of virus to almost 76%.
On the other hand, there was no such effect noted with Cathepsin B [162]. This confirms the potential of Cathepsin L
as a key target as it plays a key role in the entry, via priming the S protein in the viral particles into lysosomes.

11.5.3.5 Adaptor-associated kinase 1 and cyclin G-associated kinase

The serine 2 threonine protein kinases present in the host cell are known to regulate the intracellular viral trafficking
during entry, assembly, and release of multiple unrelated RNA viruses such as rabies, Ebola, dengue, or hepatitis C
virus [163,164]. They target by covalent inhibition and therefore can be employed to develop selective covalent
inhibitors.

FIGURE 11.13 Schematic representation of the main pathways of the innate immune response to SAR-CoV, SARS-CoV-2 and MERS. SAR-CoV,
SARS-CoV-2 and MERS infection leads to the activation of nuclear factor kappa B (NF-kB), AP-1, and interferon (IFN), resulting in the secretion of
pro-inflammatory cytokines and interferons and the activation of a cellular immune response. The inhibitory proteins are depicted in red, while the
activating proteins are in green. From Gil C, Ginex T, et al. COVID-19: drug targets and potential treatments. J Med Chem 2020;63:21. https://doi.
org/10.1021/acs.jmedchem.0c00606.
204 Pandemic Outbreaks in the 21st Century

11.5.3.6 Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve)

This protein plays an important role in the trafficking events associated with endocytic pathway [165]. Therefore it is a
suitable target to modulate infection of viruses that typically enter through endocytosis, including the coronavirus.
However, thus far, there is no 3D structure of this protein available.

11.5.3.7 Two-pore channel (TPC2)

The chief function of the two-pore channels (TPC1 2 3) is regulating conduction of ions like Na and Ca across the cel-
lular membranes [166]. However, the participation of TPC2 in COVID-19 is affirmed by the reduction in the corona-
virus entry, on using tetrandrine, a potent calcium blocker [167].

11.5.3.8 Immune response

Many SARS-CoV-2 structural and non-structural viral proteins are involved in the immune response modulation.
Typically, the immune response is exaggerated in the severe COVID-19 patients. As stated by the WHO, these include crit-
ical pneumonia, lung inflammation, trouble in respiration, and a potentially fatal cytokine storm/circulatory shock [167].
The alternative strategy involves the therapeutic targeting of the immune response. Clinical studies demonstrate that
inflammatory response in COVID-19 results in a fatal pulmonary inflammation. Therefore, a relevant strategy includes
the counterattack on the virus via the repurposing of host-based therapeutics to control the immune response [167].
This is achieved through the use of recombinant IFN-α and IFN-β as therapeutics to inhibit viral replication in tar-
geted cells. It is noted that the activation of NF-kB, AP-1, and IFN result in the secretion of pro-inflammatory cytokines
and interferons and the activation of a cellular immune response [167].
As surmised in Fig. 11.13, most of the viral proteins are typically inhibitory (red) toward several pathways of the
immune response. Remarkably, the coronavirus ORF9b and nsp15 activate (green) the IFN route, while other HCoVs
proteins have the opposite effect, leading to destruction of both the cell and the virus. In addition, the SARS-CoV-2
activates PKR, while MERS and several other viruses typically inhibit this enzyme. Thus, we have the requisite targets
that prove to be selective to design therapeutics for the treatment of COVID-19.

11.6 Concluding remarks

The global burden of COVID-19 is formidable and will likely spread further. In the absence of antiviral drugs, treat-
ment remains merely symptomatic. Currently, the vaccines for COVID-19 are under different stages of development,
and yet, there are no therapeutic interventions. Developing an attenuated vaccine for COVID-19 in a short duration of
time is really challenging, as it requires nonvirulent strain and sufficient knowledge about the virulence factors of coro-
navirus. Some of the vaccines which are in preclinical development include: (1) Formaldehyde-inactivated vaccine
being developed by Sinovac Biotech in China, (2) Vaccine being developed between the United States (Codagenix) and
India (Serum Institute). To date, three COVID-19 vaccines were authorized by the national authorities but yet, none of
them received the WHO EUL/PQ authorization. Among these three, we can expect the authorization for the Pfizer vac-
cine by the end of December 2020.
Our understanding of the SARS-CoV-2 life cycle and the interactions of the virus with host cell components and path-
ways is increasing rapidly. With the advent of efficient high-throughput techniques, large data sets are being generated, but
computational tools to process this information flow are a challenge. Thus the integration of data obtained from genomics,
transcriptomics, and proteomics studies into a complete map of SARS-CoV-2
host interactions that promote or restrict
virus replication and contribute to disease looks very promising. As illustrated in this chapter, many druggable host cell
dependency factors and viral proteins have already been identified. While these results are promising, evaluation of these
host and viral factors and potential therapeutic drug candidates in cell line, preclinical and clinical studies are necessary.
COVID-19 therapy may benefit from a synergistic effect by merging conventional antiviral drugs with novel host-directed
antivirals, thus reducing virus replication and pathogenesis while minimizing the risk of rapid resistance development.

References
[1] Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. In: Maier HJ, Bickerton E, Britton P, editors.
Coronaviruses: methods and protocols, 2015. New York, NY: Springer New York; 2015. p. 1
23.
[2] Rehman SU, Shafique L, Ihsan A, Liu Q. Evolutionary trajectory for the emergence of novel coronavirus SARS-CoV-2. Pathogens 2020;9:240.
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 205

[3] Ye Z-W, Yuan S, Yuen K-S, Fung S-Y, Chan C-P, Jin D-Y. Zoonotic origins of human coronaviruses. Int J Biol Sci 2020;16:1686
97.
[4] Halaji M, Farahani A, Ranjbar R, Heiat M, Dehkordi FS. Emerging coronaviruses: first SARS, second MERS and third SARS-CoV-2: epidemi-
ological updates of COVID-19. Le Infezioni Medicina 2020;28:6
17.
[5] Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of
a report of 72 314 cases from the Chinese Center for Disease Control and Prevention. JAMA 2020;323:1239
42.
[6] Zhou P, Yang X-L, Wang X-G, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat ori-
gin. Nature 2020;579:270
3.
[7] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med
2020;382(8):727
33.
[8] Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al. The species Severe acute respiratory syndrome-related coro-
navirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol 2020;5:536
44.
[9] WHO. Naming the coronavirus disease (COVID-19) and the virus that causes it; 2020. Available from: https://www.who.int/emergencies/dis-
eases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-the-virus-that-causes-it.
[10] WHO. Novel Coronavirus (2019-nCoV) Situation report
6; 2020. Available from: https://www.who.int/docs/default-source/coronaviruse/situ-
ation-reports/20200126-sitrep-6-2019-ncov.pdf?sfvrsn 5 beaeee0c_4.
[11] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia.
N Engl J Med 2020;382:1199
207.
[12] WHO. WHO Director-General’s statement on IHR Emergency Committee on Novel Coronavirus (2019-nCoV); 2020. Available from: https://
www.who.int/director-general/speeches/detail/who-director-general-s-statement-on-ihr-emergency-committee-on-novel-coronavirus-(2019-ncov).
[13] WHO. WHO Director-General’s opening remarks at the media briefing on COVID-19
11 March 2020; 2020. Available from: https://www.
who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19-11-march-2020.
[14] WHO. WHO coronavirus disease (COVID-19) dashboard; 2020. Available from: https://covid19.who.int/.
[15] Weiss SR, Navas-Martin S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol
Mol Biol Rev 2005;69:635
64.
[16] Chan KH, Poon LLM, Cheng VCC. Detection of SARS coronavirus (SCoV) by RTPCR, culture and serology in patients with severe acute
respiratory syndrome (SARS). Emerg Infect Dis 2003;10(2):294
9.
[17] Woo PCY, Huang Y, Lau SKP, Yuen K-Y. Coronavirus genomics and bioinformatics analysis. Viruses 2010;2:1804
20.
[18] Woo PCY, Lau SKP, Lam CSF, Lau CCY, Tsang AKL, Lau JHN, et al. Discovery of seven novel mammalian and Avian coronaviruses in the
genus Deltacoronavirus bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and Avian coronaviruses as the gene
source of Gammacoronavirus and Deltacoronavirus. J Virol 2012;86:3995
4008.
[19] Tang D, Comish P, Kang R. The hallmarks of COVID-19 disease. PLOS Pathog 2020;16:e1008536.
[20] Fouchier RAM, Hartwig NG, Bestebroer TM, Niemeyer B, de Jong JC, Simon JH, et al. A previously undescribed coronavirus associated with
respiratory disease in humans. Proc Natl Acad Sci U S A 2004;101:6212
16.
[21] Woo PCY, Lau SKP, Chu C-m, Chan K-h, Tsoi H-w, Huang Y, et al. Characterization and complete genome sequence of a novel coronavirus,
coronavirus HKU1, from patients with pneumonia. J Virol 2005;79:884
95.
[22] Chen Y, Liu Q, Guo D. Emerging coronaviruses: genome structure, replication, and pathogenesis. J Med Virol 2020;92:418
23.
[23] Hui DSC, Zumla A. Severe acute respiratory syndrome: historical, epidemiologic, and clinical features. Infect Dis Clin North Am 2019;33:869
89.
[24] Mona F, Ali T, Shokouh G. Comparison of the COVID-2019 (SARS-CoV-2) pathogenesis with SARS-CoV and MERS-CoV infections. Future
Virol 2020;15:317
23.
[25] Cui J, Li F, Shi Z-L. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17:181
92.
[26] Hui DS. Epidemic and emerging coronaviruses (Severe Acute Respiratory Syndrome and Middle East Respiratory Syndrome). Clin Chest Med
2020;38:71
86.
[27] Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends
Microbiol 2016;24:490
502.
[28] Azhar EI, Hui DSC, Memish ZA, Drosten C, Zumla A. The middle east respiratory syndrome (MERS). Infect Dis Clin North Am
2019;33:891
905.
[29] Chu DKW, Poon LLM, Gomaa MM, Shehata MM, Perera RAPM, Abu Zeid D, et al. MERS coronaviruses in dromedary camels, Egypt. Emerg
Infect Dis 2014;20:1049
53.
[30] Dudas G, Carvalho LM, Rambaut A, Bedford T. MERS-CoV spillover at the camel-human interface. eLife 2018;7:e31257.
[31] Sikkema RS, Farag EABA, Islam M, Atta M, Reusken CBEM, Al-Hajri MM, et al. Global status of Middle East respiratory syndrome coronavi-
rus in dromedary camels: a systematic review. Epidemiol Infect 2019;147:e84.
[32] Lu H, Stratton CW, Tang Y-W. Outbreak of pneumonia of unknown etiology in Wuhan, China: the mystery and the miracle. J Med Virol
2020;92:401
2.
[33] Zheng J. SARS-CoV-2: an emerging coronavirus that causes a global threat. Int J Biol Sci 2020;16:1678
85.
[34] Li F, Li W, Farzan M, Harrison SC. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science
2005;309:1864
8.
[35] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China. N Engl J Med
2019;382:727
33.
206 Pandemic Outbreaks in the 21st Century

[36] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneu-
monia in Wuhan, China: a descriptive study. Lancet 2020;395:507
13.
[37] Hossain MF, Hasana S, Mamun AA, Uddin MS, Wahed MII, Sarker S, et al. COVID-19 outbreak: pathogenesis, current therapies, and poten-
tials for future management. Front Pharmacology 2020;11:563478.
[38] Srivastava N, Baxi P, Ratho RK, Saxena SK, Global trends in epidemiology of coronavirus disease 2019 (COVID-19). In: Saxena SK, editor.
Coronavirus disease 2019 (COVID-19): epidemiology, pathogenesis, diagnosis, and therapeutics. Singapore: Springer Singapore. p. 9
21.
[39] WHO. Novel Coronavirus–China. 2020. https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/.
[40] Helmy YA, Fawzy M, Elaswad A, Sobieh A, Kenney SP, Shehata AA. The COVID-19 pandemic: a comprehensive review of taxonomy, genet-
ics, epidemiology, diagnosis, treatment, and control. J Clin Med 2020;9:1225.
[41] Andersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF. The proximal origin of SARS-CoV-2. Nat Med 2020;26:450
2.
[42] Menachery VD, Yount BL, Debbink K, Agnihothram S, Gralinski LE, Plante JA, et al. A SARS-like cluster of circulating bat coronaviruses
shows potential for human emergence. Nat Med 2015;21:1508
13.
[43] World Health Organization. Consensus document on the epidemiology of severe acute respiratory syndrome (SARS). Geneva: World Health
Organization; 2003.
[44] Riou J, Althaus CL. Pattern of early human-to-human transmission of Wuhan 2019 novel coronavirus (2019-nCoV), December 2019 to January
2020. Eurosurveillance 2020;25(4):2000058.
[45] Coroneo MT. The eye as the discrete but defensible portal of coronavirus infection. Ocul Surf 2020.
[46] Peiris JSM, Chu CM, Cheng VCC, Chan KS, Hung IFN, Poon LLM, et al. Clinical progression and viral load in a community outbreak of
coronavirus-associated SARS pneumonia: a prospective study. Lancet 2003;361:1767
72.
[47] Varia M, Wilson S, Sarwal S, McGeer A, Gournis E, Galanis E. Investigation of a nosocomial outbreak of severe acute respiratory syndrome
(SARS) in Toronto. Can CMAJ 2003;169:285
92.
[48] Poutanen SM, Low DE, Henry B, Finkelstein S, Rose D, Green K, et al. Identification of severe acute respiratory syndrome in Canada. N Engl
J Med 2003;348:1995
2005.
[49] Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J
Med 2003;348:1986
94.
[50] Guan W-j, Ni Z-y, Hu Y, Liang W-h, Ou C-q, He J-x, et al. Clinical characteristics of coronavirus disease 2019 in China. N Engl J Med
2020;382:1708
20.
[51] Chams N, Chams S, Badran R, Shams A, Araji A, Raad M, et al. COVID-19: a multidisciplinary review. Front Public Health 2020;8:383.
[52] Zhang D-m, Lu J-h, Zhong N-s. Pathogenesis of severe acute respiratory syndrome. Chin Med J 2008;121:1722
31.
[53] Mason RJ. Pathogenesis of COVID-19 from a cell biology perspective. Eur Respir J 2020;55.
[54] Li J, Guo M, Tian X, Wang X, Yang X, Wu P, et al. Virus-host interactome and proteomic survey reveal potential virulence factors influencing
SARS-CoV-2 pathogenesis. Medicine 2020;2(1):99
112.e7.
[55] Navas-MartÃn S, Weiss SR. Coronavirus replication and pathogenesis: implications for the recent outbreak of severe acute respiratory syn-
drome (SARS), and the challenge for vaccine development. J Neurovirol 2004;10:75
85.
[56] Mao L, Jin H, Wang M, Hu Y, Chen S, He Q, et al. Neurologic manifestations of hospitalized patients with coronavirus disease 2019 in
Wuhan. China JAMA Neurol 2020;77:683
90.
[57] Ahmed MU, Hanif M, Ali MJ, Haider MA, Kherani D, Memon GM, et al. Neurological manifestations of COVID-19 (SARS-CoV-2): a review.
Front Neurol 2020;11:518.
[58] Baig AM. Neurological manifestations in COVID-19 caused by SARS-CoV-2. CNS Neurosci Ther 2020;26:499.
[59] Esfandiarei M, McManus BM. Molecular biology and pathogenesis of viral myocarditis. Annu Rev Pathol Mech Dis 2008;3:127
55.
[60] Shi S, Qin M, Shen B, Cai Y, Liu T, Yang F, et al. Association of cardiac injury with mortality in hospitalized patients with COVID-19 in
Wuhan. China JAMA Cardiol 2020;5:802
10.
[61] Naicker S, Yang C-W, Hwang S-J, Liu B-C, Chen J-H, Jha V. The novel coronavirus 2019 epidemic and kidneys. Kidney Int 2020;97:824
8.
[62] Mahumud RA, Kamara JK, Renzaho AMN. The epidemiological burden and overall distribution of chronic comorbidities in coronavirus
disease-2019 among 202,005 infected patients: evidence from a systematic review and meta-analysis. Infection 2020;48:813
33.
[63] Leung JM, Niikura M, Yang CWT, et al. COVID-19 and COPD. Eur Respir J 2020;56:2002108.
[64] Thomopoulos C, Michalopoulou H. Renin
angiotensin system blockers and the risk of critical or fatal coronavirus disease 2019 in African
Americans. J Hypertens 2020;38:2384
6.
[65] Mehta N, Kalra A, Nowacki AS, Anjewierden S, Han Z, Bhat P, et al. Association of use of angiotensin-converting enzyme inhibitors and
angiotensin II receptor blockers with testing positive for coronavirus disease 2019 (COVID-19). JAMA Cardiol 2020;5:1020
6.
[66] Reynolds HR, Adhikari S, Pulgarin C, Troxel AB, Iturrate E, Johnson SB, et al. Renin-angiotensin-aldosterone system inhibitors and risk of
COVID-19. N Engl J Med 2020;382:2441
8.
[67] Bansal M. Cardiovascular disease and COVID-19. Diabetes Metab Syndr 2020;14:247
50.
[68] Lippi G, Lavie CJ, Sanchis-Gomar F. Cardiac troponin I in patients with coronavirus disease 2019 (COVID-19): evidence from a meta-analysis.
Prog Cardiovasc Dis 2020;63:390
1.
[69] Odegaard JI, Chawla A. Connecting type 1 and type 2 diabetes through innate immunity. Cold Spring Harb Perspect Med 2020;2:a007724.
[70] To Sing F, Xiang L Ding. Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 2019;73:529
57.
[71] Shang J, Ye G, Shi K, Wan Y, Luo C, Aihara H, et al. Structural basis of receptor recognition by SARS-CoV-2. Nature 2020;581:221
4.
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 207

[72] Hamming WT, Bulthuis MLC, Lely AT, Navis GJV, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coro-
navirus. A first step in understanding SARS pathogenesis. J Pathol: A J Pathol Soc Gt Br Irel 2004;203:631
7.
[73] Walls AC, Park Y-J, Tortorici MA, Wall A, McGuire AT, Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycopro-
tein. Cell 2020;181:281
92 e6.
[74] Bosch BJ, Bartelink W, Rottier PJM. Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein
upstream of rather than adjacent to the fusion peptide. J Virol. 2008;82:8887
90.
[75] Belouzard S, Chu VC, Whittaker GR. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct
sites. Proc Natl Acad Sci U S A 2009;106:5871
6.
[76] Andrews RJ, Peterson JM, Haniff HS, Chen J, Williams C, Grefe M, et al. An in silico map of the SARS-CoV-2 RNA structurome. bioRxiv
2020.
[77] Yoshimoto FK. The proteins of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2 or n-COV19), the cause of COVID-19.
Protein J 2020;39:198
216.
[78] Perlman S, Netland J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 2009;7:439
50.
[79] Sawicki SG, Sawicki DL, Siddell SG. A contemporary view of coronavirus transcription. J Virol 2007;81:20
9.
[80] Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, Rottier PJ. Coronavirus M proteins accumulate in the Golgi complex beyond
the site of virion budding. J Virol 1994;68:6523
34.
[81] Fung TS, Liu DX. Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 2019;73:529
57. Available from: https://doi.org/
10.1146/annurev-micro-020518-115759.
[82] Masters PS. The molecular biology of coronaviruses. Advances in Virus Research, 2006. Academic Press; 2006. p. 193
292.
[83] Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LLM, et al. Unique and conserved features of genome and proteome of
SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 2003;331:991
1004.
[84] Parker MM, Masters PS. Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain
structure for the nucleocapsid protein. Virology 1990;179:463
8.
[85] Laude H, Masters PS. The coronavirus nucleocapsid protein. The coronaviridae, 1995. Springer; 1995. p. 141
63.
[86] Ryu WS. Virus life cycle. Molecular virology of human pathogenic viruses. 2017;31
45. Available from: https://10.1016/B978-0-12-
800838-6.00003-5.
[87] P. V’kovski A, Kratzel S, Steiner H, Stalder, Thiel V. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol
2020;19:155
70.
[88] Daly JL, Simonetti B, Klein K, Chen K-E, Williamson MK, Antón-Plágaro C, et al. Neuropilin-1 is a host factor for SARS-CoV-2 infection.
Science 2020;370:861
5.
[89] Cantuti-Castelvetri L, Ojha R, Pedro LD, Djannatian M, Franz J, Kuivanen S, et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and infec-
tivity. Science 2020;13:856
60.
[90] Thoms M, Buschauer R, Ameismeier M, Koepke L, Denk T, Hirschenberger M, et al. Structural basis for translational shutdown and immune
evasion by the Nsp1 protein of SARS-CoV-2. Science 2020;369:1249
55.
[91] Blanco-Melo D, Nilsson-Payant BE, Liu WC, Uhl S, Hoagland D, Møller R, et al. , Imbalanced host response to SARS-CoV-2 drives devel-
opment of COVID-19. Cell 2020;181:1036
45 .e9.
[92] Taiaroa G RD, Featherstone L, Pitt M, Caly L, Druce J, Purcell D, et al. Direct RNA sequencing and early evolution of SARS-CoV-2.
bioRxiv 2020.
[93] Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The architecture of SARS-CoV-2 transcriptome. Cell 2020;181:914
21 e10.
[94] Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, White KM, et al. A SARS-CoV-2 protein interaction map reveals targets for drug
repurposing. Nature 2020;583:459
68.
[95] Bouhaddou M, Memon D, Meyer B, White KM, Rezelj VV, Marrero MC, et al. The global phosphorylation landscape of SARS-CoV-2 infec-
tion. Cell 2020;182:685
712 e19.
[96] Stukalov GV, Grass A, Bergant V, Karayel V, Urban O, Haas C, et al. Multi-level proteomics reveals host-perturbation strategies of SARS-
CoV-2 and SARS-CoV. bioRxiv 2020.
[97] Bojkova D, Klann K, Koch B, Widera M, Krause D, Ciesek S, et al. Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.
Nature 2020;583:469
72.
[98] Grenga L, Gallais F, Pible O, Gaillard JC, Gouveia D, Batina H, et al. Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it
can optimize whole viral particle antigen production for vaccines. Emerg Microbes Infect 2020;9:1712
21.
[99] Davidson AD, Williamson MK, Lewis S, Shoemark D, Carroll MW, Heesom KJ, et al. Characterisation of the transcriptome and proteome of
SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein. Genome Med
2020;12:68.
[100] Messina F, Giombini E, Agrati C, Vairo F, Ascoli Bartoli T, Al Moghazi S, et al. COVID-19: viral-host interactome analyzed by network
based-approach model to study pathogenesis of SARS-CoV-2 infection. J Transl Med 2020;18:1
10.
[101] Sadegh S, Matschinske J, Blumenthal DB, Galindez G, Kacprowski T, List M, et al. Exploring the SARS-CoV-2 virus-host-drug interactome
for drug repurposing. Nat Commun 2020;11:3518.
[102] Daniloski Z, Jordan TX, Wessels HH, Hoagland DA, Kasela S, Legut M, et al. Identification of required host factors for SARS-CoV-2 infec-
tion in human cells. Cell 2020;84(1):92
105 .e16.
208 Pandemic Outbreaks in the 21st Century

[103] Zhu FF, Hu Y, Wang G, Yu Y, Zhu Y, Xu Y, et al. The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell 2 entry pathways and
transmission. bioRxiv 2020.
[104] Wei J, Alfajaro MM, DeWeirdt PC, Hanna RE, Lu-Culligan WJ, Cai WL, et al. Genome-wide CRISPR screens reveal host factors critical for
SARS-CoV-2 infection. Cell 2020.
[105] Schneider WM, Luna JM, Hoffmann HH, Sãnchez-Rivera FJ, Leal AA, Ashbrook AW, et al. Genome-scale identification of SARS-CoV-2
and pan-coronavirus host factor networks. Cell. 2021; Jan 7;184(1):120
132.e14.
[106] Hoffmann HH, Schneider WM, Rozen-Gagnon K, Miles LA, Schuster F, Razooky B, et al. TMEM41B is a pan-flavivirus host factor. Cell.
2020; Jan 7;184(1):133
148.e20.
[107] Xiong Y, Liu Y, Cao L, Wang D, Guo M, Jiang A, et al. Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood
mononuclear cells in COVID-19 patients. Emerg Microbes Infect 2020;9:761
70.
[108] Zhou Z, Ren L, Zhang L, Zhong J, Xiao Y, Jia Z, et al. Heightened innate immune responses in the respiratory tract of COVID-19 patients.
Cell Host Microbe 2020;27:883
90 e2.
[109] Bui WN, Chung LT, Joseph MI, Gutierrez C, Habermann AJ, Adams AC, et al. Banovich NE Single-cell RNA-sequencing reveals dysregula-
tion of molecular programs associated with SARSCoV-2 severity and outcomes in patients with chronic lung disease. bioRxiv 2020.
[110] Singh M, Bansal V, Feschotte C. A single-Cell RNA expression map of human coronavirus entry factors. Cell Rep 2020;32.
[111] Shen B, Yi X, Sun Y, Bi X, Du J, Zhang C, et al. Proteomic and metabolomic characterization of COVID-19 patient sera. Cell
2020;182:59
72 e15.
[112] Akgun E, Tuzuner MB, Sahin B, Kilercik M, Kulah C, Cakiroglu HN, et al. Proteins associated with neutrophil degranulation are upregulated
in nasopharyngeal swabs from SARS-CoV-2 patients. PLoS One 2020;15:e0240012.
[113] Li Y, Wang Y, Liu H, Sun W, Ding B, Zhao Y, et al. Urine proteome of COVID-19 patients. medRxiv 2020.
[114] Tay MZ, Poh CM, RÃrnia L, MacAry PA, Ng LFP. The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol
2020;20:363
74.
[115] Tang Y, Liu J, Zhang D, Xu Z, Ji J, Wen C. Cytokine storm in COVID-19: the current evidence and treatment strategies. Front Immunol
2020;11:1708.
[116] Cron RQ, Behrens EM. Cytokine storm syndrome. Springer; 2019.
[117] Tisoncik JR, Korth MJ, Simmons CP, Farrar J, Martin TR, Katze MG. Into the eye of the cytokine storm. Microbiol Mol Biol Rev
2012;76:16
32.
[118] Gao YM, Xu G, Wang B, Liu BC. Cytokine storm syndrome in coronavirus disease 2019: a narrative review. J Intern Med 2010;289
147
61.
[119] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Lancet 2020;395:497
506.
[120] Clerkin KJ, Fried JA, Raikhelkar J, Sayer G, Griffin JM, Masoumi A, et al. COVID-19 and cardiovascular disease. Circulation
2020;141:1648
55.
[121] Cao M, Zhang D, Wang Y, Lu Y, Zhu X, Li Y, et al. Clinical features of patients infected with the 2019 novel coronavirus (COVID-19) in
Shanghai, China. medRxiv 2020.
[122] Qi D, Yan X, Tang X, Peng J, Yu Q, Feng L, et al. Epidemiological and clinical features of 2019-nCoV acute respiratory disease cases in
Chongqing municipality, China: a retrospective, descriptive, multiple-center study. medRxiv 2020.
[123] Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus infected
pneumonia in Wuhan, China. JAMA 2020;323:1061
9.
[124] Chen G, Wu D, Guo W, Cao Y, Huang D, Wang H, et al. Clinical and immunological features of severe and moderate coronavirus disease
2019. J Clin Invest 2020;130:2620
9.
[125] Chen L, Liu HG, Liu W, Liu J, Liu K, Shang J, et al. Analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia.
Chin J Tuberculosis Respir Dis 2020;43:E005.
[126] Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, et al. Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin
Infect Dis 2020;71:762
8.
[127] Tan M, Liu Y, Zhou R, Deng X, Li F, Liang K, et al. Immunopathological characteristics of coronavirus disease 2019 cases in Guangzhou,
China. Immunology 2020;160(3):261
8.
[128] Guohua L, Ling L, Min H, Haibiao L, Peifeng K, Zishao Z. Value of various inflammatory markers combined with lymphocyte subsets on
clinical diagnosis of different clinical types of COVID-19. J Chong Med Univ 2020;10.
[129] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of COVID-19 associated with acute respiratory distress syn-
drome. Lancet Respir Med 2020;8:420
2.
[130] Channappanavar R, Perlman S. Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology.
Seminars in immunopathology. Springer; 2017. p. 529
39.
[131] Sarkar, Sen S. A comparative analysis of the molecular interaction techniques for in silico drug design. Int J Peptide Res Ther 2019;26
209
23.
[132] Cavasotto CN, Aucar MG, Adler NS. Computational chemistry in drug lead discovery and design. Int J Quantum Chem 2019;119:e25678.
 SARS-CoV-2—host cell interactions and pathways Chapter | 11 209

[133] Malik N, Dhiman P, Khatkar A. In silico and 3D QSAR studies of natural based derivatives as xanthine oxidase inhibitors. Curr Top Med
Chem 2019;19:123
38.
[134] Irwin JJ, Shoichet BK. ZINC—a free database of commercially available compounds for virtual screening. J Chem Inf Model 2005;45
177
82.
[135] Rismanbaf A. Potential treatments for COVID-19; a narrative literature review. Arch Acad Emerg Med 2020;8 e29.
[136] Singh S, Florez H. Coronavirus disease 2019 drug discovery through molecular docking. F1000Research 2020;9:502.
[137] Torres MDT, de la Fuente-Nunez C. Toward computer-made artificial antibiotics. Curr Opmicrobiol 2019;51:30
8.
[138] Xu Y, Lin K, Wang S, Wang L, Cai C, Song C, et al. Deep learning for molecular generation. Future Med Chem 2019;11:567
97.
[139] Maltarollo VG, Kronenberger T, Espinoza GZ, Oliveira PR, Honorio KM. Advances with support vector machines for novel drug discovery.
Expert Opin Drug Discov 2019;14:23
33.
[140] Pérez-Sianes J, Pérez-Sánchez H, Dı́az F. Virtual screening meets deep learning. Curr Comput Drug Des 2019;15:6
28.
[141] Paul SM, Mytelka DS, Dunwiddie CT, Persinger CC, Munos BH, Lindborg SR, et al. How to improve R&D productivity: the pharmaceutical
industry’s grand challenge. Nat Rev Drug Discov 2010;9:203
14.
[142] Zhou Y, Hou Y, Shen J, Huang Y, Martin W, Cheng F. Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2.
Cell Discov 2020;6:1
18.
[143] Mohanty S, Rashid MHA, Mridul M, Mohanty C, Swayamsiddha S. Application of artificial intelligence in COVID-19 drug repurposing.
Diabetes Metab Syndr Clin Res Rev 2020;14(5):1027
31.
[144] Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2
and is blocked by a clinically proven protease inhibitor. Cell 2020;181(2):271
80 .e8.
[145] V’Kovski P, Gerber M, Kelly J, Pfaender S, Ebert N, Lagache SB, et al. Determination of host proteins composing the microenvironment of
coronavirus replicase complexes by proximity-labeling. Elife 2019;8:e42037.
[146] Wang YS, Chang C-k, Hou MH. Crystallographic analysis of the N-terminal domain of Middle East respiratory syndrome coronavirus nucleo-
capsid protein. Acta Crystallogr Sect F Struct Biol Communicat 2015;71:977
80.
[147] Chang C-k, Sue S-C, Yu T-h, Hsieh C-M, Tsai C-K, Chiang Y-C, et al. Modular organization of SARS coronavirus nucleocapsid protein.
J Biomed Sci 2006;13:59
72.
[148] Yu IM, Oldham ML, Zhang J, Chen J. Crystal structure of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein
dimerization domain reveals evolutionary linkage between corona-and arteriviridae. J Biol Chem 2006;281:17134
9.
[149] Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh C-L, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion con-
formation. Science 2020;367:1260
3.
[150] Patel M, Rajendran SB, Pakala A, Shah H, Patel, Karyala P. Virtual screening of curcumin and its analogs against the spike surface glycopro-
tein of SARS-CoV-2 and SARS-CoV. J Biomol Struct Dyn 2020;1
9.
[151] Liu W, Morse JS, Lalonde T, Xu S. Learning from the past: possible urgent prevention and treatment options for severe acute respiratory
infections caused by 2019-nCoV. Chembiochem 2020;21(5):730
8.
[152] McDonald SM. RNA synthetic mechanisms employed by diverse families of RNA viruses. Wiley Interdiscip Revi RNA 2013;4:351
67.
[153] Lehmann KC, Snijder EJ, Posthuma CC, Gorbalenya AE. What we know but do not understand about nidovirus helicases. Virus Res
2015;202:12
32.
[154] Zhang H, Penninger JM, Li Y, Zhong N, Slutsky AS. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular
mechanisms and potential therapeutic target. Intens Care Med 2020;46:586
90.
[155] Smith M, Smith JC. Repurposing therapeutics for COVID-19: supercomputer-based docking to the SARS-CoV-2 viral spike protein and viral
spike protein-human ACE2 interface. Biol Med Chem 2020.
[156] Batra R, Chan H, Kamath G, Ramprasad R, Cherukara MJ, Sankaranarayanan SKRS. Screening of therapeutic agents for COVID-19 using
machine learning and ensemble docking studies. J Phys Chem Lett 2020;11:7058
65.
[157] Rothlin RP, Vetulli HCM, Duarte M, Pelorosso FGN. Telmisartan as tentative angiotensin receptor blocker therapeutic for COVID-19. Drug
Dev Res 2020.
[158] Kuba K, Imai Y, Ohto-Nakanishi T, Penninger JM. Trilogy of ACE2: A peptidase in the reninâh“angiotensin system, a SARS receptor, and a
partner for amino acid transporters. Pharmacol Ther 2010;128:119
28.
[159] Stopsack KH, Mucci LA, Antonarakis ES, Nelson PS, Kantoff PW. TMPRSS2 and COVID-19: serendipity or opportunity for intervention?
Cancer Discov 2020;10:779
82.
[160] Couture Fdr, Kwiatkowska A, Dory YL, Day R. Therapeutic uses of furin and its inhibitors: a patent review. Expert Opin Ther Pat
2015;25:379
96.
[161] Ivanova T, Hardes K, Kallis S, Dahms SO, Than ME, Künzel S, et al. Optimization of substrate-analogue furin inhibitors. ChemMedChem
2017;12:1953
68.
[162] Ou X, Liu Y, Lei X, Li P, Mi D, Ren L, et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-
reactivity with SARS-CoV. Nat Commun 2020;11:1
12.
[163] Wang C, Wang J, Shuai L, Ma X, Zhang H, Liu R, et al. The serine/threonine kinase AP2-associated kinase 1 plays an important role in rabies
virus entry. Viruses 2020;12:45.
[164] Bekerman E, Neveu G, Shulla A, Brannan J, Pu S-Y, Wang S, et al. Anticancer kinase inhibitors impair intracellular viral trafficking and exert
broad-spectrum antiviral effects. J Clin Invest 2017;127:1338
52.
210 Pandemic Outbreaks in the 21st Century

[165] He K, Marsland Iii R, Upadhyayula S, Song E, Dang S, Capraro BR, et al. Dynamics of phosphoinositide conversion in clathrin-mediated
endocytic traffic. Nature 2017;552:410
14.
[166] Grimm C, Chen C-C, Wahl-Schott C, Biel M. Two-pore channels: catalyzers of endolysosomal transport and function. Front Pharmacol
2017;8:45.
[167] Gil C, Ginex T, Maestro I, Nozal V, Barrado-Gil L, Cuesta-Geijo MA, et al. COVID-19: drug targets and potential treatments. J Med Chem
2020;63:21.
Chapter 12

Importance of in silico studies on the

design of novel drugs from medicinal
plants against 21st-century pandemics:
past, present, and future
Mallikarjuna Nimgampalle1, Vasudharani Devanathan1 and Ambrish Saxena2
1
Department of Biology, Indian Institute of Science Education and Research Tirupati (IISER T), Tirupati, India, 2Center for Sponsored Research and
Consultancy, Indian Institute of Technology (IIT) Tirupati, Tirupati, India

12.1 Introduction
Drug treatment is the central paradigm responsible for the recovery of the human population from any diseases. Some
of the newly identified human diseases are propagating rapidly within the region and causing severe mortality rate in
the population known as an epidemic. In contrast, pandemics are deadly diseases spreading worldwide very quickly,
affecting many people, and leading to a vast mortality rate [1]. Most pandemics are contagious diseases, usually caused
by new viral or bacterial strains that are easily transmissible between humans. Pandemic outbreaks affect public health
and their mortality and alter human psychological conditions, lifestyle, social distance, unemployment, destruction in
the education system, food scarcity, and the country’s economic conditions.
Consequently, it is also a big challenge for scientists to discover novel potential drugs to treat the new disease dur-
ing a pandemic outbreak. Pandemic diseases are typically managed for emergency purposes with symptomatic treatment
using available medications until finding out new drugs. The origin, risks, and impact of pandemics have been
described as follows.
Most of the pandemic infectious diseases in human beings are originated from animals through the cross-species
transmission of microbes [2]. As such, the animal pathogen (disease-causing agents of wild and domestic animal spe-
cies) itself cannot transmit disease in humans due to the “species barrier.” To cross the “species barrier,” the pathogen
needs to undergo mutations and evolve further so that it can infect humans and retain the pathogenicity to transmit
between humans without the original animal. This process includes five progressive phases [2,3] (Fig. 12.1). Phase 1
describes that animal pathogens are not present in humans (examples include malarial plasmodia). During phase 2, the
pathogen has evolved and transmitted to humans in normal conditions but cannot sustain pathogenicity to transmit
between humans (e.g., Nipah virus, Rabies virus, Tularemia bacilli, and West Nile viruses). An evolution from phase 2
to phase 3 is known as secondary transmission between humans. In phase 3, pathogens undergo only a few secondary
transmission cycles between humans (e.g., Ebola virus, Human monkey-pox viruses, and Marburg virus). In phase 4,
pathogens undergo several secondary transmission cycles between humans without animal involvement (e.g., Vibrio
cholera, Influenza A, and Dengue virus).
In contrast to all the above phases, phase 5 especially explains the pandemic diseases that are rapidly transmitted
between humans, for example, Severe acute respiratory syndrome (SARS), smallpox, human immunodeficiency virus
infection, and tuberculosis [3]. The risk of pandemic diseases is driven by the consolidated impacts of spark risk (where
a pandemic is probably going to emerge) and spread risk (that it is to diffuse comprehensively through the human popu-
lace) [2]. Moreover, the risk of currently deadly pandemic disease, that is, COVID-19 caused by a novel coronavirus, is
quickly spreading globally. It is a risk for the resurgence of long-standing illnesses (e.g., tuberculosis) and

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00013-6

© 2021 Elsevier Inc. All rights reserved. 211
212 Pandemic Outbreaks in the 21st Century

FIGURE 12.1 Transmission of the pandemic pathogen from animals to humans.

demonstrations of bioterrorism or bioweapon. The impact of pandemic diseases includes increasing morbidity and mor-
tality, creating fear in public at workplaces and other meetings, causing social distance, reducing economic growth, and
enhancing political stresses and tensions [4].
Human beings have been faced with devastating pandemic diseases in history. In ancient days, people have also
majorly depended on plant-based remedies against deadly diseases. Additionally, plant-derived compounds have shown
an essential role as therapeutic drug molecules in various infectious diseases [5]. Recently, it has been reviewed that tra-
ditional plant-based therapeutics were used to treat some of the pandemic diseases in the past; some interesting exam-
ples include the treatment of Middle Ages Black Death with plant-based lotion (the four thieves vinegar), Americans
used carnivorous plant species for the treatment of Smallpox, garlic is known to be useful in the treatment of tuberculo-
sis, consumption of onions is recommended for the management of Spanish flu, treatment of malaria with the plant
compound, quinine (the compound extracted from Peruvian tree) [6]. Usually, plants are the primary sources of poten-
tial therapeutics against various diseases. Moreover, conventional plant-derived compounds have shown the benefit of
extended time usage, revival, safer, and healthier than synthetic drugs [6]. It can be stated with certainty that plant-
based compounds will be the most significant wellspring of new medications later in the future [7].
In silico study is one of the best ways in scientific research to screen, design, and predict the possibilities of the thera-
peutic potential of novel drug molecules more effectively without the requirement of lab work and clinical preliminaries.
In the last decade computational strategies have emerged and became a part of all disciplines of biology [8]. In silico
studies to design promising therapeutic drug candidates are significantly cost-effective [9]. This has led to quicker meth-
ods to arrive at a very precise candidate molecule much faster. In silico methods include virtual screening, homology
modeling, sequence analysis, quantitative structure
activity relationships (QSAR), pharmacophore modeling, molecular
modeling approaches, molecular dynamic (MD) simulations, genomics, proteomics, gene network analysis, data analysis,
phylogenetic analysis, receptor-binding analysis, computer-aided drug design, and drug discovery [10,11]. The signifi-
cance of all the above in silico strategies on the design of novel therapeutics using plant derivative compounds against
pandemic outbreaks will be explored in more detail in upcoming sections. In this perspective the present chapter is essen-
tially focused on the importance of in silico studies and medicinal plant’s role in discovering new drugs for pandemic
outbreaks.

12.2 Pandemic outbreaks of 21st century

Pandemic outbreaks have been ravaged human beings and lead to changing the course of history. Ancient people have
faced the worst pandemic diseases, including smallpox, tuberculosis, Black Death, Spanish flu, and malaria [6]. In the
21st century there is a list of pandemic diseases that have been spread across the world, which include SARS, Avian
influenza, Middle East respiratory syndrome (MERS), swine flu, Ebola virus disease (EVD), and Zika [12] (Fig. 12.2).
Currently, Coronavirus disease 2019 (COVID-19) is an ongoing pandemic outbreak caused by severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2); first identified in December 2019 in Wuhan city, China [13]. The aforemen-
tioned pandemics will be briefly discussed as follows.
 Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 213

FIGURE 12.2 Pandemic outbreaks during the 21st century.

12.2.1 Severe acute respiratory syndrome

SARS was the first pandemic outbreak (mid-2003) in the 21st century that figured out how to get public consideration.
It is caused by the SARS coronavirus (SARS-CoV); it began in China and affected less than 10,000 people with a 10%
mortality rate (China and Hong Kong), 251 cases in Canada (Toronto), and spread to different countries of the world
[14,15]. The symptoms of SARS include difficulty breathing with fever, dry cough, and body pains; there is no specific
treatment but supportive or symptomatic treatment has been recommended.

12.2.2 Avian influenza

Avian influenza, also known as “bird flu,”’ occurred in wild aquatic birds and can also infect poultry, other birds, and
animal species. It is caused by influenza A virus subtype H5N1 (A/H5N1), commonly known as highly pathogenic
avian influenza virus; this virus is a subtype of the influenza A virus [16]. Influenza A virus is a causative agent of
influenza in human beings and some animals. In February 2004 the Avian influenza virus was first identified in birds in
Vietnam country. It has spread to several Southeast Asia countries (India, Cambodia, China, Bangladesh, Indonesia,
Laos, Thailand, and Myanmar) and finally caused the death of 250 million birds [17]. The Avian influenza virus is not
a pandemic because it has not sustained pathogenicity to transmit from humans to humans directly. However, some
cases so far are perceived to have been transmitted from birds to humans, responsible for human
human transmission,
and caused sporadic human infections. Generally, humans get infected with the Avian influenza virus (H5N1) by expo-
sure to sick or dead poultry. The symptoms of illness vary from person to person, including feverishness, cough, head-
ache, sore throat, abdominal pain, vomiting, and diarrhea. Various signs have appeared based on age, immunity, and
genetic factors. Furthermore, the illness caused by H5N1 has been treated with various antiviral, antiinflammatory, and
immune therapeutic agents [18,19].

12.2.3 The Middle East respiratory syndrome

MERS is a viral respiratory infection caused by MERS-coronavirus known as “Camel flu” because humans are infected
either from direct or indirect contact with sick camels. It was first identified in human beings in Saudi Arabia and
Jordan in April 2012 [20]. Globally, 2499 MERS-infected cases and 858 death cases (34.3% mortality) have been
reported from April 2012 to December 2019 [21]. The symptoms of this pandemic outbreak are fever, cough, diarrhea,
and shortness of breath. Till now, there is no specific vaccine or drug proven for the management of this pandemic
[22]. Usually, this disease is more severe in people with other health complications.

12.2.4 Swine flu

Swine flu is a respiratory infection in humans caused by anyone strain of swine influenza viruses (H1N1, H1N2, H2N1,
H3N1, H3N2, and H2N3) that are originated in pigs. It began in Mexico in April of 2009 and became a pandemic out-
break within weeks, infected 10% of global populations (of about 6.8 billion), caused around five lakhs of death cases
[23,24]. This virus is a subtype of influenza A virus that generally shows influenza-like symptoms, namely, rhinitis,
fever, chills, body pains, severe headache, weakness, shortness of breathing, lack of appetite, sore throat, and intestinal
diarrhea [25]. The Centers for Disease Control and Prevention, USA, has recommended antiviral drugs, namely, oselta-
mivir (Tamiflu) and zanamivir (Relenza), for the treatment and prevention of infected people with swine flu [26].
214 Pandemic Outbreaks in the 21st Century

12.2.5 Ebola virus disease

Ebola is a deadly infectious epidemic; a viral hemorrhagic fever occurred in humans and primates caused by
Ebolaviruses. It is also known as Ebola hemorrhagic fever or EVD. Usually, the Ebola virus spreads through the body
fluids (blood, semen, and breast milk) of humans who have perished due to EVD. The EVD has an incubation period
between 2 and 21 days to show symptoms in the infected patients. According to the WHO, the initial symptoms of
EVD include sudden fever, muscle pain, sore throat, headache, and reduced kidney and liver functions, subsequently
leading to death. The disease was first identified in 1976 and 1977 in the African Countries of Zaire and Sudan. Its out-
break is also reported in other African countries till 2019, with an average mortality rate of about 50% [27]. In
December 2019 Food and Drug Administration, USA approved the vaccine to prevent EVD, and two treatment strate-
gies, namely, REGN-EB3 and mAb114, have better improvement in patients [28,29].

12.2.6 Zika fever

Zika fever is an epidemic known as Zika or Zika virus disease or Zika viral infection caused by Zika virus belongs to
the viral family of Flaviviridae. Generally, this disease is spread through daytime-active mosquitoes, namely, Aedas
aegypti and Aedas albopictus. Furthermore, it can also be potentially transmitted between humans through blood trans-
fusion, sexual transmission, and infected pregnant women can also spread it to the fetus. The symptoms of Zika fever
include fever, headache, joint pains, red eyes, and maculopapular rashes [30]. The Zika virus has been reemerging once
a while from 1947 to till now for every 2
5 years in the globe as an outbreak [31]. There is no specific treatment for
Zika fever, symptomatic treatment can be supportive, and the vaccines are under clinical trials. Reducing the mosqui-
toes biting in infected areas is recommended as a preventive measure for Zika fever.

12.2.7 COVID-19
COVID-19 is a pandemic outbreak caused by SARS-CoV-2. It is spreading rapidly throughout the world, claiming lakhs of
lives and creating a significant public health problem this year. The mild to moderate symptoms of COVID-19 include
fever, dry cough, dyspnea, headache, body pains, and general body weakness, whereas, in severe cases, difficulty breathing,
chest pain, and loss of speech are also observed [32,33]. Till now, there is no specific drug or vaccine available for the
complete cure of COVID-19. However, this disease is symptomatically treated with various available medicines, and it is
managing in urgent situations by different pharmacologically active drugs such as chloroquine phosphate, hydroxychloro-
quine, lopinavir, remdesivir, favipiravir, and tocilizumab [34]. The present crisis has challenged researchers to discover
potent drugs or vaccines to combat COVID-19. Consequently, scientists are conducting enormous experiments in diverse
scientific research fields to bring potent therapeutic medicines to fight against COVID-19 successfully.

12.3 Plant-derived compounds as source of drugs to treat pandemics

It is a big challenge for researchers to discover potential drugs against pandemic outbreaks during disease-emerging condi-
tions. At that moment, researchers use available medications for other diseases to find out repurposing drugs for the treat-
ment of pandemics in emergency conditions. Similarly, plant-derived compounds are also another option to search for
suitable drug and plant-based vaccines for pandemics. In ancient days most people have used medicinal plants for emerging
diseases and their medicinal needs. Research findings have recently described that numerous antiviral compounds exist in
medicinal plants and show potential activities, including inhibition of viral replication, prevention of viral attachment to
host cells, and inhibiting various signaling and metabolic activities of viruses. Consequently, researchers have been trying
to screen and find potential antiviral drugs from medicinal plants to combat pandemic outbreaks. The compounds from
plant sources with potential therapeutic activities against each pandemic outbreak will be described as follows.

12.3.1 Plant-derived antiviral compounds as therapeutics for coronaviruses (SARS, MERS,

SARS-CoV-2)
Plant compounds are the essential source of drug discovery to treat all kinds of emerging diseases. As a result, plant-
based therapeutics have been reported on coronaviruses, starting from the outbreak of SARS to the current COVID-19
pandemic [35]. The details of each reported therapeutic compound, its plant source, and its mode of action against pan-
demics are given in Table 12.1.
Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 215

TABLE 12.1 Antiviral activity of plant-derived compounds against coronaviruses (SARS, MERS, and SARS-CoV-2)

S. no. Plant-derived Name of the plant Pandemic Mode of action

compound outbreak
1. Chalcones and coumarins Angelica keiskei SARS-CoV Xanthoangelol E inhibits cysteine
protease
2. Diarylheptanoids Alnus japonica SARS-CoV Hirsutenone inhibits papain-like
protease of SARS coronavirus
3. Indigo, sinigrin, Isatis indigotica SARS 3C-like protease inhibition
aloeemodin, and coronavirus
hesperetin
4. Flavonoids, tomentin A, B, Paulownia tomentosa SARS-CoV Papain-like protease (PLpro) inhibition
C, D, and E papain-like
protease
(PLpro)
5. Diterpenoids, biflavonoids Torreya nucifera SARS-CoV Plant extract inhibits virus
(Biflavone amentoflavone, 3CLpro
apigenin, luteolin, and
quercetin)
6. Allyl disulfide, allyl Allium sativum L. SARS-CoV-2 Inhibition of the ACE2
trisulfide, allyl (E)-1-
propenyl disulfide, allyl
methyl trisulfide, and
diallyl tetrasulfide
7. Torilin Torilisfructus SARS-CoV Decreases intracellular components of
the virus
8. Lycorine Lycorisradiata SARS-CoV Inhibits virus-induced CPE
9. Acanthosis, chiisanoside, Acanthopanacis cortex SARS-CoV Decreases intracellular components of
and phytosterol the virus
10. Carvacrol and α-pinene Anthemis hyaline SARS-CoV Reduces replication
11. Quinone-methide- Tripterygium regelii SARS-CoV 3CLpro inhibitors
triterpenes (celastrol,
pristimerin, tingenone, and
iguesterin)
12. Brazilein, Brazilin Caesalpinia sappan SARS-CoV-2 Inhibits viral enzymes (SARS-CoV-2
protease, Spike glycoprotein-RBD,
and PD-ACE2)
13. Anthraquinones including Cassia tora SARS-CoV Inhibits the 3CLp
emodin, physcion, and
rhein
14. Flavonoids (bavachinin, Psoralea corylifolia SARS-CoV Papain-like protease inhibition
neobavaisoflavone,
isobavachalcone, 40-O-
methylbavachalcone,
psoralidin, and corylifol A)
15. Triterpenoids Gentianascabra SARS-CoV Inhibits the 3CL protease activity of
SARS-CoV
16. Procyanidin A2 and Cinnamomum verum SARS-CoV Inhibits the internalization of TfR
procyanidin B1
17. Luteolin and quercetin Taxillus chinensis SARS-CoV Inhibits the 3CL protease activity of
SARS-CoV
18. Emodin (anthraquinone) Polygonum multiflorum SARS-CoV Blocked the S protein and ACE2
interaction
19. Emodin (anthraquinone) Rheum officinale SARS-CoV Blocked the S protein and ACE2
interaction

(Continued )
216 Pandemic Outbreaks in the 21st Century

TABLE 12.1 (Continued)

S. no. Plant-derived Name of the plant Pandemic Mode of action

compound outbreak
20. Anthraquinones Rheum palmatum SARS-CoV High level of anti-SARS-CoV 3CL
protease activity
21. Thymoquinone, qsimen, Nigella sativa SARS-CoV Decrease replication through the
karvakrol, t-anetol, 4- involvement of TRP genes family
terpineol, and longifoline
22. Flavonoids, limonene, and Citrus sinensis SARS-CoV Decrease replication through the
linalool involvement of TRP genes family
23. Nafamostat, lopinavir Alpinia galanga SARS-CoV-2 High binding affinities with SARS-
CoV-2 protease (PDB:6LU7), Spike
glycoprotein-RBD (PDB:6LXT), and
PD-ACE2 (PDB:6VW1)
24. Glucoside, curcumin, Capsicum annuum, Curcuma Coronavirus Inhibition of COVID-19 Mpro protein
oleuropein, luteolin-7, longa, Mentha longıfolia L., (CoV)
epicatechingallate, Olea europaea L., Curcuma
catechin, longa, Phoenix hanceana,
demethoxycurcumin, and Camellia sinensis
glucoside, and apigenin-7
25. Hypericin Hypericum perforatum L. 2019-nCoV C-terminal and N-terminal domains of
NSP 14 2019-nCoV NSP14 were found to be
bound by hypericin
26. Tylophorine Tylophora indica Coronaviruses Growth retardation of coronaviruses
27. ALS-1 and ALS-2 Alangium salvifolium SARS-CoV-2 Inhibition of spike glycoprotein

12.3.2 Plant-derived compounds as antiviral drugs for influenza viruses (swine flu and Avian flu)
As similar to coronaviruses, plant-derived compounds have also been shown significant therapeutic activity against
influenza viruses. The details of plant-derived compounds and their mode of action against swine flu and Avian flu are
shown in Table 12.2.

12.3.3 Antiviral activity of plant-derived compounds against Ebola and Zika viruses
There is limited research on the discovery of plant-based therapies for both Ebola and Zika viruses. It is reported that
some of the herbal compounds, namely, belladonna, arsenic, nitric acid, aconite, gelsemium, bryonia, have been used
for the symptomatic treatment of the Ebola virus [36]. Moreover, plant-derived compounds (eugenol, silvestrol, mesua-
ferrone B, euphorbianin, rutoside, 30 -O-methylmaysin, kuwanon C, 4,5-dicaffeoyl quinic acid, dacinostat, 30 -O-methyl
sappanol, morindolin, and quercetin-3-O-glucuronide) have stopped replication and inhibited viral proliferation of
Ebola virus in cellular and computational approaches [37
39]. Furthermore, the compounds from medicinal plants,
namely, gossypol, curcumin, digitonin, conessine, terrestrial, marine, berberine, and emodin have shown anti-Zika viral
activity.

12.4 Computational approaches in identifying novel drugs using

plant-derived compounds
The discovery of potential drugs using in silico methods for any disease mainly depends on drug target identification in
the pathogen, which is the foremost task. Drug targets include biomolecules, which may consist of DNA, RNA, and
protein (enzymes, receptors, ion channels, transporters, etc.). Drug targets play an essential role in “pathogens” life
cycle, proliferation, pathogenicity, metabolic and molecular events, attachment to host cells, and so on. Most of the
drugs will act as inhibitors, blockers, and promoters against drug targets of pathogens. The critical drug targets for cur-
rent pandemic SARS-CoV-2 include host receptor angiotensin-converting enzyme 2 (ACE2), spike glycoprotein, viral
Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 217

TABLE 12.2 Antiviral activity of plant-derived compounds against influenza viruses (swine flu and Avian flu)

S. no. Plant-derived compound Name of the Influenza viruses Mode of action

plant (swine flu and
Avian flu)
1. Aurantiamide acetate Baphicacanthus Influenza A virus Inhibition of NF-κB
cusia signaling pathway
2. Ginsenosides, polysaccharides, and Panax Influenza A H1N1 Natural killer cell activity
essential oils notoginseng virus and enhancement of
antiviral activity
3. Biaron C Aloe Influenza A and Inhibition of viral
arborescens influenza B viruses replication
4. Flavonoid glycosides and caffeoylquinic Eupatorium Influenza A virus Stops attachment of the
acids perfoliatum (IAV) H1N1 virus to host cells, inhibition
of virus-induced
hemagglutination
5. Polyphenolic compound Taraxacum Influenza virus type Inhibits replication
(epigallocatechin-gallate) officinale A, H1N1
6. Diarylheptanoids Alnus japonica Influenza virus Inhibits replication of the
KBNP-0028 (H9N2) virus
7. Polyphenolic compound Canarium Influenza A virus Inhibiting neuraminidase
album (Lour.) (IAV) activity
8. Flavonoids (quercetin, isoquercetin, and Capparis sinaica Avian influenza The antiviral activity and
rutin) strain H5N1 inhibition of replication
9. Salacinol, kotalanol, and catechins Salacia Influenza A virus Reduces clinical symptoms
reticulata H1N1
10. Lignans, diterpenes, flavonoids, Taxodium Influenza A and B Act against hemagglutinin
proanthocyanidins, and sterols distichum viruses
11. Diterpenoids, jatrophane-type Jatropha Influenza A H1N1 Inhibits viral binding to host
diterpenoids, and coumarino-type lignoids, multifida virus cells
lathyrane-type diterpenoids, multifidone,
multifidanol, and multifidenol
12. Flavonoid and polyphenol Acacia arabica Influenza A virus Inhibits H9N2 virus
H9N2 replication
13. Gallic acid, protocatechuic acid, corilagin, Geranium Influenza virus Neuraminidase inhibition
geraniin, ellagic acid, kaempferitrin, thunbergii (H1N1, H3N2,
kaempferol 7-O-rhamnoside, quercetin, Influenza type B)
kaempferol
14. Tannins (prodelphinidins and Ribes nigrum Influenza A virus Virucidal activity and stops
proanthocyanidins) the virus replication
15. Hydrolyzable tannins and pseudotannins, Hamamelis Influenza A virus and Receptor binding (but not
gallic acid, epigallocatechin gallate or virginiana human neuraminidase) inhibition
hamamelitannin papillomavirus
16. Terpenoid and polyphenol Ocimum Influenza A virus Inhibits viral replication
sanctum H9N2
17. Polyphenol Punica Influenza A virus Inhibits the virus
granatum proliferation
18. Paeoniflorin, monoterpene glycosides, Paeonia Influenza virus Antineuraminidase (NA)
albiflorin, benzoylpaeoniflorin, gallic acid, delavayi activity
ethyl gallate
19. Oxypaeoniflorin, albiflorin, paeoniflorin, Paeonia Influenza virus A/ Inhibition of
benzoic acid, and paeonol lactiflora WSN/33 (H1N1) hemagglutination and
prevention of viral
penetration

(Continued )
218 Pandemic Outbreaks in the 21st Century

TABLE 12.2 (Continued)

S. no. Plant-derived compound Name of the Influenza viruses Mode of action

plant (swine flu and
Avian flu)
20. Plumbagin, allicin, carbohydrates, Plumbago Influenza A (H1N1) Obstruct the viral
flavonoids, proteins, saponins, fats and indica adsorption to cells
oils, alkaloids, steroids, phenols, and
tannins
21. Benzoquinones and embelin Embelia ribes Influenza virus A/ Suppression of viral
Puerto Rico/8/34 replication
(H1N1)
22. Flavonoids (catechin, hyperoside, Agrimonia Influenza viruses Inhibition of replication
quercitrin, quercetin and rutin), tannins pilosa (H1N1 and H3N2) process
and triterpenoids
23. Diarylheptanoids, monoterpenes, Alpinia Influenza virus Stops viral binding to the
sesquiterpenoid, flavonoids, and chalcones katsumadai type A cell receptor and viral
replication inhibition

structural and nonstructural proteins (membrane, envelope and nucleocapsid protein), RNA-dependent RNA polymer-
ase, 3CLPRO main proteinase, membrane proteases, ADP-ribose-1 monophosphatase, chimeric receptor-binding domain,
replicase protein, helicase, endoribonuclease, and so on [40,41], whereas membrane proteases and cathepsin L as drug
targets of the SARS and MERS. The enzyme neuraminidase is considered a potential drug target for swine flu and
Avian flu. The structural proteins of Ebola, viral proteins (24, 30, 35, and 40), heat shock protein 90, glycoproteins, and
Niemann-Pick Type C1 proteins act as drug targets. Zika virus consists of the following therapeutic target, namely,
ZIKV helicase protein, envelope protein domain, ZIKV protease, methyltransferase, and RNA-dependent RNA poly-
merase, NS2B-NS3 protease, envelope protein, and capsid protein.
Essentially, the following in silico methods are routinely used for drug discovery, (1) virtual screening: It is a
computational technique used to identify small molecule structures that are most likely fit on to a drug target known as
docking studies. (2) MD simulations: It is a computer simulation employed to analyze the physical movement of inter-
action between drug and drug-target complex within a fixed period, resulting in the view of the system’s dynamic evo-
lution. Furthermore, MD simulations are also majorly used to calculate the binding free energy, the number of
hydrogen bonds, root mean square deviation, root mean square fluctuation, the radius of gyration, structural conforma-
tional changes, and compactness of protein
ligand complexes. (3) QSAR: It is computational modeling, reveals the
relationship between the structural properties of the drug and biological activities. (4) Pharmacophore modeling: It can
be used to study the possible interactions between drug and drug targets with the matching of equivalent chemical
groups of the well-known drug molecule. The importance of in silico studies on drug designing from plant-derived anti-
viral compounds against pandemic outbreaks will be explained in detail as follows.

12.4.1 In silico screening of plant-derived antiviral compounds against pandemics

of the 21st century
As we discussed in previous sections about the importance of plant-derived compounds, recent literature has also been
reported that various phytochemicals have shown potential therapeutic activities against pandemic outbreaks to manage
disease propagation. Moreover, these plant compounds consist of diverse chemical groups with fewer side effects than
chemically synthesized drugs. Furthermore, the production of plant compounds is also budget-wise low cost than that
of industrial chemical synthesis. Hence, here we describe the plant compounds as therapeutics to pandemics, which
have been significantly validated through in silico strategies.

12.4.2 SARS and MERS

These two diseases show respiratory problems in human beings caused by coronaviruses. We found few research arti-
cles on in silico studies of plant-derived compounds against coronaviruses. A molecular simulation study has
 Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 219

demonstrated that a natural compound (MOL376) from a Traditional Chinese Medicine Database (TCMD) is a lead
drug molecule for cathepsin-L inhibition for SARS therapy [42]. In the same way, the natural compounds namely, Wu-
2 and AG7088 from TCMD, have strongly exhibited protease of SARS-CoV [43]. The plant compounds, namely, psora-
lidin, scutellarein, silvestrol, tryptanthrin, caffeic acid, quercetin, myricetin, saikosaponin B2, griffithsin (lectins), and
isobavachalcone, have shown antiviral activity against coronaviruses [44].

12.4.3 Swine flu and Avian flu

The subtypes of influenza A viruses cause both swine flu and Avian flu. Various in silico studies have been performed
on plant compounds to find out lead drug molecules against viruses different therapeutic targets. Molecular docking of
phytochemicals from Ocimum Sanctum has revealed that the compound apigenin has shown vigorous inhibitory activity
against neuraminidase and hemagglutinin H1N1 proteins [45]. Another study has shown that the compounds, namely,
stilbenoids_19, stilbenoids_23, stemonine, and tuberospironine from Stemonaceae have strongly inhibited neuramini-
dase of H1N1 [46]. Molecular docking has demonstrated that the compound theaflavin, found in green tea, has also sig-
nificantly inhibited neuraminidase [47]. Docking study on flavonoids has described that the compounds, namely,
quercetin, catechin, naringenin, luteolin, hispidulin, vitexin, chrysin, and kaempferol have effectively inhibited the
active site of neuraminidase [48]. One more in silico study has stated that the compound andrographolide from
Andrographis paniculata has shown effective inhibition against neuraminidase of H1N1 [49].
The compounds, namely, rubraxanthone, α-mangostin, and garcinone C from G. mangostana have shown the best
inhibition against neuraminidase of H5N1 (Avian flu) in virtual screening approaches [50]. Docking studies have
reported various plant compounds as potential inhibitors against the H5N1 influenza virus’s neuraminidase, including
artesunate 4-hydroxypanduratin A and Guajavin B [51
53].

12.4.4 Ebola virus disease

Ebola virus is a contagious disease; epidemiologically, it has reemerged in history. Accordingly, researchers have
developed a vaccine to manage this disease. Besides, few in silico studies were also reported some potential plant
compounds for the development of drug molecules to combat Ebola. Virtual screening of plant-phenolic compounds
against VP24-Ebola virus membrane-associated protein has revealed few potential inhibitors (1,2,3,6-tetragalloyl glu-
cose, epigallocatechin gallate, chlorogenic acid, oleuropein, and miquelianin) against this drug target [54]. Various
docking studies reported few plant compounds as potential inhibitors against different drug targets of the Ebola virus
(Table 12.3).

12.4.5 Zika fever

Zika fever is considered a reemerging outbreak since 1947; it has also warned researchers to find a suitable drug to
manage this disease worldwide. Numerous in silico researches have been reported on the Zika virus to design in vitro
and in vivo experiments to develop specific drug candidates. A study with MD simulations and pharmacokinetics has
demonstrated that tannic acid from Terminalia arjuna has interacted significantly with envelope protein of Zika virus
and suggested this compound to control Zika viral infection [58]. Another study has also proved in both in silico and

TABLE 12.3 Plant compounds as lead molecules against different drug targets of Ebola.

S. Plant compound as drug molecule Drug target of Ebola References

no.
1. Lominin Viral protein24 (VP24) [55]
2. Lominin, cosmosiin, and molludistin Viral protein35 (VP35)
3. Curcumin Viral protein30 (VP30)
4. Mesuaferrone B, euphorbianin, and mahanin Viral protein40 (VP40) [38]
5. 1-O-galloyl-6-O-luteoyl-α-D-glucose, euphorbianin, and scutellarein 7- Heat shock protein 90 [56]
neohesperidoside (Hsp90)
6. Neoandrographalide, fumaric acid, vasicoline, andrographalide, and GPs and NPC1s proteins [57]
andrograpanine
220 Pandemic Outbreaks in the 21st Century

in vitro studies that the three phytochemicals out of 5550 compounds from various databases, namely, eleutheroside B,
neoandrographolide, and apigenin, have strongly inhibited two targets, ZIKV helicase protein and envelope protein
domain III; moreover, these compounds also reduced Zika viral infection in the cellular model at noncytotoxic concen-
trations [59]. Likewise, the same authors have reported that the plant compounds, namely, galloylquinic acid, bacopa-
side III, and bacopaside A, were identified as lead molecules against multiple Zika virus targets [60]. In silico screening
of 2263 plant-derived compounds against ZIKV protease, methyltransferase, and RNA-dependent RNA polymerase, 43
compounds have shown anti-Zika viral activities inhibition of the above drug targets [61]. One more study with docking
and MD simulations has also stated that the plant compounds chicoric acid, luteone, reserpine, and rosmarinic acid
have strongly inhibited various drug targets of Zika, namely, ZIKA NS2B-NS3 protease, envelope protein, Capsid pro-
tein, and NS5 RNA-dependent RNA polymerase [62].

12.4.6 COVID-19
Currently, COVID-19 is a devastating pandemic outbreak globally. Since the beginning of this outbreak, researchers
have been hard working in various aspects of science to discover a potential therapeutic to combat COVID-19.
Accordingly, computational biologists have also been tried to find out the best drug molecules against this virus using
plant-derived compounds. From the recent findings, we found that researchers have reported various plant compounds
as therapeutics against different drug targets of COVID-19. Newly, docking and MD simulations work has been
reported that plant compounds, namely, bisdemethoxycurcumin, demethoxycurcumin, scutellarin, quercetin, and myri-
cetin, have exhibited potential inhibition against 3CLpro and endoribonuclease of SARS-CoV-2 [63]. Similarly, the
compounds lopinavir, amodiaquine, and theaflavin digallate have shown the best inhibition against the main protease
(Mpro) [64]. Other docking results have also reported that the phytochemicals including glycyrrhizin, bicylogerme-
crene, tryptanthrine, β-sitosterol, Indirubin, indican, indigo, hesperetin, crysophanic acid, rhein, berberine,
β-caryophyllene, epicatechin, and apoquine have exhibited potential inhibition against the Mpro of SARS-CoV-2 [65].
Furthermore, docking and network pharmacology studies reveal that plant compounds, namely, rocymosin B, verbasco-
side, rutin, caftaric acid, luteolin 7-rutinoside, fenugreekine, and cyaniding have shown best inhibition against the thera-
peutic targets of CL protease, PL protease, and RNA-dependent RNA polymerase [66]. One more study with docking,
MD simulations, and quantum computations has reported that the phytochemicals, namely, EryvarinM, Silydianin,
Osajin, and Raddeanine, have potentially inhibited methyltransferase, In contrast, TomentodiplaconeB, Osajin,
Sesquiterpene Glycoside, Rhamnetin, and Silydianin have strongly inhibited helicase of SARS-CoV-2 [67].
Another in silico study states that bonducellpin D acts as a broad-spectrum inhibitor against SARS-CoV M protease
and MERS-CoV M protease [68]. Another study in silico and in vitro has reported that specific plant compounds
including abietane-type and labdane-type diterpenes, lupane-type triterpenes, liganoids, curcumin, and so on have
shown precisely anti-SARS-CoV activity [69]. One more study with docking and MD simulation has demonstrated that
the phytochemicals, namely, qingdainone, edgeworoside C, and adlumidine have potentially inhibited the protease
(TMPRSS2) of SARS-CoV-2. Simultaneously, the compounds including ararobinol, (1)-oxoturkiyenine, and 3α,17α-
cinchophylline have also strongly inhibited the cathepsin protease [70]. Similarly, the plant compounds, namely, bisde-
methoxycurcumin, demethoxycurcumin, scutellarin, quercetin, and myricetin have shown strong inhibition activity
against main proteases 3CLpro and endoribonuclease [63]. In the same way, in silico studies (docking and MD simula-
tions) on hundred plant compounds of ten medicinal plants reveals that the compounds, namely, alizarin, aloe-emodin,
and anthrarufin have shown potential inhibition against the N terminal domain of RNA binding domain of nucleocapsid
protein of SARS-CoV-2 [71]. A study with the virtual screening on 318 phytochemical of 11 medicinal plants has dem-
onstrated that the plant compounds, namely, piperolactam A, quercetin 3-glucuronide-7-glucoside, quercetin
3-vicianoside, schaftoside, chrysoeriol 8-C-glucoside, isosakuranetin 7-O-neohesperidoside, delphinidin 3-O-glucoside,
petunidin 3-O-glucoside, riboflavin oleanolic acid, 3-0-caffeoylquinic, absinthin, anabsinthin, and dicaffeoylquinic acids
and quercetin-7-O-galactoside, 3,5-dicaffeoylquinic acid, and 3,4,5-tricaffeoylquinic acid have confirmed strong binding
affinity against the Mpro and ACE2 [72].

12.5 Future prospective and limitations of in silico studies

The 21st century has brought severe pandemic outbreaks with high mortality rates globally. Accordingly, the current
pandemic, COVID-19, has taught us many things about its severe impact on health and economic conditions globally.
For future pandemics, there will be urgent need to identify symptoms, and to develop rapid molecular diagnostics, so
that the disease may be detected in a short time. Consequently, necessary safety measures have to be taken before the
 Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 221

situation gets out of control. In general, from the experiences of the current pandemic situation, globally, every human
has to act responsibly to take necessary precautions to prevent the spreading of any pandemic in the future. Besides, the
government has to encourage researchers with enough funds to initiate rapid projects in all biomedical disciplines to
discover potential drugs or vaccines to combat pandemics before spreading disease throughout the world. From the past
pandemics, it is clear that coronavirus subtypes have been remerging as outbreaks, including SARS, MERS, and SARS-
CoV-2. Hence, there will be a chance again for the recurrence of coronavirus kind of outbreak in the future.
Accordingly, the public health agency (WHO) and scientific research communities should be alert to public health
issues.
Following the recognition of a new threat to public health, researchers initially have to identify the causative agent’s
structure and its therapeutic targets. Then computational biologists must work on the modeling of various drug targets
using bioinformatics tools. Computer-aided drug designing is a powerful strategy to identify and recommend novel
drug molecules to carry out in vitro, in vivo. Additionally, clinical trial research is also necessary to discover
suitable therapy against new diseases. Advanced in silico techniques have to be suggested to carry out research to find
potential lead molecules, including target-centric drug design methods and emerging compound-centric virtual targeting
profile strategies [73]. Target-centric drug design consists of structure-based drug design (docking, scoring, and de
novo design) and ligand-based drug design (similarity search, pharmacophore mapping, shape comparison, and
machine/deep learning) with the filtering of drug-likeness properties. However, compound-centric drug design consists
of high-throughput screening, binding site comparison, shape comparison, pharmacophore mapping, interaction finger-
printing, and machine/deep learning or hybrid approaches. In silico strategies are highly useful to find out repurposing
drug molecules against pandemics from plant/chemical compound libraries.

12.6 Conclusion
Pandemics are contagious diseases causing global health emergencies due to their severity on health and mortality. For
instance, drug discovery, plant-derived compounds, or existing medications are the primary sources for finding a poten-
tial drug molecule against the outbreak. Furthermore, in silico studies are the best ways than experimental research to
screen, design, and predict lead drug molecules against pandemic diseases using plant/chemical compound libraries.
Advanced strategies, namely, target-centric drug design and emerging compound-centric drug design methods, have
been suggested as novel approaches for future drug discovery.

Acknowledgment
The authors would like to thank IISER Tirupati and IIT Tirupati for their financial support.

References
[1] Porta M, editor. A Dictionary of epidemiology. 6th (ed.) Oxford: Oxford University Press; 2014.
[2] Hughes JM, Wilson ME, Pike BL, Saylors KE, Fair JN, LeBreton M, et al. The origin and prevention of pandemics. Clin Infect Dis 2010;50
(12):1636
40.
[3] Wolfe ND, Dunavan CP, Diamond J. Origins of major human infectious diseases. Nature 2007;447(7142):279
83.
[4] Madhav N, Oppenheim B, Gallivan M, Mulembakani P, Rubin E, Wolfe N. Pandemics: risks, impacts, and mitigation; 2017.
[5] Butler MS, Robertson AA, Cooper MA. Natural product and natural product derived drugs in clinical trials. Nat Prod Rep 2014;31
(11):1612
61.
[6] Garcia S. Pandemics and traditional plant-based remedies. A historical-botanical review in the era of COVID19. Front Plant Sci
2020;11:571042. Available from: https://doi.org/10.3389/2Ffpls.2020.571042.
[7] Atanasov AG, Waltenberger B, Pferschy-Wenzig E-M, Linder T, Wawrosch C, Uhrin P. Discovery and resupply of pharmacologically active
plant-derived natural products: a review. Biotech Adv 2015;33:1582
614. Available from: https://doi.org/10.1016/j.biotechadv.2015.08.001.
[8] Nussinov R. Advancements and challenges in computational biology. PLoS Comput Biol 2015;11(1):e1004053.
[9] Brogi S, Ramalho TC, Kuca K, Medina-Franco JL, Valko M. In silico methods for drug design and discovery. Front Chem 2020;8:612.
Available from: https://doi.org/10.3389/fchem.2020.00612.
[10] Ekins S, Mestres J, Testa B. In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Br J Pharmacol
2007;152(1):9
20.
[11] Mishra D, Mishra A, Chaturvedi VK, Singh MP. An overview of COVID-19 with an emphasis on computational approach for its preventive
intervention. 3 Biotech 2020;10:1
3.
[12] Huremović D. Brief history of pandemics (pandemics throughout history). Psychiatry of pandemics. Cham: Springer; 2019. p. 7
35.
222 Pandemic Outbreaks in the 21st Century

[13] Ciotti M, Angeletti S, Minieri M, Giovannetti M, Benvenuto D, Pascarella S, et al. COVID-19 outbreak: an overview. Chemotherapy 2019;64
(5
6):215
23.
[14] LeDuc JW, Barry MA. SARS, the first pandemic of the 21st century. Emerg Infect Dis 2004;10(11):e26.
[15] Nishiura H, Brockmann SO, Eichner M. Extracting key information from historical data to quantify the transmission dynamics of smallpox.
Theor Biol Med Model 2008;5:20. Available from: https://doi.org/10.1186/1742-4682-5-20.
[16] Martins NR. An overview on avian influenza. Braz J Poult Sci 2012;14(2):71
87.
[17] OIE - Office International des Epizooties.World organization for animal health. Highly pathogenic avian influenza Aetiology epidemiology
diagnosis prevention and control references; 2012. Available from: http://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/
pdf/AVIAN_INFLUENZA_FINAL.pdf. [cited 03 July 2012].
[18] Lu DY, Lu TR, Wu HY. Avian flu; pathogenesis and therapy. Anti-Infect Agents 2012;10(2):124
9.
[19] Uyeki TM. Human infection with highly pathogenic avian influenza A (H5N1) virus: review of clinical issues. Clin Infect Dis 2009;49
(2):279
90.
[20] WHO. Middle East respiratory syndrome coronavirus (MERS-CoV). Available from: https://www.who.int/emergencies/mers-cov/en/ [Accessed
12 February 2020).
[21] Zumla A, Hui DS, Perlman S. Middle East respiratory syndrome. Lancet 2015;386(9997):995
1007.
[22] Yong CY, Ong HK, Yeap SK, Ho KL, Tan WS. Recent advances in the vaccine development against Middle East respiratory syndrome-
coronavirus. Front Microbiol 2019;10:1781.
[23] Dawood FS, Iuliano AD, Reed C. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus cir-
culation: a modelling study. Lancet Infect Dis 2012;12(9):687
95. Available from: https://doi.org/10.1016/S1473-3099(12)70121-4.
[24] McNeil Jr DG. In new theory, swine flu started in Asia, not Mexico. The New York Times; 2009.
[25] Sebastian MR, Lodha R, Kabra SK. Swine origin influenza (swine flu). Indian J Pediatr 2009;76(8):833
41.
[26] Rewar S, Mirdha D, Rewar P. Treatment and prevention of pandemic H1N1 influenza. Ann Glob health 2015;81(5):645
53.
[27] Tseng CP, Chan YJ. Overview of Ebola virus disease in 2014. J Chin Med Assoc 2015;78(1):51
5.
[28] United States Food and Drug Administration. sirst FDA-approved vaccine for the prevention of Ebola virus disease, marking a critical milestone
in public health preparedness and response. Press Announcements; 2019.
[29] Lucey DR. New treatments for Ebola virus disease; 2019.
[30] Musso D, Nilles EJ, Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infect 2014;20(10):O595
6.
[31] Galan-Huerta KA, Rivas-Estilla AM, Martinez-Landeros EA, Arellanos-Soto D, Ramos-Jiménez J. The Zika virus disease: an overview.
Medicinauniversitaria 2016;18(71):115
24.
[32] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.
Lancet 2020;395:497
506.
[33] Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, et al. Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a
descriptive study. Lancet Infect Dis 2020;20:425
34.
[34] Sanders JM, Monogue ML, Jodlowski TZ, Cutrell JB. Pharmacologic treatments for coronavirus disease 2019 (COVID-19): a review. JAMA
2020;323(18):1824
36.
[35] Bhuiyan FR, Howlader S, Raihan T, Hasan M. Plants metabolites: possibility of natural therapeutics against the COVID-19 pandemic. Front
Med 2020;7(7):444. Available from: https://doi.org/10.3389/fmed.2020.00444.
[36] Yogesh S, Amol J, Rakeshkumar T, Chetan S. Review on-herbal drug treatment on Ebola virus. J Pharmacogn Phytochem 2016;5(2):47.
[37] Lane T, Anantpadma M, Freundlich JS, Davey RA, Madrid PB, Ekins S. The natural product eugenol is an inhibitor of the ebola virus in vitro.
Pharm Res 2019;36(7):104.
[38] Nasution MA, Alkaff AH, Tambunan United States. Discovery of Indonesian natural products as potential inhibitor of Ebola virus VP40
through molecular docking simulation. AIP Conf Proc 2018;2023(1):020055.
[39] Biedenkopf N, Lange-Grünweller K, Schulte FW, Weißer A, Müller C, Becker D, et al. The natural compound silvestrol is a potent inhibitor of
Ebola virus replication. Antivir Res 2017;137:76
81.
[40] Nimgampalle M, Devanathan V, Saxena A. Screening of chloroquine, hydroxychloroquine and its derivatives for their binding affinity to multi-
ple SARS-CoV-2 protein drug targets. J Biomol Struct Dyn 2020;23:1
3.
[41] Saxena A. Drug targets for COVID-19 therapeutics: ongoing global efforts. J Biosci 2020;45(1):1
24.
[42] Wang SQ, Du QS, Zhao K, Li AX, Wei DQ, Chou KC. Virtual screening for finding natural inhibitor against cathepsin-L for SARS therapy.
Amino Acids 2017;33(1):129
35.
[43] Liu B, Zhou J. SARS-CoV protease inhibitors design using virtual screening method from natural products libraries. J Comput Chem 2005;26(5):484
90.
[44] Attia YA, Alagawany MM, Farag MR, Alkhatib FM, Khafaga AF, Abdel-Moneim AM, et al. Phytogenic products and phytochemicals as a can-
didate strategy to improve tolerance to coronavirus. Front Vet Sci 2020;7:573159. Available from: https://doi.org/10.3389/fvets.2020.573159.
[45] Alhazmi MI. Molecular docking of selected phytocompounds with H1N1 Proteins. Bioinformation 2015;11(4):196.
[46] Dammalli M, Chandramohan V, Biradar MI, Nagaraju N, Gangadharappa BS. In silico analysis and identification of novel inhibitor for new
H1N1 swine influenza virus. Asian Pac J Trop Dis 2014;4:S635
40.
[47] Sahoo M, Jena L, Rath SN, Kumar S. Identification of suitable natural inhibitor against influenza A (H1N1) neuraminidase protein by molecular
docking. Genomics Inform 2016;14(3):96.
 Importance of in silico studies on the design of novel drugs from medicinal plants against 21st-century Chapter | 12 223

[48] Sadati SM, Gheibi N, Ranjbar S, Hashemzadeh MS. Docking study of flavonoid derivatives as potent inhibitors of Influenza H1N1 virus neur-
aminidase. Biomed Rep 2019;10(1):33
8.
[49] Seniya C, Shrivastava S, Singh SK, Khan GJ. Analyzing the interaction of a herbal compound Andrographolide from Andrographis paniculata
as a folklore against swine flu (H1N1). Asian Pac J Trop Dis 2014;4:S624
30.
[50] Ikram NK, Durrant JD, Muchtaridi M, Zalaludin AS, Purwitasari N, Mohamed N, et al. A virtual screening approach for identifying plants with
anti H5N1 neuraminidase activity. J Chem Inf Model 2015;55(2):308
16.
[51] Parikesit AA, Ardiansah B, Handayani DM, Tambunan US, Kerami D. Virtual screening of Indonesian flavonoid as neuraminidase inhibitor of
Influenza A subtype H5N1. IOP Conf Ser Mater Sci Eng 2016;107:12053
62.
[52] Pratama MR, Gusdinar T. Between artemisinin and derivatives with neuraminidase: a docking study insight. Asian J Pharm Clin Res 2017;10(8):304
8.
[53] Syafrudin S, Septiadi L, Alfaruqi NT, Wahyudi D, Kharisma VD. The in silico analysis and identification of possible inhibitor of H5N1 virus.
Bioinforma Biomed Res J 2018;1(2):33
9.
[54] Plesko S, Volk H, Lukšič M, Podlipnik Č. In silico study of plant “polyphenols” interactions with VP24
Ebola virus matrix protein. Acta
Chim Slovenic 2015;62(3):555
64.
[55] Setlur AS, Naik SY, Skariyachan S. Herbal lead as ideal bioactive compounds against probable drug targets of Ebola virus in comparison with
known chemical analogue: a computational drug discovery perspective. Interdiscip Sci Comput Life Sci 2017;9(2):254
77.
[56] Haikal MC, Nasution MA, Saputro L, Tambunan United States. In silico identification of potent inhibitors of heat shock protein 90 (Hsp90)
from Indonesian natural product compounds as a novel approach to treat ebola virus disease. IOP Conf Ser: Mater Sci Eng 2019;509
(1):012082.
[57] Jeyaram C, Hemaiswarya S, Jaihari KN, Ramasamy MS. GP and NPC1 targeted herbal compound
an in silico evidence for Ebola drugs. Ann
Pharmacol Pharm 2017;2(13):1133.
[58] Priya S, Kumar NS, Hemalatha S. Antiviral phytocompounds target envelop protein to control Zika virus. Comput Biol Chem 2018;77:402
12.
[59] Sangeetha K, Martı́n-Acebes MA, Saiz JC, Meena KS. Molecular docking and antiviral activities of plant derived compounds against zika virus.
Microb Pathog 2020;149:104540.
[60] Kothandan S, Purushothaman I, George SP, Purushothaman SR, Sulochana MK. Molecular docking and density function theory studies of com-
pounds from Euphorbia hirta and Bacopa monnieri to zika virus structural and nonstructural proteins. Pharmacogn Mag 2018;14(57):481.
[61] Byler KG, Ogungbe IV, Setzer WN. In-silico screening for anti-Zika virus phytochemicals. J Mol Graph Model 2016;69:78
91.
[62] Ahmed SR, Banik A, Anni SM, Chowdhury MM. Plant derived bioactive compounds as potential inhibitors of ZIKA virus: an in silico investi-
gation. bioRxiv 2020.
[63] Sharma A, Goyal S, Yadav AK, Kumar P, Gupta L. In-silico screening of plant-derived antivirals against main protease, 3CLpro and endoribo-
nuclease, NSP15 proteins of SARS-CoV-2. J Biomol Struct Dyn 2020;1
15.
[64] Peele KA, Chandrasai P, Srihansa T, Krupanidhi S, Sai AV, Babu DJ, et al. Molecular docking and dynamic simulations for antiviral com-
pounds against SARS-CoV-2: a computational study. Inform Med Unlocked 2020;11:100345.
[65] Narkhede RR, Pise AV, Cheke RS, Shinde SD. Recognition of natural products as potential inhibitors of COVID-19 main protease (Mpro): in-
silico evidences. Nat Prod Bioprospect 2020;10(5):297
306.
[66] Shawky E, Nada AA, Ibrahim RS. Potential role of medicinal plants and their constituents in the mitigation of SARS-CoV-2: identifying related
therapeutic targets using network pharmacology and molecular docking analyses. RSC Adv 2020;10(47):27961
83.
[67] Kousar K, Majeed A, Yasmin F, Hussain W, Rasool N. Phytochemicals from selective plants have promising potential against SARS-CoV-2:
investigation and corroboration through molecular docking, MD simulations, and quantum computations. BioMed Res Int 2020;2020:6237160.
Available from: https://doi.org/10.1155/2020/6237160.
[68] Gurung AB, Ali MA, Lee J, Farah MA, Al-Anazi KM. Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro
enzyme through in silico approach. Life Sci 2020;22:117831. Available from: https://doi.org/10.1016/j.lfs.2020.117831.
[69] Wen CC, Kuo YH, Jan JT, Liang PH, Wang SY, Liu HG, et al. Specific plant terpenoids and lignoids possess potent antiviral activities against
severe acute respiratory syndrome coronavirus. J Med Chem 2007;50(17):4087
95.
[70] Vivek-Ananth RP, Rana A, Rajan N, Biswal HS, Samal A. In silico identification of potential natural product inhibitors of human proteases key
to SARS-CoV-2 infection. Prepr arXiv:2006 00652; 2020.
[71] Rolta R, Yadav R, Salaria D, Trivedi S, Imran M, Sourirajan A, et al. In silico screening of hundred phytocompounds of ten medicinal plants as
potential inhibitors of nucleocapsid phosphoprotein of COVID-19: an approach to prevent virus assembly. J Biomol Struct Dyn 2020;1
8.
[72] Joshi T, Joshi T, Sharma P, Mathpal S, Pundir H, Bhatt V, et al. In silico screening of natural compounds against COVID-19 by targeting Mpro
and ACE2 using molecular docking. Eur Rev Med Pharmacol Sci 2020;24(8):4529
36.
[73] Du J, Guo J, Kang D, Li Z, Wang G, Wu J, et al. New techniques and strategies in drug discovery. Chin Chem Lett 2020;31(7):1695
708.
Available from: https://doi.org/10.1016/j.cclet.2020.03.028.
This page intentionally left blank
Chapter 13

Recent developments in the diagnosis of

COVID-19 with micro- and nanosystems
Manpreet Singh1, Kamal Kishore1 and Seshadri Reddy Ankireddy2
1
Department of Chemistry, Akal College of Basic Sciences, Eternal University, Sirmour, India, 2Department of Chemical and Biological Sciences,
Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr. Buddolla’s Educational Society, Tirupati, India

13.1 Introduction
Since after inception of the SARS-CoV-2 victims in 2019, morbidity and mortality rates have been increasing on daily
basis, and still, situations are not controlled adequately. The major symptoms like fever, dry cough, dehydration,
fatigue, headache, loss of smell, taste, cognition, shortness of breath or difficulty breathing, and diarrhea are commonly
observed among the SARS-CoV-2-affected patients [1]. The mortality rate can be increased and led to serious outbreaks
if the patient fails to maintain the proper medical precautions that are suggested by the concerned physicians. After a
1-year struggle, in the year 2021 January, the virus victims have been dropped down fruitfully. However, the second
phase was started again from march 2021 onward not only in India but also across the globe. As per the WHO medical
reports, approximately 3,017,109 outbreaks have been recorded worldwide and 176,745 deaths were identified in India
as of April 2021 due to negligence and violating the medical precautions [2]. Although having a breath of recovery
rate, the virus has continued its prevalence in many countries via a variety of clinical manifestations. During its tenure,
Remdesivir is the only drug that was approved by the FDA and it has been shown affirmative results on the SARS-
CoV-2 virus [3]. In addition, the Covaxin vaccine was developed by Bharat Biotech
Indian Council of Medical
Research associated with the National Institute of Virology against the COVID 19 virus [4,5].
Despite the great advancements for the treatment of COVID-19 using the aforementioned medications, COVID-19
active cases were also still rising dramatically due to the formation of more mutants as the second wave of infection
which are highly susceptible to the developed vaccine and drugs as well. Interestingly, it is also quite difficult to distin-
guish the new strains using existing technologies including conventional and advanced methodologies. Before going for
the treatment, accurate identification methodologies are in great demand for the COVID-19. Currently, molecular
genetic material level detection assay approaches like reverse transcription-polymerase chain reaction (RT-PCR) are a
more prominent and accurate method for the identification of the virus. However, a few drawbacks are also associated
with this RT-PCR technique. Primarily, the instrument itself is quite expensive and not affordable to everyone who is
working in the research areas. Second, instrumentation, procedure, protocols, and reaction conditions are also too
sophisticated to maintain the reaction results of every cycle. Third and more importantly, more accurate results can be
obtained 6
10 h later once the reaction is started. During the reaction tenure, several reaction steps, incubation time,
and addition of required reagents are crucial steps and are tedious protocols to follow up the corresponding specialized
techniques. On the other hand, the major disadvantage is that it is unable to produce the results within the given framed
time for the proper treatments due to the rapid increase in cases per day. Henceforth, there is additional room for alter-
native approaches to detect the virus’s existence, efficacy, load, and infection rate with more accurate results compared
to the conventional approach within less time [6].
To overcome the aforementioned key issues, nanotechnology- or nanomaterials-mediated detection assays as biosen-
sors can offer amazing challenges to detect the COVID-19 virus at initial stages with low concentration levels.
Interestingly, divergent nanotechnology-mediated approaches like colorimetric, electrochemical, acoustic, microfluidic
chip-based, and fluorescence-based detection assays are available. Wherein, gold nanoparticles (AuNPs) [7], carbon
dots [8], graphene oxide (GO) [9], copper oxide (Cu2O) [10], titanium dioxide (TiO2) [11], nanohybrid platforms [12],

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00017-3

© 2021 Elsevier Inc. All rights reserved. 225
226 Pandemic Outbreaks in the 21st Century

metallic Quantum Dots [13], iron nanoparticles (Fe3O4) [14], and polymer-based nanoformulations matrixes [15] act as
the active platforms where corresponding reactions can be accomplished. In addition to these approaches, different
organic and inorganic complexes mediated approaches are also available for the detection COVID-19 virus with the
help of different enzymatic or fluorophore modifications via chemical reactions.
The above-stated approaches are involved in different working principles with molecular mechanisms that can
enhance the trapping of the COVID-19 virus in respective platforms. As a result, corresponding results can be recog-
nized using various transducers based on the working reaction conditions. Interestingly, some of the approaches were
also developed with the microfluidic chip-based point-of care-detection kits, where these kits are involved with either
enzyme-linked or nucleic acid-based methodologies. Finally, nanotechnology-mediated biosensors are also currently
proving their significant importance by providing accurate results quickly compared to the conventional approaches and
these approaches. As per their performances, these techniques are robust, cheap, efficient, and much faster to produce
results. Therefore many authorized laboratories and research centers are using this technology as a preliminary confir-
mative tool for the detection of the COVID-19 virus in biological samples.

13.2 SARS CoV-19 structure

Coronaviruses are spherical, which come under the family of Coronaviridae in the order Nidovirales. These viruses are
broadly classified in the genera of alpha, beta, gamma, and delta viruses. Structurally, it has several components like a
membrane, integrated transmembrane proteins, envelopes, and spike proteins as presented in Fig. 13.1. Nucleocapsid
protein with a single standard positive-sense RNA as genetic material is also present inside the cell. These components
are crucial for attacking of host molecules either directly or indirectly. However, among them, spike proteins are more
important to bind the host molecules. Above-stated classifications of viruses, the Middle East respiratory syndrome
(MERS), severe acute respiratory syndrome (SARS) virus (SARS-CoV), and COVID-19 causative agent SARS-CoV-2
virus, are coming under the category of beta viruses. According to the medical reports, glycosylated spike protein is
responsible for the major booster of immune response that can be encoded by the betacoronavirus genome. As a result,
both SARS-CoV and SARS-CoV-2 viruses are ready to attack the host cell through angiotensin-converting enzyme 2
located on the surface membrane of host cells via invasion process. After initiation of this process, the genetic material
of the SARS-CoV-2 virus is entered into the host cell and subsequently encoded with the host cell intracellular proteins
like papain-like protease (PLpro), coronavirus main protease (3CLpro), and RNA-dependent RNA polymerase (RdRp)
[16].

13.3 Micro- and nanosystems for the diagnosis of COVID-19

Despite the existence of conventional detection and diagnosis approaches for the COVID-19, micro- and
nanotechnology-based assays are also gaining special focus for the diagnosis of the COVID-19 virus even in low con-
centration levels. However, most of the nanomaterials-mediated approaches are also again linked with corresponding
antibody and antigen interactions. Interestingly, prominent COVID-19 detection approaches are mainly classified into
three types including nucleic acid (genetic material), serological, and nanomaterial-based sensor technologies. On the
other hand, nanomaterial-based biosensors are showing affirmative results based on their structural and chemical prop-
erties of antigen and antibody interactions with functional nanomaterials through filtration process by functional nano-
materials inserted face masks, colorimetric naked eye detection, fluorescence, and electrochemical assays as illustrated
in Fig. 13.2.

FIGURE 13.1 Schematic representation of SARS-CoV-19 structure with its struc-

tural components.
 Recent developments in the diagnosis of COVID-19 with micro- and nanosystems Chapter | 13 227

Li and his coworkers have developed a point-of-care chip-mediated microsystem-based detection kit. In this
approach the working principle is lateral flow immunoassay where COVID-19 was detected against immunoglobulin M
(IgM) and immunoglobulin G (IgG) antibodies simultaneously. They have developed a strip-based material that consists
of five components including plastic backing, sample pad, conjugate pad, absorbent pad, and nitrocellulose membrane
layer. The antihuman IgM, antihuman IgG, and anti-rabbit-IgG were immobilized on the nitrocellulose membrane and
represented as M, G, and C lines, respectively. This layer was bounded with the plastic backing layer for the supportive
purpose. In a separate reaction chamber, AuNP-COVID-19 recombinant antigen conjugate and AuNP-rabbit-IgG were
sprayed on the conjugate pad. After preparation of the strip, approximately 20 μL of the blood sample or 20 μL serum/
plasma samples were mixed with the 70
100 μL of elusion buffer to drive the capillary action. Finally, corresponding
bands have appeared when the surface antigen from SARS-CoV-2 is specifically interacted with the antihuman IgM,
antihuman IgG. The colorization bands appeared due to the conjugation of AuNPs with the corresponding antibodies as
represented in Fig. 13.3 [17]. In another report, Hussain et al. have reported a flexible, removable, and reusable N95
surgical grade mask for the effective trapping of the SARS-CoV-19 virus. As normally developed cloth or cellulose-
based face masks for the protection from SARS-CoV-19 virus is a tedious and difficult process due to the pore size of
the masks. Usually, the size of the SARS-CoV-19 virus is between 50 and 140 nm, which is almost twice or thrice
smaller than the commercially available face masks ($300 nm). Based on the above information, the authors have
developed a lesser pore size masks than the COVID-19 virus size. Herein, a Si-based flexible nanoporous template has
been designed using KOH etching on a silicon-on-insulator wafer. Later the mask design was transferred on to a flexi-
ble polymeric membrane, which facilitates the pore size between 5 and 10 nm. The developed mask was much effective
and efficient to capture and control the COVID-19 viruses with less porous-sized face masks. Interestingly, the outer
polymeric flexible layer is easy to remove and reusable after gentle wash [18].
On the other hand, colorimetric naked eye sensors are also getting special interest in the medical research field for
robust and effective measurements. For this approach, nanomaterials have been used with the surface modification by
nucleic acid or antigen and antibodies. In this regard, Kang and his coworkers have developed a colorimetric detection
of MERS-CoV using surface-modified nucleic acid materials via thiolated ligands. The developed strategy is much effi-
cient to detect the viral molecules via localized surface plasmon resonance shift and color changes of AuNPs in the
presence of 1 pmol μL21 of 30 bp MERS-CoV [19]. Nanomaterial-based fluorescence turns on/off and on the mecha-
nism is also one of the scalable and convenient approaches for the detection of the SARS-CoV-2 virus. Herein,
DNA-stabilized silver nanoclusters (DNA-AgNCS) were synthesized. Based on the stabilization of genetic material, the
fluorescent emission properties have been changing for the detection of viral entities. Interestingly, blue color emission
DNA-AgNCS were synthesized using a sol-gel approach and as-prepared nanomaterials, blue color fluorescence has
been quenched upon addition of SARS-CoV-2 virus molecules [20].
Apart from the colorimetric and fluorometric approaches, electrochemical biosensors are also providing excellent
information on the detection of SARS-CoV-2 from real samples. In this approach, generally, conductive nanomaterials
can be used on the supporting platform. Furthermore, these nanomaterials surface is also modified with respective

FIGURE 13.2 Possible detection

approaches for the COVID-19 virus
via nucleic acid, serological and
nanomaterials-based biosensors.
228 Pandemic Outbreaks in the 21st Century

FIGURE 13.3 (A) Schematic representation of preparation of strip-based point-of-care lateral immune flow cytometry for the detection of COVID-
19 virus. (B) Corresponding test results in the form of band formation. Adapted from Li Z, Yi Y, Luo X, Xiong N, Liu Y, Li S, et al. Development and
clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J Med Virol 2020;92(9):1518
24.

human antibodies or RNA molecules. After that, proper electrochemical signals can be observed after the addition of
the corresponding SARS-CoV-2 virus sample into the electrochemical platform. Based on this strategy, several effective
methodologies have been developed for the detection of the SARS-CoV-2 virus in real samples. Kim and his team
developed a GO oxide-functionalized field-effect transistor (FET) platform. In this approach, the FET platform was
functionalized with the GO molecules and followed by the addition of corresponding human anti-IgG and IgM antibo-
dies which were against the SARS-CoV-2 virus antivirus samples. Using this approach, 1 and 100 fg mL21 of virus
samples were detected from phosphate buffer saline and clinical transport medium, respectively [21].
Based on similar strategies, many other types of methodologies were developed for the low concentration working
load levels of the SARS-CoV-2 virus in real samples. Keeping in view the severity of the COVID-19, effective strate-
gies need to be explored to expand the testing capacities, to develop potent therapeutics and safe vaccines to provide
long-lasting immunity. Apart from various strategies and techniques that are being explored for the detection of
COVID-19, nanotechnology could be one of the best alternatives. In this subtopic, we are going to explore various
nanomaterials that are effective in detecting coronaviruses. Nanomaterials have already been successfully employed in
various applications such as medical, biosensing, drug delivery, and antimicrobial treatment [22
25]. The applicability
of nanoparticles in tackling this pandemic situation depends on their various properties such as wide range of chemical
composition, size, and shape of nanoparticles, biocompatibility, tunable biological and physical properties along with
ease of production [26].
There is no denying the fact that nanomaterial-based vaccines or antiviral agents are presently far from implementa-
tion because of lengthy and strict regulatory reforms [27]. Therefore viral detection seems to be a better alternative for
nanomaterials to have a closer impact on the current pandemic situation in very little time. Moreover, it would be bene-
ficial if the detection methods are reliable, accurate, less expensive, easy to handle and use. Although RT-PCR methods
are one of the best known standard methods its limitations have led to the incorporation of nanoparticles not only in
RT-PCR methods but also for other detection methods including an enzyme-linked immune-sorbent assay and reverse
 Recent developments in the diagnosis of COVID-19 with micro- and nanosystems Chapter | 13 229

transcription loop-mediated isothermal amplification (RT-LAMP) [28,29]. Nanoparticles such as metal nanoparticles,
carbon nanotubes, quantum dots, silica nanoparticles, and polymeric nanoparticles have been widely studied for virus
detections [30]. On the other hand, metal nanoparticles, metal nanoislands, magnetic nanoparticles, and quantum dots
have been extensively used for the detection of coronaviruses. Most of the diagnostic procedures for detection are
broadly based on four major techniques, namely, colorimetric, electrochemical, fluorescence, and optical detection [31].
Apart from the noble metal nanoparticles, Au nanoparticles are the most common metal nanoparticles that have
gained importance for coronavirus diagnostic detection due to their unique stability, biocompatibility, and optical prop-
erties [12,32,33]. Li and Rothberg reported the detection of SARS-CoV using Au nanoparticles. In this study, the differ-
ence in electrostatic properties of ssDNA and dsDNA were used to design a sample oligonucleotide recognition assay
using commercially available materials. The results were observed within 10 min without the need for complicated
apparatus. The formation of dsDNA was confirmed with a target concentration of 4.3 mM [34]. Recently, Alafeef et al.
reported the development of a low-cost, easy to implement, and quantitative paper-based electrochemical sensor chip
made from the conjugation of graphene AuNPs and antisense oligonucleotides for the detection of SARS-COV-2 viral
RNA. The sensor chip was tested on Vero cells infected with the SARS-CoV-2 virus and clinical samples. A significant
improvement has been observed in the output signal only in the presence of SARS-CoV-2 RNA in less than 5 min of
incubation along with a sensitivity of 231 (copies μL21)21 and limit of detection 6.9 copies μL21 without any further
amplification [35]. In one recent study by Moitra et al. a colorimetric assay based on AuNPs capped with thiol-
modified antisense oligonucleotides specific for N-gene (Nucleocapsid phosphoprotein) of SARS-CoV-2 was developed
and used for naked-eye detection of COVID-19 virus. The results were obtained in less than 10 min after the isolation
of RNA samples with a reported detection limit of 0.18 ng μL21 [36].
The ability of nanoparticles to functionalize with biomolecules further modifies their surface properties. Huang et al.
studied Au nanoparticles combined with streptavidin to use in RT-LAMP combined with a vertical flow visualization
strip (RT-LAMP-VF) assay for the detection of MERS-CoV-2 nucleic acid. The reported results were visible to the
naked eye within 5 min. The RT-LAMP-VF assay was able to detect 2 3 101 copies μL21 of synthesized RNA tran-
script and 1 3 101 copies μL21 of MERS-CoV RNA. No cross-reactivities were observed with multiple CoVs including
SARS-related (SARS-CoV), HKU4, HKU1, OC43, and 229E thereby showing high specificity [37]. Further Au nano-
particles have also been reported for the electrochemical detection of coronaviruses. Layqah and Eissa studied carbon
electrodes modified with Au nanoparticles for electrochemical detection of HcoV and MERS-CoV. The biomarker used
for MERS-CoV is recombinant spike protein S1. The method is still able to detect both HCoV and MERS-CoV with
detection limits as 0.4 and 1.0 pg mL21, respectively. The method is highly selective and gives results within 20 min.
Apart from Au nanoparticles, researchers have suggested other nanoparticles for detection such as silver nanoparticles.
Teengam et al. proposed a paper-based colorimetric DNA sensor for the detection of MERS-CoV. The device is also
helpful in the screening of mycobacterium tuberculosis (MTB) and human papillomavirus (HPV). Ag nanoparticles
were used as a colorimetric reagent for DNA detection based on acpcPNA-induced nanoparticle aggregation. The sensor
was found to have high selectivity against a single-base mismatch, two-base mismatch, and noncomplementary target
DNA with a detection limit of 1.53, 1.27, and 1.03 nM for MERS-CoV, MTB, and HPV, respectively [38].
Magnetic nanoparticles have drawn the interest of the scientific community due to their property of separating viral
RNA from solution by applying an external magnetic field [39,40]. Due to high magnetization and simple synthetic pro-
cedures, magnetic nanoparticles especially iron oxide nanoparticles are considered important in biological applications
[41,42]. Gong et al. studied the detection of the SARS-CoV gene. In the study, silica-coated superparamagnetic nano-
particles were used in PCR-based assay to improve the selectivity of the target cDNA of SARS-COV. Viral target
cDNAs were captured by conjugating superparamagnetic nanoparticles with oligonucleotide probes to produce a
magnetic-conjugated dsDNA complex. Magnetic separation was used to separate magnetic-conjugated dsDNA from
other components. Sandwich hybridization assay with silica-coated fluorescent nanoparticle-based signaling probes was
used to detect the final amplified viral target cDNA. The reported detection of limit for this method was 2.0 3 103 cop-
ies within the time of 6 h [43]. Recently, the properties of magnetic nanoparticles are further explored for the detection
of SARS-CoV-2. The literature shows various studies reporting RNA extraction from patient samples using silica-
coated iron oxide nanoparticles [44,45].
Besides, several studies have reported the use of other functional groups due to their affinity for viral RNAs. Zhao
et al. reported the synthesis of poly(amino ester) with carbonyl groups (PC)-coated magnetic nanoparticles (pcMNPs)
followed by the development of pcMNPs viral RNA extraction method for SARS-COV-2 detection. The external mag-
netic field was used to extract pcMNP
RNA complexes from the solution. The extracted complex can be instan-
taneously treated in the RT-PCR process for viral RNA amplification without any need for an elution step [46].
Somvanshi et al. proposed a multifunctional nanomagnetic particle assisted
viral RNA extraction protocol for detection
230 Pandemic Outbreaks in the 21st Century

of COVID-19. They have synthesized silica and carboxyl-modified polyvinyl alcohol surface-functionalized zinc ferrite
nanoparticles via the sol-gel auto combustion route. To reduce the time of detection, adsorption steps could be removed.
The protocol could be automated as it does not require any centrifugation process [47].
Other types of nanoparticles that have gained substantial interest among the community of researchers regarding the
fight against viral infections are quantum dots. The main reason to use quantum dots may be attributed to their sensitiv-
ity in presence of a specific wavelength of light [48]. Also, the desired size and shape make them appropriate materials
to penetrate or target SARS-CoV-2 having a size between 60 and 140 nm [49]. Furthermore, the S protein of SARS-
CoV-2 could be disabled by the positive charge of carbon-based quantum dots [50]. Various reports have already been
published showing the antiviral effect of quantum dots [51
53]. However, less literature is available where quantum
dots are used for the detection of COVID-19. In one of the reported studies, optical quantum dots
conjugated aptamers
were used to detect the N protein of SARS-CoV via a chip system. Using this chip, the N protein of SARS-CoV could
be detected at concentrations as low as 0.1 pg mL21. The fluorescent emission intensity was detected by confocal laser
scanning microscopy [54].
Bao et al. developed a novel method for clustered regularly interspaced short palindromic repeats and their associ-
ated proteins (CRISPR-Cas) nucleic acid detection in which quantum dots act as a reporter. Magnetic beads coated with
streptavidin were used to bind the probe molecules after target recognition and Cas-mediated cleavage of biotinylated
ssDNA probe molecules, followed by the addition of a complementary ssDNA oligonucleotide quantum dot conjugate
hybridizing only with uncleaved probes on the magnetic beads. Magnetic sequestration was used to separate the quan-
tum dot conjugates (hybridized and unhybridized). Activity regarding cleavage and hence, the presence of target nucleic
acid was measured through fluorometric signals. The detection ability of the method was studied using a DNA target
resembling part of the African swine fever virus. The observed limit of detection was B0.5 nM in buffer and
B1.25 nM in porcine plasma [55]. In a different approach, Ahmed et al. synthetically synthesized chiral zirconium
quantum dots (Zr QDs) for optical detection of coronavirus. Methylthiazolyldiphenyl-tetrazolium assay was used to
examine the cytotoxicity of Zr QDs against glioma C6 cells of rat brain. Furthermore, conjugation of as-synthesized
quantum dots with antiinfectious bronchitis virus (IBV) antibodies of coronavirus led to an immune link at the presence
of target analyte and anti-IBV antibody-conjugated magnets-plasmonic nanoparticles (MPNPs). The external magnetic
field was used to separate the immune-conjugated QD-MPNPs nanohybrids for the study of fluorescence properties and
the reported detection of limit was 79.15 EID/50 μL [56].
Graphene-field-effect transistors (Gr-FET) are one of the important class of sensors that hold the potential of early
prediction and fast diagnosis of viral diseases. A high performance, solution compatible Gr-FET was fabricated by
Zhang et al. using an “upside-down” technique followed by combining it with highly selective antibody
antigen inter-
action, that is, SARS-CoV spike S1 subunit protein antibody (CSAb)
COVId-19 spike S1 subunit protein (containing
RBD) to develop an immunosensor for the detection of coronaviruses. It was found that Gr-FET immunosensors can
accurately identify COVID-19 spike protein S1 in a very short period (B2 min) with a reported limit of detection of
0.2 pM in a real-time and label-free manner. Furthermore, the developed immunosensor has found application in screen-
ing of high-affinity antibodies having a binding constant in the range of 2 3 1011 M21 against RBD at concentrations as
low as 0.1 pM [57].
It is further reported that the use of nanoparticles has improved the efficacy of PCR assays which are one of the
most reliable and renowned methods for the detection of CoV. Zhu et al. developed a sensitive duplex nanoparticle-
assay PCR for the identification of porcine epidemic diarrhea virus (PEDV) and porcine transmissible gastroenteritis
virus (TGEV). The reported detection limit of the duplex nano-PCR under optimized conditions was 7.6 3 101 and
8.5 3 101 copies μL21 for PEDV and TGEV, respectively, and the sensitivity was found to be 10 times higher than a
conventional PCR method [58]. A similar report has been published by Huang et al. in which they developed an ultra-
sensitive nanoparticle DNA probe-based PCR assay (duplex UNDP-PCR) for the detection of DNA and RNA viruses in
the same reaction system. However, the sensitivity of duplex UNDP-PCR was found to be similar to single UNDP-
PCR, that is, 20 copies each for porcine circovirus type 2 (PCV2) and TGEV in the serum but it was almost 250-fold
more than conventional duplex PCR/RT-PCR methods. Even DNA/RNA extraction, purification, and reverse transcrip-
tion of RNA were not required for the detection of PCV2 and TGEV from large-scale samples via duplex UNDP-PCR
[59].

13.4 Limitations and future prospectus

The genetic material-based detection approaches like RT-PCR and RT-qPCR producing more accurate results for the
finding of exact results at molecular levels are in great demand currently. It is also a very necessary situation to find the
 Recent developments in the diagnosis of COVID-19 with micro- and nanosystems Chapter | 13 231

exact number of COVID-19 confirmed cases currently to know the status of COVID-19 victims to treat them with
suitable medications, thereby which can provide proper solutions to treatment and mitigate strategies. However, screen-
ing, identification, and diagnosis of COVID-19 victims using conventional approaches are challenging tasks to perform
at a time at the peak moment, since more cases are increasing unpredictably by every day. Hence, handling all cases
within a given time frame is not a convenient way to produce the results since the RT-PCR approach itself is a time-
consuming technique to produce the results. Besides, other drawbacks from TR-PCR are also discussed in earlier sec-
tions. Therefore robust, fast, accurate, and less time-consuming assay technologies are always required to overcome the
above disadvantages. In this regard, nanomaterials-mediated naked eye detection via colorimetric approach, nanomater-
ial mediated antigen
antibody colorimetric reactions, fluorescence quenching approaches, and electrochemical detec-
tion methods are enabling accurate results compared to the conventional approaches.
These methodologies are alternative ways to do the detection and diagnosis of SARS-CoV-19 viruses. However,
few disadvantages are also interconnected with these methods although having amazing performances. Initially, SARS-
CoV-19 viruses are changing their genetic material via various degree of mutations, which eventually facilitates the
structural changes in corresponding antigen and antibody structural evolution. The structural evolutions are more impor-
tant to form the proper bindings between antigen and antibody. However, due to the formation of various mutants, these
binding interactions may not be in a proper way to recognize the viruses, since most of the nanomaterials have been
immobilized and functionalized with these antibodies and nucleic acids. Second, as-synthesized nanomaterials stability,
existing functional groups, working mechanisms are also considerable parameters to execute the accurate results.
Therefore every procedure and protocol have their own merits and demerits in the actions of the mechanism. Hence,
based on the necessity and demand it is quite certain to select the way of approach for the diagnosis of COVID-19
viruses. More importantly, detection and diagnosis of the COVID-19 virus would not be sufficient to control and eradi-
cate the present COVID-19 grave period. Finally, we are in great demand to find the proper solutions to save human
life against SARS-CoV-19 not only by the detection and diagnosis approaches but also with the help of proper precau-
tions that are given by the doctors and scientists. Existing technologies would have an amazing capability to find the
possible proper solutions to completely eradicate the COVID-19 grave situations and would have the COVID-19 virus-
free globe.

13.5 Conclusion
Currently, the entire globe is facing serious problems for the detection, diagnosis, tackling, and treatment measures for
COVID-19 virus eradication. However, it is also a challenging task to detect and diagnose these virus species using
conventional and advanced technologies due to the molecular changes via various mutations. Based on these views, we
have thoroughly discussed and symmetrized the advantages of using conventional approaches for the rapid detection
and diagnosis of COVID-19 virus using molecular level RT-PCR technique. Owing to the presence of time consump-
tion, sophisticated protocol, instrumentation, and evaluation of results, this technique is facing some unsolved false
results. To overcome the aforementioned drawbacks, nanomaterials-mediated biosensor technologies can have a
suitable open space in the detection and diagnosis of the SARS-CoV-19 virus. We have also discussed numerous recent
advancements in nanomaterials-mediated bio-sensing approaches with possible mechanisms. As per the available infor-
mation, these approaches would have enough capabilities to detect and diagnose the COVID-19 virus at a low concen-
tration load. Also, these approaches are not only involved in the detection and diagnosis of the COVID-19 virus but
also can have affirmative treatment measures to eradicate the viruses via divergent nanoformulations. Finally, we need
a technology that can simultaneously detect and kill viruses irrespective of their structural, morphological, and molecu-
lar changes. The existing and future technologies would have the proper solutions against the COVID-19 grave period
with fruitful days ahead with enrichment of great health and wealth across the globe.

Acknowledgments
The authors are indebted to Dr. Buddolla’s Institute of Life Sciences, and Eternal University, India, for their support and to all the researchers
whom we cited in this chapter for their significant and valuable research. No funding was received to perform this review.

Conflict of interest
The authors declare no relevant competing financial interests to disclose.
232 Pandemic Outbreaks in the 21st Century

References
[1] Carfı̀ A, Bernabei R, Landi F. Persistent symptoms in patients after acute COVID-19. JAMA 2020;324(6):603
5.
[2] Kim JH, Marks F, Clemens JD. Looking beyond COVID-19 vaccine phase 3 trials. Nat Med 2021;27(2):205
11.
[3] Eastman RT, Roth JS, Brimacombe KR, Simeonov A, Shen M, Patnaik S, et al. Remdesivir: a review of its discovery and development leading
to emergency use authorization for treatment of COVID-19. ACS Central Sci 2020;6(5):672
83.
[4] Bagcchi S. The world’s largest COVID-19 vaccination campaign. Lancet Infect Dis 2021;21(3):323.
[5] Bhuyan A. India begins COVID-19 vaccination amid trial allegations. Lancet 2021;397(10271):264.
[6] Jeong H, Rogers JA, Xu S. Continuous on-body sensing for the COVID-19 pandemic: gaps and opportunities. Sci Adv 2020;6(36) eabd4794.
[7] Pramanik A, Gao Y, Patibandla S, Mitra D, McCandless MG, Fassero LA, et al. The rapid diagnosis and effective inhibition of coronavirus
using spike antibody attached gold nanoparticles. Nanoscale Adv 2021;3(6):1588
96.
[8] Ehtesabi H. Application of carbon nanomaterials in human virus detection. J Sci Adv Mater Devices 2020;5(4):436
50.
[9] Ali MA, Hu C, Jahan S, Yuan B, Saleh MS, Ju E, et al. Sensing of COVID-19 antibodies in seconds via aerosol jet nanoprinted reduced-gra-
phene-oxide-coated 3D electrodes. Adv Mater 2021;33(7) 2006647.
[10] Raha S, Mallick R, Basak S, Duttaroy AK. Is copper beneficial for COVID-19 patients? Med Hypotheses 2020;142:109814.
[11] Vadlamani BS, Uppal T, Verma SC, Misra M. Functionalized TiO2 nanotube-based electrochemical biosensor for rapid detection of SARS-
CoV-2. Sensors 2020;20(20):5871.
[12] Medhi R, Srinoi P, Ngo N, Tran H-V, Lee TR. Nanoparticle-based strategies to combat COVID-19. ACS Appl Nano Mater 2020;3
(9):8557
80.
[13] Manivannan S, Ponnuchamy K. Quantum dots as a promising agent to combat COVID-19. Appl Organomet Chem 2020;34(10):e5887.
[14] Islam MA, Ahsan MZ. Plausible approach for rapid detection of SARS-CoV-2 virus by magnetic nanoparticle based biosensors. Am J Nanosci
2020;6(2):6
13.
[15] Van Tran V, Tran NHT, Hwang HS, Chang M. Development strategies of conducting polymer-based electrochemical biosensors for virus bio-
markers: potential for rapid COVID-19 detection. Biosens Bioelectron 2021;182:113192.
[16] Huang Y, Yang C, Xu X-f, Xu W, Liu S-w. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug develop-
ment for COVID-19. Acta Pharmacol Sin 2020;41(9):1141
9.
[17] Li Z, Yi Y, Luo X, Xiong N, Liu Y, Li S, et al. Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-
CoV-2 infection diagnosis. J Med Virol 2020;92(9):1518
24.
[18] El-Atab N, Qaiser N, Badghaish H, Shaikh SF, Hussain MM. Flexible nanoporous template for the design and development of reusable anti-
COVID-19 hydrophobic face masks. ACS Nano 2020;14(6):7659
65.
[19] Kim H, Park M, Hwang J, Kim JH, Chung D-R, Lee K-s, et al. Development of label-free colorimetric assay for MERS-CoV using gold nano-
particles. ACS Sens 2019;4(5):1306
12.
[20] Li D, Chen H, Gao X, Mei X, Yang L. Development of general methods for detection of virus by engineering fluorescent silver nanoclusters.
ACS Sens 2021;6(3):613
27.
[21] Seo G, Lee G, Kim MJ, Baek S-H, Choi M, Ku KB, et al. Rapid detection of COVID-19 causative virus (SARS-CoV-2) in human nasopharyn-
geal swab specimens using field-effect transistor-based biosensor. ACS Nano 2020;14(4):5135
42.
[22] Farka Z k, Jurik T, Ková ř D, Trnková L e, Skládal P. Nanoparticle-based immunochemical biosensors and assays: recent advances and chal-
lenges. Chem Rev 2017;117(15):9973
10042.
[23] Han X, Xu K, Taratula O, Farsad K. Applications of nanoparticles in biomedical imaging. Nanoscale 2019;11(3):799
819.
[24] Patra JK, Das G, Fraceto LF, Campos EVR, del Pilar Rodriguez-Torres M, Acosta-Torres LS, et al. Nano based drug delivery systems: recent
developments and future prospects. J Nanobiotechnol 2018;16(1):1
33.
[25] Canaparo R, Foglietta F, Giuntini F, Della Pepa C, Dosio F, Serpe L. Recent developments in antibacterial therapy: focus on stimuli-responsive
drug-delivery systems and therapeutic nanoparticles. Molecules 2019;24(10):1991.
[26] Singh L, Kruger HG, Maguire GE, Govender T, Parboosing R. The role of nanotechnology in the treatment of viral infections. Ther Adv Infect
Dis 2017;4(4):105
31.
[27] Chen L, Liang J. An overview of functional nanoparticles as novel emerging antiviral therapeutic agents. Mater Sci Eng C 2020;112:110924.
[28] Itani R, Tobaiqy M, Al Faraj A. Optimizing use of theranostic nanoparticles as a life-saving strategy for treating COVID-19 patients.
Theranostics 2020;10(13):5932.
[29] Zhang Y, Qu S, Xu L. Progress in the study of virus detection methods: the possibility of alternative methods to validate virus inactivation.
Biotechnol Bioeng 2019;116(8):2095
102.
[30] Park JE, Kim K, Jung Y, Kim JH, Nam JM. Metal nanoparticles for virus detection. ChemNanoMat 2016;2(10):927
36.
[31] Mokhtarzadeh A, Eivazzadeh-Keihan R, Pashazadeh P, Hejazi M, Gharaatifar N, Hasanzadeh M, et al. Nanomaterial-based biosensors for
detection of pathogenic virus. Trends in Anal Chem 2017;97:445
57.
[32] Draz MS, Shafiee H. Applications of gold nanoparticles in virus detection. Theranostics 2018;8(7):1985.
[33] Dykman L, Khlebtsov N. Gold nanoparticles in biology and medicine: recent advances and prospects. Acta Nat 2011;3(2):34
55.
[34] Khan MS, Vishakante GD, Siddaramaiah H. Gold nanoparticles: a paradigm shift in biomedical applications. Adv Colloid Interface Sci
2013;199:44
58.
[35] Li H, Rothberg L. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc Natl
Acad Sci U S A 2004;101(39):14036
9.
 Recent developments in the diagnosis of COVID-19 with micro- and nanosystems Chapter | 13 233

[36] Alafeef M, Dighe K, Moitra P, Pan D. Rapid, ultrasensitive, and quantitative detection of SARS-CoV-2 using antisense oligonucleotides
directed electrochemical biosensor chip. ACS Nano 2020.
[37] Moitra P, Alafeef M, Dighe K, Frieman MB, Pan D. Selective naked-eye detection of SARS-CoV-2 mediated by N gene targeted antisense oli-
gonucleotide capped plasmonic nanoparticles. ACS Nano 2020;14(6):7617
27.
[38] Zhang X, Qi Q, Jing Q, Ao S, Zhang Z, Ding M, et al. Electrical probing of COVID-19 spike protein receptor binding domain via a graphene
field-effect transistor. arXiv preprint:2003.12529; 2020.
[39] Ma C, Li C, Wang F, Ma N, Li X, Li Z, et al. Magnetic nanoparticles-based extraction and verification of nucleic acids from different sources.
J Biomed Nanotechnol 2013;9(4):703
9.
[40] Teengam P, Siangproh W, Tuantranont A, Vilaivan T, Chailapakul O, Henry CS. Multiplex paper-based colorimetric DNA sensor using pyrroli-
dinyl peptide nucleic acid-induced AgNPs aggregation for detecting MERS-CoV, MTB, and HPV oligonucleotides. Anal Chem 2017;89
(10):5428
35.
[41] Chen Y-T, Kolhatkar AG, Zenasni O, Xu S, Lee TR. Biosensing using magnetic particle detection techniques. Sensors 2017;17(10):2300.
[42] Borlido L, Azevedo A, Roque A, Aires-Barros M. Magnetic separations in biotechnology. Biotechnol Adv 2013;31(8):1374
85.
[43] Rocha-Santos TA. Sensors and biosensors based on magnetic nanoparticles. Trends Anal Chem 2014;62:28
36.
[44] Gong P, He X, Wang K, Tan W, Xie W, Wu P, et al. Combination of functionalized nanoparticles and polymerase chain reaction-based method
for SARS-CoV gene detection. J Nanosci Nanotechnol 2008;8(1):293
300.
[45] Lee AH, Gessert SF, Chen Y, Sergeev NV, Haghiri B. Preparation of iron oxide silica particles for Zika viral RNA extraction. Heliyon 2018;4
(3):e00572.
[46] Wang J, Ali Z, Si J, Wang N, He N, Li Z. Simultaneous extraction of DNA and RNA from hepatocellular carcinoma (Hep G2) based on silica-
coated magnetic nanoparticles. J Nanosci Nanotechnol 2017;17(1):802
6.
[47] Zhao Z, Cui H, Song W, Ru X, Zhou W, Yu X. A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of
SARS-CoV-2. BioRxiv 2020.
[48] Somvanshi SB, Kharat PB, Saraf TS, Somwanshi SB, Shejul SB, Jadhav KM. Multifunctional nano-magnetic particles assisted viral RNA-
extraction protocol for potential detection of COVID-19. Mater Res Innov 2021;25(3):169
74.
[49] Jha S, Mathur P, Ramteke S, Jain NK. Pharmaceutical potential of quantum dots. Artif Cells Nanomed Biotechnol 2018;46(suppl 1):57
65.
[50] Prajapat M, Sarma P, Shekhar N, Avti P, Sinha S, Kaur H, et al. Drug targets for corona virus: a systematic review. Indian J Pharmacol
2020;52(1):56.
[51] Huang S-L. Liposomes in ultrasonic drug and gene delivery. Adv Drug Deliv Rev 2008;60(10):1167
76.
[52] Ting D, Dong N, Fang L, Lu J, Bi J, Xiao S, et al. Multisite inhibitors for enteric coronavirus: antiviral cationic carbon dots based on curcumin.
ACS Appl Nano Mater 2018;1(10):5451
9.
[53] Tong T, Hu H, Zhou J, Deng S, Zhang X, Tang W, et al. Glycyrrhizic-acid-based carbon dots with high antiviral activity by multisite inhibition
mechanisms. Small 2020;16(13):1906206.
[54] Lin F, Bao Y-W, Wu F-G. Carbon dots for sensing and killing microorganisms. J Carbon Res 2019;5(2):33.
[55] Roh C, Jo SK. Quantitative and sensitive detection of SARS coronavirus nucleocapsid protein using quantum dots-conjugated RNA aptamer on
chip. J Chem Technol Biotechnol 2011;86(12):1475
9.
[56] Bao M, Jensen E, Chang Y, Korensky G, Du K. Magnetic bead-quantum dot (MB-Qdot) CRISPR assay for instrument-free viral DNA detec-
tion. BioRxiv 2020.
[57] Ahmed SR, Kang SW, Oh S, Lee J, Neethirajan S. Chiral zirconium quantum dots: a new class of nanocrystals for optical detection of coronavi-
rus. Heliyon 2018;4(8):e00766.
[58] Zhu Y, Liang L, Luo Y, Wang G, Wang C, Cui Y, et al. A sensitive duplex nanoparticle-assisted PCR assay for identifying porcine epidemic
diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens. Virus Genes 2017;53(1):71
6.
[59] Huang Y, Xing N, Wang Z, Zhang X, Zhao X, Du Q, et al. Ultrasensitive detection of RNA and DNA viruses simultaneously using duplex
UNDP-PCR Assay. PloS One 2015;10(11):e0141545.
This page intentionally left blank
Chapter 14

Recent trends in the development of

vaccine technologies to combat pandemic
outbreaks and challenges
Gayathri Chellasamy1, Rose Mary Kiriyanthan2, Saravanan Govindaraju1, A. Radha2 and Kyusik Yun1
1
Department of Bionanotechnology, Gachon University, Seongnam, South Korea, 2PG and Research Department of Botany, Bharathi Women’s
College, Tamil Nadu, India

14.1 Introduction
The advancement of human life on the planet Earth indirectly defines insecurity, complexity, uncertainty now and then.
It means that on one side of the world, we humans and technology are developing rapidly, simultaneously, on the other
hand, the world has been affected by enormous sufferings like natural disasters, pandemic outbreaks, terroristic attacks,
and climatic changes. These pandemic diseases have moved the world to a new normality state, with a significant
impact in the upcoming centuries. The pandemic attacks have resulted in greater responsibility and cooperation of every
individual all over the world [1]. Perhaps, the people committed to the spread of pandemics need medication to treat
and eradicate to safeguard further life on Earth altogether. Multilinguistic research efforts have been carried out and
thus results in the development of the vaccine.
Starting from the first vaccine for smallpox eradication to the current COVID outbreak, vaccines play a significant
role in combating pandemics. Vaccination is a better remedy necessary for a multifaceted general wellbeing reaction to
the future rise of a pandemic ailment. Notwithstanding various estimates intended to react and control a pandemic, for
example, reconnaissance, correspondence plans, isolation, and illness treatment, organization of successful antibodies
can ensure lives and limit sickness spread. However, the pandemic preparedness differs among the nations in which the
developing countries face the most difficulties to combat the pandemics due to their lower clinical infrastructure.
Above all the pandemic control measures, there is a risk of a further new pandemic outbreak due to the environmental
changes that impact the distribution, abundance, and prevalence of microorganism-bearing vectors. However, not all ill-
ness dangers have a comparing immunization, and for those that do, there are significant challenges to their fruitful use
in a pandemic [2]. Even though immunology has not contributed much to combat the pandemics, in that the more sig-
nificant part of the antibodies we use today was created and tried observationally, obviously there are significant diffi-
culties ahead to grow new immunizations for complex to-target microbes, for which we critically need a superior
comprehension of defensive invulnerability. Also, acknowledgment of the immense potential and difficulties for immu-
nizations to control sickness episodes and secure the people on Earth, along with the accessibility of a variety of inno-
vations, make it the ideal time for researchers and scientists around the world to be associated together to design and
develop the next generation of potent vaccines and medications to combat such pandemic outbreaks and challenges [3].

14.2 Pandemic outbreaks and challenges in vaccine development

Emerged and emerging pandemic diseases (epidemic diseases that spread over a broad region) have shown the truth of
worldwide pandemic dangers over the years to date. The pandemic diseases have killed billions of people, starting from
the bubonic plague to COVID-19. From the tremendous historical period to the current fast developing world, they
always prevail the vaccine development challenges due to the causative organism’s severity and effectiveness. The
Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00004-5
© 2021 Elsevier Inc. All rights reserved. 235
236 Pandemic Outbreaks in the 21st Century

emergence of resistant microorganisms, which causes pandemic diseases, requires new approaches and ways to prevent
them.
The plague disease caused by the most virulent pathogen, which killed 200 million people in human history, has no
effective licensed vaccine to date. The most formidable challenge in developing a vaccine for plague is its short incuba-
tion period of about 1
3 days with a high fatality rate and bacterium’s intrinsic genetic plasticity. As a countermeasure
against the above situations, lots of research are under study to build up a protected and solid immunization against pla-
gue [4]. Next comes smallpox that caused a devastating pandemic outbreak until it was declared eradicated in 1980.
The great mind behind discovering a vaccine for smallpox is Edward Jenner, who accidentally found a low percentage
of smallpox among a cow’s caretakers having lesions. Coined the cross-protective immunization among orthopox-
viruses by exposing a young child’s skin to liquid recovered from the lesions. He eventually showed the child
immune to smallpox, which eventually leads to the invention of the first-ever vaccine to the world. Since then,
World Health Organization (WHO) established a worldwide campaign to combat the smallpox disease and success-
fully eradicate it [5].
The influenza virus outbreak in 1918
19 killed almost 40
70 million individuals worldwide, resulting in an appar-
ent decline in life expectancy in many countries. To the astonishment of researchers and the people worldwide, influ-
enza became annual seasonal outbreaks, as it subsequently emerged in 1957
58, 1968, and 2009. The challenges in
finding a vaccine for influenza are their capacity to taint a wide range of animal varieties and their high genomic incon-
stancy. Furthermore, it bears the steady danger of zoonosis presenting an infection with totally new immunogenic prop-
erties into the human population. In all the emergence of the influenza virus, scientists created flu immunizations
focused explicitly on the circulating virus; however, unpredictability nature of the viruses leads to discovering licensed
seasonal vaccine specific for predefined viral strains, which cannot protect against a future pandemic and firm shreds of
evidence that production of vaccine against influenza [6].
Sooner the world faced another pandemic in 2002 is the emergence of a severe acute respiratory syndrome (SARS);
it has spread to nearly 29 countries, which resulted in 774 deaths with 8096 infected individuals. The SARS vaccine
development’s primary challenge is that the SARS animal models do not mimic human disease, as the virus kills the
mice in less than 1 week. And also, coronaviruses induce a short-lived immunity, which results in multiple infections
with coronaviruses that cause the common cold [7]. Above all the challenges, researchers had carried out various stud-
ies to produce the vaccine, and at some point, the efficacy of containment measures halted the spread of disease.
Following this, progressing endeavors to create a vaccine against SARV-CoV were stopped [8]. In 2012 another
COVID showed up in Saudi Arabia, causing Middle East respiratory condition (MERS). Like SARS-CoV, the infection
begun in bats and likely spread to people through tainted dromedary camels. As per the WHO, there have been 2143
affirmed instances of MERS, with 750 deaths in 27 nations since 2012. An assortment of examination exercises right
now continues to build up an immunization against MERS-CoV. Notwithstanding, an authorized immunization is not
yet accessible [9].
The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is later named coronavirus disease
2019 (COVID-19). Everyone knows that the COVID-19 pandemic is affecting billions of people around the world. At
the initial stage of the emergence of COVID-19, various treatments like antibiotic therapy including moxifloxacin, levo-
floxacin, cephalosporins, hydroxychloroquine azithromycin, and chloroquine, antibody therapy, oxygen therapy, conva-
lescent plasma transfusion have been administered to the infected individuals. These treatments were carried out only to
avoid worsening of patient condition and prevent further secondary infections [10]. There is a critical need to design
and produce a vaccine to treat and eradicate COVID-19, potentially to overcome the pandemic. There were many chal-
lenges in the vaccine development for SARS-CoV-2 as there is a lack of information on its biological properties, epide-
miology, and specific immune responses against it. Adding on the genetic instability of SARS-CoV-2 is also
challenging as it gets mutated and becoming a more virulent strain. COVID-19 lacks an appropriate animal model to
test the developed vaccine’s safety and efficacy, which is also a major obstacle faced in developing the vaccine toward
COVID-19 [11].
Above all the obstacles and challenges in vaccine development of COVID-19, enormous research is undergoing
worldwide; few were already released to the nations to start its dosage to people. Various types of vaccine approaches
are used to develop a potential vaccine toward COVID-19, which include whole-virus vaccines, viral-protein-based vac-
cines, and nucleic acid-based vaccines [12]. Due to the severity and fast-spreading nature of COVID-19, there lies an
urgent need for a vaccine to combat this, the estimated timeline for one complete production of vaccine candidate has
been reduced, which is not usual in the actual process of vaccine development [13].
Vaccine development and vaccination ordinarily require long periods of exploration and testing before arriving
at the facility, yet in 2020, researchers left on a competition to deliver protected and potent COVID antibodies in
 Recent trends in the development of vaccine technologies to combat pandemic outbreaks and challenges Chapter | 14 237

TABLE 14.1 Leading vaccines for COVID-19

Developer Developing Vaccine Phase Status

country technology
Pfizer-BioNTech America and mRNA Phase 2, 3 Approved in several countries.Emergency use in the
Germany United States, E.U., and other countries
Moderna America mRNA Phase 3 Approved in Switzerland.Emergency use in the United
States, United Kingdom, E.U., and others
Gamaleya Russia Ad26, Ad5 Phase 3 Early use in Russia.Emergency use in other countries
Oxford- Britain and ChAdOx1 Phase 2, 3 Emergency use in United Kingdom, E.U., and other
AstraZeneca Sweden countries
CanSino China Ad5 Phase 3 Limited use in China
Johnson & America and Ad26 Phase 3 Emergency use in U.S., E.U., other countries
Johnson Belgium
Vector Institute Russia Protein Phase 3 Early use in Russia
Novavax America Protein Phase 3
Sinopharm China Inactivated or Phase 3 Approved in China, U.A.E., and Bahrain.Emergency
attenuated virus use in Egypt and other countries
Sinovac China Inactivated or Phase 3 Approved in China.Emergency use in Brazil and other
attenuated virus countries
Sinopharm- China Inactivated or Phase 3 Limited use in China and U.A.E.
Wuhan attenuated virus
Bharat Biotech India Inactivated or Phase 3 Emergency use in India
attenuated virus

Modifies from The New York Times-Coronavirus vaccine Tracker.

record time. Specialists are presently trying 70 vaccine candidates in clinical preliminaries on people, and
20 have arrived at the last phases of testing. At any rate, 89 preclinical vaccines are under dynamic examination in
animal models [14]. The available leading vaccines for COVID-19 are tabulated in Table 14.1. COVID-19 vaccines
have been developed with the most safety and effectiveness; on the other hand, it is also rolled out that there is a
mild range of side effects. The volunteers’ significant side effects are injection-site pain, fatigue, headache, muscle
pain, fever, joint pain, chills, nausea, and swelling; these were common at both the stages of doses with slightly
increased effect at the second dose [15]. Above all the medical measures taken, the basic measures, including social
distancing, quarantine, and masks, should be followed worldwide to reduce the additional risks and spread of
COVID-19.

14.3 Vaccine technologies and its types

Traditional vaccination technologies such as live, live attenuated, and subunit vaccines are in use for centuries.
However, these technologies are highly lacking in several ways. Live vaccines can be hazardous for people with com-
promised immunity. For people with preexisting medical conditions, attenuated vaccines risk reversion and subunit
vaccines possess low immunogenicity. As these technologies are not suitable for rapid development, large scale produc-
tion, and distribution of vaccines in a short period, they are not recommended during epidemic and pandemic outbreaks
[16,17]. The nascent technologies such as viral vector vaccine, viral-like particle vaccine, and nucleic acid vaccine
were found to be promising. Until a few years back, these technologies were still in their nascent stage but due to the
present Corona pandemic, a plethora of research work had been carried out on vaccines, which led to a better under-
standing and development of these technologies, making them a potential solution to combat pandemic outbreaks and
vaccine development challenges [16].
238 Pandemic Outbreaks in the 21st Century

FIGURE 14.1 Development and mecha-

nism of viral vector vaccine.

14.3.1 Viral vector vaccines

In viral vector vaccines, harmless or attenuated virus act as vehicles to deliver immunogenic antigen’s genetic code into
the host. When the vector (virus) carries the genetic code introduced into the host, it exposes the antigen, leading to
immunogenic responses. The development and mechanism of the viral vector vaccine is illustrated in Fig. 14.1 [9,18].
A few commonly employed viral vectors are adenovirus, poxvirus, vesicular stomatitis virus, lentivirus, herpesvirus,
and measles virus. Adenovirus and poxvirus have replicating and nonreplicating forms; vesicular stomatitis virus and
measles virus are replicating vectors while the lentiviral vectors and herpesvirus are replication-incompetent [19,20].
There are several approved replicating viral vector vaccines, but currently, there are no approved nonreplicating viral
vector vaccines. However, they utilized gene therapy, and extensive research work has been carried out to develop a
nonreplicating vector vaccine against infectious diseases [21].
Like every other vaccine, the viral vector vaccine also has certain risk factors that have to be taken care of during
the development and production processes. Extra precautions have to be taken to prevent unwanted microbial contami-
nation as it can cause recombination, which may result in reactogenicity. The replication rate of the virus replaces viral
vectors, and in the attenuated vectors must be carefully assessed. All these concerns may lead to delays in clinical stud-
ies. Nevertheless, it is one of the most sought-after technologies, especially during pandemic and epidemic outbreaks,
as it shows high immunogenic response even in the absence of adjuvant and comparatively higher yields can be
obtained, facilitating large-scale production [9].
Adenovirus vector is the most widely used viral vector for vaccines due to its ability to induce robust and sustained
cellular and humoral immune responses [22]. Adenovirus is a nonpathogenic, nonenveloped DNA virus containing only
two genes replaced with foreign genes. As it is nonenveloped, it showcases high physical stability like resistance to
acid, enabling oral administration. Reports claim that the virus-infected muscle cells when administered intramuscularly
offered prolonged expression [19]. One disadvantage of adenovirus is that some individuals might have preexisting
immunity to it, as a result of which the adenovirus vector vaccine was removed immediately by the host’s immune cells
even before it could express the antigen. This setback could surpass by using adenovirus from other species like
Chimpanzees [23]. The Gam-COVID-Vac (Sputnik V COVID-19) developed in Russia, a heterologous recombinant
adenovirus-based vaccine against COVID-19, has been approved for full use in several countries. ChAdOx1 nCoV-19
(AZD1222) is another approved vaccine against COVID-19. It is a nonreplicating chimpanzee adenovirus-vectored vac-
cine expressing SARS-CoV-2 spike protein, developed by Oxford University [24,25].
Apart from the adenoviral vector, promoting several other efficient viral vectors like attenuated measles viral vector
and Vesicular stomatitis viral vector could also be utilized. Attenuated measles viral vector can accommodate large
genome inserts (exceeding 6 kb), and the tropism of the vector can be modified to target and infect APCs. They provide
stable and prolonged immunity. A report states that the measles vector vaccine-specific CD81 T cells and IgGs were
 Recent trends in the development of vaccine technologies to combat pandemic outbreaks and challenges Chapter | 14 239

detected even after 25
30 years in the vaccinees after a single dose of vaccination [26,27]. Vesicular stomatitis viral
vector is highly effective in the induction of CD41, CD81, and antibodies. It has a high growth rate of invalidated cell
lines and does not require DNA intermediate during viral replication [28]. The rVSV-ZEBOV (ERVEBO) vaccine
against Ebola is constructed using the vesicular stomatitis viral vector. The gene encoding for vector’s envelope glyco-
protein was removed and replaced with the 1995 Zaire strain Ebola virus glycoprotein. Though it is a replicating vector,
the virus replication gets attenuated due to the genome replacement and is the first Ebola vaccine to get approval from
the FDA [29,30].

14.3.2 Viral-like particles

Subunit vaccines consist of purified viral proteins that act as antigen and induce immunity in the host. Though subunit
vaccines are safer than live and attenuated vaccines, it is not a preferred technology as it is less immunogenic and so, it
requires adjuvants and repeated booster shots to maintain the immunity. The viral-like particles (VLP) vaccine is an
advanced version of the subunit vaccine [16]. They are single or multiprotein structures that mimic an authentic virus
configuration but lack a viral genome [31]. It generally consists of high-density displays of viral surface proteins, which
present as viral epitopes that can elicit strong T cell and B cell immune responses [32]. They possess the self-
adjuvating ability, but it was reported that the addition of adjuvants like aluminum salts could enhance the immunoge-
nicity of the vaccine [33]. The expression systems like mammalian cell cultures, transgenic plants, bacteria, yeast, and
insect cells can produce VLP-based vaccines. Fig. 14.2 represents the development and mechanism of the viral-like par-
ticle vaccine.
Even though the VLP-based vaccine is utilized to develop a safe and highly immunogenic vaccine, some areas need
to improve, like the cost and ease of construction. Nevertheless, the VLP vaccine is advantageous over other vaccine
platforms in several ways, such as induction of high and stable immunogenic response due to the structural similarity
with the pathogenic virus. They are replication-deficient; hence, safe use in immunocompromised individuals and the
consistency of production in stable expression systems facilitate upscaling and commercialization [34,35].
There are several commercially available VLP-based vaccines like Cervari and Gardasil for human papillomavirus,
Sci-B-Vac and Heplisav-B for Hepatitis B virus (HBV), and Mosquirix for Malaria [36]. Sci-B-Vac is a third-
generation vaccine against HBV, consisting of three HBV surface antigens, namely, S, pre-S1, and pre-S2, produced in
mammalian Hamster ovary cell lines containing major S and minor M and L proteins. Repeated monthly administration
of this vaccine, intramuscularly, along with daily oral Lamivudine treatment, can suppress HBV replication in chronic
HBV patients [37
39]. Heplisav-B is a recently approved vaccine. It is similar to the formerly approved three genera-
tions of vaccines, but additionally, it contains a CpG sequence 1018, which codes for an adjuvant. Over 4 weeks, two
doses are administered intramuscularly, and it induces high seroprotection rates than any other commercially available
vaccine for HBV [38,40].

FIGURE 14.2 Development and mechanism

of viral-like particle vaccine.
240 Pandemic Outbreaks in the 21st Century

FIGURE 14.3 Development and mechanism of nucleic acid vaccine (i. DNA vaccine, ii. mRNA vaccine).

14.3.3 Nucleic acid vaccine

Nucleic acid vaccines include DNA and RNA vaccines. The antigen coding plasmid DNA, RNA, or mRNA are intro-
duced into the host, and it utilizes the host’s transcription and translation machinery to produce viral proteins or anti-
gens, which would lead to the elicitation of the immune responses (Fig. 14.3) [9].

14.3.3.1 DNA vaccine

The DNA vaccine’s unique feature is that it stimulates more cellular immune responses than most vaccines that address
humoral immunity. This type of vaccine can provide effective prophylaxis against viruses with high antigenic variabil-
ity like the influenza virus and HIV [41].
DNA vaccines are often associated with their inefficiency to produce high immunogenic responses because previ-
ously, the most studied routes for DNA vaccines are intramuscular and intradermal. In both cases, the vaccine showed a
high immunogenic response at the administration site but low immunogenicity in other regions because the plasmid
DNA must cross the plasma membrane and the nuclear membrane to reach the cell’s nucleus and begin the transcription
process, which infers that these vaccines are highly immunogenic; however, the problem lies with the route of adminis-
tration. This problem can be solved using a gene gun or electroporation for administration, thus aiding the vaccine’s
penetration into the cell and inducing high immunogenicity at low dosages [42]. Encapsulation of the plasmid into lipid
nanoparticles or adsorption onto polymers can increase cellular uptake. Molecular adjuvants like pattern recognition
receptors, ligands, or various cytokines like IL-12 can encode the plasmid along with the antigen, which increases the
tropism of the antigen to the APC or any other cells or cellular components.
The drawbacks of DNA vaccines include the generation of antibodies against the host’s DNA and the expression of
cytokines, which may lead to adverse reactions. These vaccines have the risk of long-term persistence and genomic
integration into the host is also a possibility that is not proven yet, but if it happens, it may result in the incorporation
of unwanted bacterial elements like antibiotic resistance into the host. Due to its stability, ease of development and pro-
duction, absence of infectious agents, and flexibility, this technology is still in demand, especially during emergencies
like pandemic outbreaks. Currently, there is no approved DNA vaccine. The ZYCoV-D vaccine against SARS-CoV-2,
developed by Zydus Cadila, India, is in phase 3 clinical trials. This vaccine comprises a DNA plasmid vector carrying
the gene encoding the virus’s spike protein [43,44].
 Recent trends in the development of vaccine technologies to combat pandemic outbreaks and challenges Chapter | 14 241

14.3.3.2 RNA vaccine

There are two types of RNA vaccines, namely, nonreplicating and self-amplifying mRNA. The nonreplicating mRNA
consists of a straightforward structure, including the 50 and 30 untranslated region and the antigen encoding region. The
self-amplifying mRNA structure is similar, but additionally, it contains regions that encode for viral translational
machinery that enables RNA amplification and protein expression [45]. RNA vaccines are more immunogenic than
DNA vaccines due to the presence of multiple pathways that activate an innate immune response against foreign RNA-
like Toll-like receptors and RIG-I-like receptors. These vaccines do not require sophisticated administration methods
like the DNA vaccine, as RNA has better cellular uptake because it only has to cross the plasma membrane to begin the
translation process [16].
Naked mRNA gets degraded by the ribonuclease enzyme. Complexation by lipid and polymer nanoparticles can pro-
tect the mRNA and also increase the efficiency of the vaccine. The administration route can be selected based on the
nature of the infectious disease. Previous studies report that administration by intravenous targets the liver during intra-
muscular and intradermal cause prolonged expression at the site of injection. The IV route showed the highest expres-
sion, whereas the duration was longest for intradermal, followed by intramuscular [46,47].
In the preclinical trials, the mRNA vaccine always gave encouraging results. But when it comes to clinical trials,
the results are entirely discouraging. The reasons being decreased immunogenicity due to reduction in mRNA stability
leading to a reduction in the translation rate impact mRNA’s metabolism due to concomitant administration of other
drugs [48]. Therefore this technology remained disdained until 2019. In 2020 during the advent of the Corona pan-
demic, the mRNA vaccine became the leading technology in the COVID-19 vaccine race [49]. Pfizer-BioNTech
COVID-19 vaccine sold under the brand name Comirnaty is the first RNA vaccine and the first COVID-19 vaccine to
get approval from the FDA. It is a lipid nanoparticle formulated, nucleoside-modified nonreplicating mRNA vaccine
encoding the perfusion spike glycoprotein of SARS-CoV-2 [50]. The overall development mechanism of the nucleic
acid vaccine is presented in Fig. 14.3.

14.4 Challenges in the success of vaccination toward pandemic outbreaks

In that case, a successful vaccine to the market faces a range of challenges like poor infrastructure, lack of funding at
the stage of its development, access limitation, the antivaccination movement, commercial viability, and immunological
challenges [3]. Immunization has been perhaps the best general wellbeing measure since the presentation of essential
disinfection. Considerable decrease in mortality and morbidity of pandemic outbreaks have been accomplished through
the mass vaccination programs, and the rundown of infections that are possibly controllable by immunizations is devel-
oping consistently decade by decade. Every illness is an immunological issue in itself: even today, with all the informa-
tion available to one, it is hard to anticipate what sort of immunization can be genuinely compelling. This trouble is
much more noteworthy for COVID-19, another infection in which continuous investigations in research facilities overall
are adding new information at a colossal speed [51]. In such circumstances, the vaccine development pathway has
altered according to the disease’s severity, which happened in COVID-19. Few of the vaccines were approved and sent
to people in most countries.

14.5 Conclusion and future perspectives

Immunization procurement, conveyance, and take-up issues are generously unique in the creating scene. Less affluent
nations regularly do not broadly utilize flu antibodies for an assortment of reasons, maybe the most unmistakable of the
need to commit wellbeing assets to additional squeezing concerns. In case of a lethal flu pandemic or other infection
episode requiring mass inoculation, developing nations’ legislatures confronting critical difficulties, for example,
addressing supply needs, subsidizing immunization procurement, and guaranteeing take-up of antibody in spots where
flu inoculation is not customarily practiced.
Under the direction of the WHO and various governments’ help, many developing nations have set up vaccine pro-
duction limits or are gaining ground to build up this limit. Above all the well-versed techniques in vaccine development
like RNA vaccines, viral vector vaccines, and the whole viral vaccine, there is still a need to complete the technologies
followed to get a better and faster vaccine candidate at the peak of the pandemic. While it appears to be impossible that
a solitary innovation capable of answering every future flare-up circumstance, the blend of present information, progres-
sing improvement, and the developing comprehension of human immunology can give instruments to effectively battle
242 Pandemic Outbreaks in the 21st Century

arising worldwide dangers. As it is as yet far shy of absolute worldwide need; however, it is clear that worldwide vacci-
nation requirement is expanding.

Acknowledgment
This research was supported by the National Research Foundation of Korea (NRF) grant which was funded by the Korea Government
(MSIP) (No. 2020R1G1A1102394).

References
[1] de Amorim Wellyngton Silva, de Andrade José Baltazar Salgueirinho Osório. Pandemics, global risks and adaptation: challenges for a changing
world. Res Globalization 2020;2:100023.
[2] Oshitani Hitoshi, Kamigaki Taro, Suzuki Akira. Major issues and challenges of influenza pandemic preparedness in developing countries.
Emerg Infect Dis 2008;14(6):875.
[3] Pollard Andrew J, Else M Bijker. A guide to vaccinology: from basic principles to new developments. Nat Rev Immunol 2020;21:80
100.
[4] Sun Wei, Singh Amit K. Plague vaccine: recent progress and prospects. npj Vaccines 2019;4(1):1
9.
[5] Melamed Sharon, Israely Tomer, Paran Nir. Challenges and achievements in prevention and treatment of smallpox. Vaccines 2018;6(1):8.
[6] Rockman Steven, Karen Laurie, Ian Barr. Pandemic influenza vaccines: what did we learn from the 2009 pandemic and are we better prepared
now? Vaccines 2020;8(2):211.
[7] Taylor Deborah R. Obstacles and advances in SARS vaccine development. Vaccine 2006;24(7):863
71.
[8] Reperant Leslie A, Osterhaus Albert DME. AIDS, Avian flu, SARS, MERS, Ebola, Zika. . . what next? Vaccine 2017;35(35):4470
4.
[9] Rauch Susanne, Jasny Edith, Schmidt Kim E, Petsch Benjamin. New vaccine technologies to combat outbreak situations. Front Immunol
2018;9:1963.
[10] Chellasamy Gayathri, Arumugasamy Shiva Kumar, Govindaraju Saravanan, Yun Kyusik. Analytical insights of COVID-19 pandemic. Trends
Anal Chem 2020;116072.
[11] Begum Jubeda, Mir Nasir Akbar, Dev Kapil, Buyamayum Bidyarani, Wani Mohd Yaqoob, Raza Meesam. Challenges and prospects of
COVID-19 vaccine development based on the progress made in SARS and MERS vaccine development. Transboundary Emerg Dis 2020;68
(3):1111
24.
[12] Forni Guido, Mantovani Alberto. “COVID-19 vaccines: where we stand and challenges ahead. Cell Death Differ 2021;1
14.
[13] Liu Yi, Wang Kai, Massoud Tarik F, Paulmurugan Ramasamy. “SARS-CoV-2 vaccine development: an overview and perspectives. ACS
Pharmacol Transl Sci 2020;3(5):844
58.
[14] Corum J, Sui-Lee W, Zimmer C. The New York Times; 2020. Available from: https://www.nytimes.com/interactive/2020/science/coronavirus-
vaccine-tracker.html. [Accessed 05 Aug 2021].
[15] COVID vaccines and safety: what the research says Ariana Remmel. Available from: https://doi.org/10.1038/d41586-021-00290-x
[16] Brisse Morgan, Sophia M Vrba, Natalie Kirk, Yuying Liang, Hinh Ly. Emerging concepts and technologies in vaccine development. Front
Immunol 2020;11:2578.
[17] Vartak Abhishek, Steven J Sucheck. Recent advances in subunit vaccine carriers. Vaccines 2016;4(2):12.
[18] Juno Jennifer A, Shelby L O’Connor. Translating viral vaccines into immunity. Science 2021;371(6528):460
1.
[19] Robert-Guroff Marjorie. Replicating and non-replicating viral vectors for vaccine development. Curr Opin Biotechnol 2007;18(6):546
56.
[20] Milone Michael C, O’Doherty Una. Clinical use of lentiviral vectors. Leukemia 2018;32(7):1529
41.
[21] Callaway Ewen. The race for coronavirus vaccines: a graphical guide. Nature 2020;580(7805):576.
[22] Afkhami Sam, Yao Yushi, Xing Zhou. “Methods and clinical development of adenovirus-vectored vaccines against mucosal pathogens”. Mol
Ther Methods Clin Dev 2016;3:16030.
[23] CDC, Understanding and explaining viral vector COVID-19 vaccines, in United States Department of Health & Human Services USA.gov;
2021.
[24] Logunov Denis Y, Inna V Dolzhikova, Shcheblyakov Dmitry V, Tukhvatulin Amir I, Zubkova Olga V, Dzharullaeva Alina S, et al. Safety and
efficacy of an rAd26 and rAd5 vector-based heterologous prime-boost COVID-19 vaccine: an interim analysis of a randomised controlled phase
3 trial in Russia. Lancet 2021;397(10275):671
81.
[25] Voysey Merryn, Sue Ann Costa Clemens Shabir A, Madhi Lily Y, Weckx Pedro M, Folegatti Parvinder K, Aley, et al. Safety and efficacy of
the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South
Africa, and the UK. Lancet 2021;397(10269):99
111.
[26] Guerbois Mathilde, Moris Arnaud, Combredet Chantal, Najburg Valérie, Ruffié Claude, Février Michèle, et al. Live attenuated measles vaccine
expressing HIV-1 Gag virus like particles covered with gp160ΔV1V2 is strongly immunogenic. Virology 2009;388(1):191
203.
[27] Naim Hussein Y. Measles virus: a pathogen, vaccine, and a vector. Hum Vaccines Immunother 2015;11(1):21
6.
[28] Humphreys Ian R, Sebastian Sarah. Novel viral vectors in infectious diseases. Immunology 2018;153(1):1
9.
[29] Medaglini Donata, Siegrist Claire-Anne. Immunomonitoring of human responses to the rVSV-ZEBOV Ebola vaccine. Curr Opin Virol
2017;23:88
94.
[30] FDA. ERVEBO; 2019. Available from: https://www.fda.gov/vaccines-blood-biologics/ervebo. [Accessed 22 February 2021].
 Recent trends in the development of vaccine technologies to combat pandemic outbreaks and challenges Chapter | 14 243

[31] Roldão António, Maria Candida M Mellado, Leda R Castilho, Manuel JT Carrondo, Paula M Alves. Virus-like particles in vaccine develop-
ment. Exp Rev Vaccines 2010;9(10):1149
76.
[32] Bogani Giorgio, Raspagliesi Francesco, Ditto Antonino, Fuente José de la. The adoption of viral capsid-derived virus-like particles (VLPs) for
disease prevention and treatments. Vaccines 2020;8:432.
[33] Lua Linda HL, Connors Natalie K, Sainsbury Frank, Chuan Yap P, Wibowo Nani, Middelberg Anton PJ. Bioengineering virus-like particles as
vaccines. Biotechnol Bioeng 2014;111(3):425
40.
[34] Garg Himanshu, Mehmetoglu-Gurbuz Tugba, Joshi Anjali. Virus like particles (VLP) as multivalent vaccine candidate against Chikungunya,
Japanese Encephalitis, Yellow Fever and Zika Virus. Sci Rep 2020;10(1):1
13.
[35] Chackerian Bryce. Virus-like particles: flexible platforms for vaccine development. Exp Rev Vaccines 2007;6(3):381
90.
[36] Mohsen Mona O, Zha Lisha, Cabral-Miranda Gustavo, Bachmann Martin F. “Major findings and recent advances in virus
like particle (VLP)-
based vaccines. Seminars in immunology, 34. Academic Press; 2017. p. 123
32.
[37] Qian Ciying, Liu Xinlin, Xu Qin, Wang Zhiping, Chen Jie, Li Tingting, et al. Recent progress on the versatility of virus-like particles. Vaccines
2020;8(1):139.
[38] Pumpens Paul, Ulrich R, Sasnauskas Kestutis, Kazaks Andris, Ose Velta, Elmars Grens. Construction of novel vaccines on the basis of the
virus-like particles: Hepatitis B virus proteins as vaccine carriers. Medicinal protein engineering. Boca Raton, FL: CRC Press Taylor & Francis
Group; 2008. p. 205
48.
[39] Shouval Daniel, Roggendorf Hedwig, Roggendorf Michael. Enhanced immune response to hepatitis B vaccination through immunization with a
Pre-S1/Pre-S2/S vaccine. Med Microbiol Immunol 2015;204(1):57
68.
[40] Jackson Sam, Lentino Joseph, Kopp James, Murray Linda, Ellison William, Rhee Margaret, et al. Immunogenicity of a two-dose investigational
hepatitis B vaccine, HBsAg-1018, using a toll-like receptor 9 agonist adjuvant compared with a licensed hepatitis B vaccine in adults. Vaccine
2018;36(5):668
74.
[41] Calina Daniela, Chandan Sarkar, Andreea Letitia Arsene, Bahare Salehi, Anca Oana Docea, Milon Mondal, et al. Recent advances, approaches
and challenges in targeting pathways for potential COVID-19 vaccines development. Immunol Res 2020;1
10.
[42] Restifo NP, Ying H, Hwang L, Leitner WW. The promise of nucleic acid vaccines. Gene Ther 2000;7(2):89
92.
[43] Dey Ayan, Rajanathan Chozhavel, Chandra Harish, Pericherla Hari PR, Kumar Sanjeev, Huzaifa S, et al. Immunogenic potential of DNA vac-
cine candidate, ZyCoV-D against SARS-CoV-2 in animal models. bioRxiv 2021.
[44] PTI, Coronavirus|Zydus Cadila gets DCGI nod to initiate Phase-3 clinical trials for COVID-19 vaccine, in THE HINDU: New Delhi, India;
2021.
[45] Pardi Norbert, Hogan Michael J, Porter Frederick W, Weissman Drew. mRNA vaccines—a new era in vaccinology. Nat Rev Drug Discov
2018;17(4):261.
[46] Akinc Akin, Querbes William, De Soma, Qin June, Frank-Kamenetsky Maria, Narayanannair Jayaprakash K, et al. Targeted delivery of RNAi
therapeutics with endogenous and exogenous ligand-based mechanisms. Mol Ther 2010;18(7):1357
64.
[47] Lutz Johannes, Lazzaro Sandra, Habbeddine Mohamed, Ellen Schmidt Kim, Baumhof Patrick, Mui Barbara L, et al. Unmodified mRNA in
LNPs constitutes a competitive technology for prophylactic vaccines. NPJ Vaccines 2017;2(1):1
9.
[48] Liu Margaret A. A comparison of plasmid DNA and mRNA as vaccine technologies. Vaccines 2019;7(2):37.
[49] Saltzman DGAJ. The story of mRNA: how a once-dismissed idea became a leading technology in the COVID vaccine race, in STAT: online;
2020.
[50] Oliver Sara E, Julia W Gargano, Marin Mona, Wallace Megan, Curran Kathryn G, Chamberland Mary, et al. The Advisory Committee on
Immunization Practices’ interim recommendation for use of Pfizer-BioNTech COVID-19 vaccine—United States, December 2020. Morb
Mortal Wkly Rep 2020;69(50):1922.
[51] Metcalf C Jessica E, Andreasen Viggo, Bjørnstad Ottar N, Ken Eames, Edmunds W John, Funk Sebastian, et al. Seven challenges in modeling
vaccine preventable diseases. Epidemics 2015;10:11
15.
This page intentionally left blank
Chapter 15

Could repurposing existing vaccines and

antibiotics help to control the COVID-19
pandemic?
Kajal Rathod1,2, Niyati Dhingra1,2, Soumya Dakshinamurthy3 and Buddolla Viswanath2
1
Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Tirupati, India, 2Dr. Buddolla’s Institute of Life Sciences, A Unit of Dr.
Buddolla’s Educational Society, Tirupati, India, 3Department of Microbiology, Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupati,
India

15.1 Introduction
The virus that causes COVID-19, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has had a significant
effect on human health around the world, infecting a large number of people, causing severe disease and long-term
health consequences, and resulting in death and excess mortality [1]. Despite the fact that physical isolation, face cover-
ings, monitoring, and tracing can slow the virus’s spread, the risk of outbreaks and economic and social damage will
almost certainly continue until effective treatments become available [2]. One of the most promising approaches to
combating the current pandemic situation is drug repurposing, which is a strategy in which existing drugs are used to
treat emerging and challenging diseases, including COVID-19. Moreover, it is a cost-effective and efficient tool [3].
Many currently approved medications may be repurposed to treat a range of diseases, which has a number of benefits
over developing an entirely new drug for a specific indication. First and foremost, the probability of failure is lowered;
since the repurposed drug has already been shown to be sufficiently effective in preclinical models and humans if
early-stage trials have been completed, it is less likely to fail in subsequent efficacy trials, at least from a safety per-
spective. Second, since most preclinical testing, safety assessments, and, in some cases, formulation development may
have already been completed, the time period for drug development can be minimized. Third, less investment is needed,
but this will vary greatly depending on the repurposing candidate’s stage and development process [4].
Outbreaks of novel emerging viruses, such as coronavirus disease 2019 (COVID-19), present unique obstacles to
health#care practitioners in terms of selecting suitable therapeutics/pharmacological drugs in a clinical setting with little
time for experimental drugs development [5]. Furthermore, developing a vaccine or drug for any disease, including
COVID-19, takes time, and even though the process is ramped up, it would take 18
20 months to get it to market as a
ready-to-use medicine. Moreover, the latest drug research process costs more than a billion dollars and takes 10
15
years to complete, with a success rate of less than 5% [6]. This causes a lag in pharmaceutical drug development, result-
ing in a gap between the need for a drug at a particular time and the effectiveness and production rate of the drug. At
that time, the pathogen may have adapted, rendering all research useless, and as a consequence, finding successful
COVID-19 therapeutic agents are crucial and urgent. Rather than suffering significant losses, it is more necessary and
cost-effective to investigate existing antiviral and other SARS-CoV2 medicines. Therefore, repurposing existing drugs
for the treatment of various diseases has recently become a common technique since it involves de-risked compounds
with well-known preclinical, pharmacokinetic, and pharmacodynamic profiles that can go straight through phase III or
IV clinical trials, allowing the drug development process theoretically low-cost and fast [7]. The benefits of drug repur-
posing over de novo medicine synthesis are shorter testing and development times, relatively lower development costs,
and most significantly, a lower chance of failure because the medication’s safety profile is typically well-established
[3,8,9]. As a result, the World Health Organization (WHO) and other health bodies have turned to reassess the effec-
tiveness of approved and experimental medicine to cure existing and evolving health issues [7,10]. Drug repurposing is
Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00020-3
© 2021 Elsevier Inc. All rights reserved. 245
246 Pandemic Outbreaks in the 21st Century

usually based on two criteria: one, a single drug interacts with multiple target sites, allowing different target sites of
activity for the identified compound to be discovered [11,12]. The other concept is that disease targets are often applica-
ble to a wide range of biological pathogenesis processes, allowing for the designation of a new indication for the target
[13,14]. Following the pandemic, drug repurposing has gained attention as a means of delivering the COVID-19 vaccine
and treatment in a timely manner.

15.1.1 An upsurge of SARS CoV-2

COVID-19 outbreak caused by SARS-CoV-2 that began in Wuhan (China) in December 2019 (Hubei province) had
already spread to 213 countries and territories and has created havoc worldwide [15]. On January 30, 2020, the novel
coronavirus epidemic was declared a Public Health Emergency of International Concern [16]. The WHO named this
disease as COVID-19 on February 11, 2020, and on March 11, 2020, the WHO declared COVID-19 a pandemic [17].
There have been approximately 136,291,755 cases and 2,941,148 deaths recorded as a result of COVID-19 as of April
13, 2021 [18]. The emergence of various strains of SARS-CoV-2 with characteristic mutations in their genome has
occurred in the last few months of 2020 [19]. The risk of a virus mutating increases as it is widely distributed in a popu-
lation and potentially causing infections [20]. The more chances a virus have to spread, the more it replicates—and the
more changes it can go through [21]. The ability of most viruses to cause infections and disease is unaffected by most
mutations. However, depending on where the changes occur in the virus’s genetic material, they can have an effect on
the virus’s properties, such as transmission or severity [22]. According to the WHO, there is currently no treatment
available for COVID-19 control. COVID-19 infection and mortality rates vary substantially between countries, which
may be due to differences in health care infrastructure, testing facilities, international travel, environmental factors, and
cultural norms [23].

15.2 Genome of coronavirus

The coronaviruses are pleomorphic, measuring between 80 and 160 nm in length, with a small genome of 27
32 kb
and a unique replication strategy [24]. Coronaviruses are classified into four classes: alphacoronaviruses, betacorona-
viruses, gammacoronaviruses, and deltacoronaviruses. They are all members of the coronaviridae family in the
Nidovirales order. Alpha and beta family of viruses’ infects only mammals while the gamma family infects only birds
and delta family infects both birds and mammals. The SARS-CoV-2 genome was found to be approximately 29.9 kb in
size [25]. According to preliminary research, the genome of SARS-CoV2 shares the most genome similarities with the
RATG13 virus found in bats, implying that SARS-CoV-2 may have originated in bats [26]. The “receptor-binding
domain (RBD)” of SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) receptors with a high affinity in
humans, cats, and other animals with inflated receptor homology, and the presence of a polybasic cleavage site at the
S1/S2 junction is indeed noteworthy genomic features [27]. Both SARS-CoV-2 and SARS-CoV use the human ACE2
receptor to enter cells [28]. By transforming the vasoconstrictor peptide angiotensin II to angiotensin 1
7 (vasodilator
peptide) bound to the cell membrane, ACE2 protects the heart and blood vessels [29]. The heart, lungs, kidneys, endo-
thelium, and other tissues may all contain ACE2. It controls blood pressure in the body and decreases the negative
effects of other Renin-Angiotensin System components by lowering angiotensin II levels and increasing angiotensin
1
7 levels [30]. A small subset of type II alveolar cells (AT2) was discovered to express the ACE2 receptor as well as
several other genes that positively control viral replication and transmission, rendering the lungs more vulnerable to the
virus [30]. When ACE2-expressing cells are found in the lungs, an immune response is triggered that can overreact,
causing lung cell damage by filling the air sacs with fluid rather than gas, resulting in pneumonia [31].
Like other coronaviruses, SARS-CoV-2 has four main structural genes that code for four major structural proteins:
the spike protein (S), the envelope protein (E), the membrane protein (M), and the nucleocapsid protein (N) [32]. The
virus’s ssRNA genome is encapsulated by the N protein, while the virus’s envelope is composed of the proteins spike
(S), small membrane (E), and membrane (M) [24]. The S protein helps the virus to bind to and amalgamate with the
host cell membrane. Virulence, tissue tropism, host range, and protective immunity are all determined by this primary
immunogenic antigen [33]. SARS-CoV-2 S protein is a trimeric type I fusion protein with two spike protein subunits,
S1 and S2. Protein receptors of type S1 are required for receptor recognition, whereas protein receptors of type S2 are
required for membrane fusion [34]. The RBD of the S1 subunit directly binds to the ACE2 receptor [35]. The S protein,
which is in a metastable prefusion state, undergoes a conformational rearrangement when it fuses with the ACE2 recep-
tor and there is a discharge of the S1 subunit [36]. This enables the S2 subunit of S protein to transit to a
stable postfusion state. The cellular protease TMPRSS2 is involved in the priming of S protein. SARS-CoV-2, like
 Could repurposing existing vaccines and antibiotics help to control the COVID-19 pandemic? Chapter | 15 247

MERS-CoV and SARS-CoV, replicates within the host cell and follows a positive-sense RNA virus’s typical life cycle
[37]. Other structural proteins involved in the viral assembly including envelope (E); membrane protein (M); nucleocap-
sid protein (N) and nonstructural proteins, such as papain-like protease, 3-chymotrypsin-like protease, RNA-dependent
RNA polymerase (RdRp), helicase, and other accessory proteins, participate in viral transcription and replication [38].

15.3 Drug repurposing

Considering the high attrition rates, high prices, and sluggish speed of new drug research and production, repurposing
“existing/old” drugs to treat COVID-19 is becoming a more appealing proposition since it requires the use of derisked
molecules, which may result in lower total development costs and shorter development timelines. Drugs that could be
repurposed to treat, prevent, or cure COVID-19 have piqued researchers’ interest [39,40], but a number of data-driven
and experimental approaches have been proposed for identifying repurposable drug candidates [4].

15.4 Therapeutic targets

G Coronaviruses inhibit antiviral immunity, allowing interferons (IFNs) to sustain an antiviral state.
G The virus moves into the cell by fusing viral spike proteins with the cellular ACE2 receptor, which causes ACE2 to
be downregulated. Angiotensin receptor blockers and angiotensin-converting enzyme inhibitors and statins increase
ACE2 expression, thereby having better efficacy.
G The virus is then endocytosed, with low endosomal pH assisting in the lysis of viral structural proteins. The antiviral
action of diprotic bases like chloroquine and hydroxychloroquine may disrupt the acidic environment.
G Nucleic acid (NA) is released into the cytoplasm.
G Translation of viral proteins with help of the host ribosomes.
G Viral protease enzyme undergoes proteolysis to form functional proteins, for example, RDRP. Inhibitors such as
lopinavir, ritonavir, and darunavir may thus be effective against the virus by inhibiting the key protease enzyme.
G The replication and transcription of viruses are dependent on the presence of RDRP. Coronaviruses may be prone to
RDRP inhibitors including remdesivir, favipiravir, ribavirin, and arbidol. Virions are formed after subsequent trans-
lation, proteolysis, and packaging of proteins, which are then exocytosed out of the cell.

15.5 Therapeutic options for COVID-19 management

No therapy has been proven to be beneficial in outpatients with mild to moderate COVID-19 who are not at high risk for
disease progression. Given the urgent need to minimize existing morbidity and mortality rates, many drugs, including
repurposed medicines, have been the focus of clinical trials (Fig. 15.1). Drug manufacturers and leading medical institu-
tions have also been working on developing a vaccine that is both effective and safe. Dexamethasone has shown promise
in the symptomatic treatment of COVID-19 patients with severe pneumonia, despite the initial hysteria around medicines
like hydroxychloroquine. Despite this, no single confirmed therapeutic option for COVID-19 patients has been

FIGURE 15.1 Repurposing drugs available to treat COVID-19.

248 Pandemic Outbreaks in the 21st Century

thoroughly elucidated to date. This emphasizes the importance of all key stakeholders implementing evidence-based
interventions rather than rushing to implement unproven therapies that could do more harm than good. Researchers are
still waiting for the results of ongoing trials, such as new vaccines, in the hopes of providing clinicians with more infor-
mation so they can make evidence-based decisions about COVID-19 treatment options.

15.5.1 Repurposed drugs that act through virus-related targets such as RNA genome
It is worth mentioning that the protease and polymerase enzymes for SARS-CoV-2 and SARS-CoV are extensively con-
served, with 96% and 97% overall identity, respectively. As a result, anti-SARS blockers could be suitable therapeutic
candidates for binding protease or polymerase sites against SARS-CoV-2.

15.5.1.1 Remdesivir
Remdesivir is an adenosine derivative prodrug with a nitrile substituent in position 1 that has shown broad-spectrum
efficacy against viruses such as SARS-CoV, MERS-CoV, and Ebola [41]. Remdesivir was created as an anti-Hepatitis
C Virus candidate at first. It functions as a chain terminator and competes with adenosine for chain inclusion within the
catalytic site of viral replicase [42]. Since the beginning of the pandemic, remdesivir has been one of the most promis-
ing candidates, and its repurposing for COVID-19 has allowed for the development of planned phase III clinical trials
in early 2020. The mild to moderate side effects of this medication included hepatocellular toxicity, nausea, anemia,
kidney damage, hypotension, respiratory failure, and constipation; as a result, clinical results were controversial and
worthy of discussion. Despite this, current evidence indicates that remdesivir can be provided to COVID-19 patients in
hospitals safely.

15.5.2 Repurposed drugs acting through polypeptide packing

Viral RNAs are converted into polypeptide fragments before being packed into virions, and then cleaved into functional
proteins [43]. The cleavage of these polypeptide chains is carried out by the viral proteases. The SARS-CoV-2 protease is
96% identical to the SARS-CoV protease. HIV-1 protease inhibitors have been shown to be effective against SARS-CoV.

15.5.2.1 Lopinavir-ritonavir combination

In the year 2000 the FDA approved the drug as an antiretroviral for the treatment of HIV patients. Since lopinavir is
rapidly destroyed by host proteases in the human body, it is given in conjunction with ritonavir (another protease inhibi-
tor) at a lower dose, which helps lopinavir last longer by inhibiting the metabolizing enzyme cytochrome P450 [44].
SARS-CoV has been discovered to be immune to nonspecific protease inhibition, which is commonly used in HIV
treatment. The binding energies of SARS-CoV-2 and HIV-1 proteases for lopinavir are similar [45]. In SARS-CoV-2
patients who were given lopinavir
ritonavir, significant virus clearance was achieved [46]. Protease inhibitors can
cause diarrhea, vomiting, diabetes, hypertriglyceridemia, and hypercholesterolemia, along with other side effects. It
may also cause significant hepatic damage, ranging from mild to severe hepatotoxicity, depending on the dose. A lot of
clinical and laboratory research is needed to make a conclusive point about the use of lopinavir
ritonavir.

15.5.3 Repurposed drugs acting through host target such as antiviral immunity
Pattern recognition receptors in immune cells identify viral pathogen-associated molecular patterns, which cause antivi-
ral IFN responses in the host [47]. The secreted IFNs activate hundreds of IFN-stimulated genes, which code for pro-
teins with important antiviral activity.

15.5.3.1 Interferons (pegylated Ifnα-2a and pegylated Ifnα-2b)

The IFNs are antiviral that play a critical role in innate immunity against viral infections [48]. By targeting B cells
via the host IFN receptor, IFNAR1, pegylated interferon alfa-2b boosts the immune response to viral infections.
Recombinant human IFN-2b had greater antiviral activity than ribavirin against respiratory viruses such as influenza B
virus, parainfluenza virus, respiratory syncytial virus, and coronavirus. China used interferon-2b in combination with
ribavirin to treat COVID-19, and it was also discovered that IFN-2b sprays reduced SARS-CoV-2 infection rates [49].
PEGIFN-2a does not require a major dosage change since it is metabolized by both the kidneys and the liver.
Dizziness, headache, depression, nausea, insomnia, alopecia, myalgia, arthralgia, pyrexia, and anorexia are the most
 Could repurposing existing vaccines and antibiotics help to control the COVID-19 pandemic? Chapter | 15 249

common constitutional symptoms seen with PEG-IFN-2a and PEG-IFN-2b therapy in different diseases. They could
necessitate prompt medical intervention, or, in the worst-case scenario, the dose will need to be decreased and ulti-
mately discontinued.

15.5.4 Repurposed drugs targeting the virus uptake pathways

The coronavirus enters the cell in two ways: endocytosis, in which the virus enters with the endosomes, and fusion of
the viral spike protein with the cell surface receptor ACE2, the latter being the most common path. Infections can be
treated by preventing viruses from entering the body [34].

15.5.4.1 Chloroquine and hydroxychloroquine

Chloroquine and hydroxychloroquine are weak diprotic bases that are mostly used to treat plasmodium infections [50].
These drugs bind to endosomes and boost the pH of the endosomal fluid in the same way as the virus does [51]. An
acidic pH in endosomes is needed for viral enzymes responsible for proteolysis and posttranslational modification of
nascent protein to function optimally [52]. The replication and life cycle of the virus are therefore interrupted as the
acidic pH is affected. The drug has also been shown to interfere with the glycosylation of the virus’s host receptor,
ACE2 [53]. Inappropriate terminal glycosylation may obstruct virus binding and ultimately entry into host cells [54]. In
spite of having some uncertainties, hydroxychloroquine has been associated with some side effects such as a prolonged
QT interval and heart failure. The WHO recently halted the Solidarity Trial of hydroxychloroquine arm in order to find
an effective COVID-19 drug, wherein hydroxychloroquine did not show any remarkable decrease in the death rate of
COVID patients when compared with normal care.
Although a high dose of chloroquine is needed for SARS-CoV-2, an overdose of chloroquine has been reported to
cause poisoning and death. Adverse effects of chloroquine/hydroxychloroquine treatment at the therapeutic doses
include retinopathy, myopathy, electrocardiographic changes, bleaching of hair, pruritus, headaches, dizziness, and gas-
trointestinal distress. Furthermore, the use of chloroquine and hydroxychloroquine in prophylaxis as well as in COVID-
19 patients with a greater number of clinical trials may be justified.

15.5.5 Drugs acting on host pro-inflammatory cytokines

Cytokines regulate infections, immune responses, inflammation, and trauma. Some cytokines promote inflammation,
while others are antiinflammatory and promote healing (antiinflammatory). Blocking cytokines, which damage the host,
has also received a lot of attention, particularly when the infection is severe.

15.5.5.1 Tocilizumab
Tocilizumab is an antiinflammatory drug used to treat rheumatoid arthritis and cytokine release syndrome/systemic
inflammatory response syndrome [55]. This treatment has no effect on the virus, but it does reduce the cytokine
response of the host. Tocilizumab is a recombinant monoclonal antibody that binds to the human IL-6 receptor and
blocks the signal transduction pathway [56]. Tocilizumab appears to be a safe treatment choice for COVID patients
who are at risk of a cytokine storm, according to preliminary findings. It is difficult to assess Tocilizumab’s effective-
ness against SARS-CoV-2 due to the small number of trials that have been performed.

15.5.6 Others probable potential retasking agents for the treatment of COVID-19
15.5.6.1 Azithromycin
Azithromycin is a macrolide antibiotic widely used to treat upper and lower respiratory tract infections, as well as odon-
tostomatological, skin, and soft tissue infections, nongonococcal urethritis, and soft ulcers. The antibacterial effects of
macrolides come from their interaction with the bacterial ribosome and the resulting inhibition of protein synthesis
[57]. Macrolides have antiinflammatory and immunomodulatory properties, making them a promising COVID-19 treat-
ment option. Coadministration of hydroxychloroquine and azithromycin resulted in greater SARS-CoV-2 clearance than
hydroxychloroquine alone [58]. In the absence of bacterial superinfections, the Italian Drug Agency (AIFA) advised
against treating COVID-19 patients with azithromycin alone or in combination with other drugs, especially hydroxy-
chloroquine [59].
250 Pandemic Outbreaks in the 21st Century

15.5.6.2 Corticosteroid
As a possible COVID-19 therapy, corticosteroids have received considerable attention. Low doses of dexamethasone or
slightly higher doses of intravenous hydrocortisone, administered orally or intravenously, were found to be highly effec-
tive in lowering mortality in patients who needed oxygen support or intrusive mechanical ventilation [60]. On the other
hand, these drugs were ineffective in patients who did not need oxygen support. The use of systemic corticosteroids for
the treatment of patients with severe or critical COVID-19 has been recommended by the WHO.

15.5.6.3 Teicoplanin
Chloroquine, remdesivir, lopinavir, ribavirin, and ritonavir have all been shown to suppress coronavirus in vitro studies.
In addition, after being shown to be active in vitro against SARS-CoV, Teicoplanin, a glycopeptide antibiotic effective
in treating bacterial infections, has been added to the list of molecules that could be used as a therapeutic tool against
COVID-19. This antibiotic is currently being used to treat Gram-positive bacterial infections, especially those caused
by Staphylococcus aureus. It has already shown efficacy against a number of viruses, including Ebola, influenza, flavi-
virus, hepatitis C, HIV, and coronaviruses including MERS-CoV and SARS-CoV. Teicoplanin works at the beginning
of the viral life cycle in coronaviruses by preventing cathepsin L from cleaving the viral spike protein at low pH in late
endosomes, preventing the release of genomic viral RNA and the progression of the viral replication cycle [61].

15.6 Limitations to drug repurposing approach

While drug repurposing has had some notable successes, it is not always effective, as many drug repurposing attempts
have failed, mostly at the phase III trial stage [62]. Any defects are inevitable in late-stage production, just as they are
in the development of brand-new products, but since the safety profiles are so high, these failures may be less likely to
be attributed to toxicity. To ensure high success rates of repositioned drugs, more in-depth understanding and integrated
approaches between computational and experimental methods are needed for better drug repositioning. Drug repurpos-
ing, on the other hand, can be used to effectively identify and produce new medicines with innovative and appropriate
therapeutic indications for COVID-19 and other diseases.

15.7 COVID-19 vaccination programs and repurposing of existing vaccines

The COVID-19 pandemic is unlikely to be over before vaccines that protect against severe disease and, hopefully, drive
herd immunity are widely spread around the world. Vaccine development for viral infections is complex and time-
consuming, similar to drug development. Because of the disease’s uncertain pathogenesis, lack of a validated animal
model, and effectiveness of human clinical trials, the approach is much more complicated in the case of COVID-19 dis-
ease. Due to the extreme disease’s uncertain pathogenesis, lack of a validated animal model, and effectiveness of human
clinical trials, the approach is much more complicated in the case of COVID-19 disease. As a result, the first step in the
research would be to find a protein that contains the dominant neutralizing epitope. Since it is likely easier to manufac-
ture whole-killed virus particles and inactivated virus as a first-generation vaccine, these vaccines represent all antigens
contained in a pathogen, including proteins, NAs, polysaccharides, lipids, and other components, and they are also capa-
ble of eliciting a strong immune response.
In immunized animals, SARS-CoV inactivated with formaldehyde, β-propiolactone, or UV light has been shown
to cause virus-neutralizing antibodies [63]. Vaccines based on fragments containing neutralizing epitopes, on the other
hand, can be used instead of the inactivated virus vaccine once the neutralizing epitopes have been detected since they
are safer and more effective [64]. Most coronavirus subunit vaccines depend on preventing the spike protein from
binding to the host ACE2 receptor to elicit immune responses against it [33]. The RBD on the spike protein of SARS-
CoV-2 (which has been shown to attach to the ACE2 receptor) is one way to block access to the entry receptor, as are
RBDs on the spike proteins of other coronaviruses, such as mouse hepatitis virus), transmissible gastroenteritis virus,
HCoV-229E, SARS-CoV, and others, which contain main antigenic determinants that can induce the development of
neutralizing antibodies [65]. Recombinant spike protein, a potent immunogen, is also being used to make virus-like
nanoparticles. Subunit vaccinations, on the other hand, have many disadvantages, including the need for multiple
booster shots and the use of suitable adjuvants [66].
Data from observational, epidemiological studies, and recent randomized controlled trials have shown that besides
the specific targeted effects, existing live-attenuated vaccines such as BCG, measles, chickenpox, and OPV also have
immune-modulating nonspecific effects (NSEs) that can influence host defense against diseases caused by unrelated
 Could repurposing existing vaccines and antibiotics help to control the COVID-19 pandemic? Chapter | 15 251

TABLE 15.1 Most promising vaccine candidates and their technologies.

Vaccines Technology
Sanofi with GlaxoSmithKline Protein subunit
Bharat Biotech Inactivated virus
Moderna mRNA
BioNTech with Pfizer mRNA
Novavax Protein subunit
SII with Max Planck Institute Live attenuated virus
Sinopharm with Beijing Institute Inactivated virus
AstraZeneca with Oxford University Nonreplicating viral vector
Clover Pharmaceuticals with Dynavax Protein subunit
CureVac mRNA
Inovio DNA
Covaxx with Nebraska University Protein subunit
SK Biosciences Protein subunit
CAMS with IMB Inactivated virus
Johnson & Johnson Nonreplicating viral vector

pathogens, thereby contributing for lower morbidity and mortality levels in children with reduced infant mortality rates
and overall improved childhood survivals [67,68]. Inactivated vaccines, such as diphtheria
pertussis
tetanus), hepatitis
B virus, and IPV, on the other hand, while protecting against the diseases they are designed to prevent, appear to have
negative side effects, increasing vulnerability to other pathogens. Established immunological mechanisms mediating
NSEs are heterologous cross-reactive T lymphocyte reactivity and induction of innate immune memory or “trained”
innate immunity through epigenetic and metabolic reprogramming of innate immune cells [69].
There were 289 experimental COVID-19 vaccines in production as of February 3, 2021, with 66 of them in different
stages of clinical trials, including 20 in phase 3 [70]. Just five of the 66 vaccines developed by AstraZeneca and Oxford
University, BioNTech and Pfizer, Gamaleya, Moderna, and Sinopharm and the Beijing Institute have obtained regula-
tory approval (as per WHO criteria). Other regulatory agencies have authorized or licensed five more vaccines for
emergency use, including those from China, India, Kazakhstan, and Russia; several pharmaceutical companies that
manufacture these vaccines have submitted documents to WHO for emergency use listing or prequalification, but these
submissions are still being reviewed. Table 15.1 summarizes the most promising vaccine candidates and their technolo-
gies all around the world.

15.8 Evolving strains of coronavirus genome and ineffectiveness of the vaccines

G Ever since scientists raised the alarm about B.1.1.7, a SARS-CoV-2 variant that first caught scientists’ attention in
England in December which is a more transmissible virus than previously circulating viruses; the emergence of
coronavirus variants have been in news.
G Since then 501Y.V2, a variant detected in South Africa, E484K, K417N variants have been emerged and are
believed to have worsened the pandemic situation.
G The findings indicate that SARS-CoV-2 variants may evolve resistance to recombinant spike protein vaccine-
induced immunity. Individual responses to the spike protein differ: for example, the EM188 mutant, which was cre-
ated after several passages in convalescent sera, displayed a wide range of ability to escape the neutralizing action
of sera from various patients, with some samples showing only a twofold reduction in activity.
G A lot of research into vaccinated sera testing across a variety of approved and candidate vaccines is required to
conclude that the current existing vaccines are ineffective against the mutant strains. Although vaccinologist Philip
252 Pandemic Outbreaks in the 21st Century

Krause, who chairs a WHO working group on COVID-19 vaccines, claims that the virus does not seem to have
developed resistance to COVID-19 vaccines so far for some of the mutant strains, the emergence of new strains may
escape the immune system.

15.9 Conclusion
When the virus SARS-CoV-2 began to spread presumed from China to other countries, there were no specific drugs or
preventive vaccines available, resulting in a staggering number of infected people and deaths worldwide, particularly
when compared to previous coronavirus outbreaks. The unprecedented pace at which the SARS-CoV-2 pandemic is
spreading across the globe is raising global fear to new heights. COVID-19 has become the source of widespread fear,
anxiety, and concern around the world. To meet these challenges, the scientific community has dedicated significant
research and technological resources to understand the molecular mechanisms of SARS-CoV-2 infection and replica-
tion, enhancing and speeding up diagnosis, and developing therapeutic options. In this regard, drug repurposing, which
entails testing a drug for a medical application other than its original indications, was one of the most profitable techni-
ques for identifying therapeutic agents for the treatment of COVID-19.
G Drug repurposing for COVID-19 therapy was undoubtedly the most fruitful technique to date and the approval of
remdesivir, a nucleotide mimetic prodrug, was the most promising outcome of this phase as it was approved as the
first specific medication for the treatment of COVID-19 patients in hospitals on October 22, 2020.
G Without interfering with SARS-CoV-2 replication, drug repurposing for COVID-19 was also successful in managing
patients’ symptoms. The clinical benefits of corticosteroids and LMWH therapy back up the repurposing strategy’s
extensive applicability, delivering results that are qualitatively comparable to antiviral medicines.
G Other antiviral compounds, such as favipiravir or lopinavir/ritonavir, were repositioned but did not achieve the
desired outcomes in clinical trials because they were unsuccessful in minimizing mortality or shortening the time to
recovery.
G Medications that can disrupt the virus replication cycle and minimize mortality and recovery time in COVID-19
patients are available since the onset of the outbreak because of drug repurposing approaches.
G Several drugs are actively being tested through clinical trials as possible repurposing candidates, but till now only
remdesivir has been approved for COVID-19 therapy. Remdesivir is currently being debated in the scientific
community.
G Early evidence suggested that remdesivir might shorten the period it takes for chronically ill patients to recover in
the hospital, so it received a lot of unnecessary attention such that the United States Food and Drug Administration
(FDA) approved the use of this drug.
However, its effectiveness in reducing COVID-19-related mortality has recently been questioned. Provisional find-
ings from a large international study involving thousands of patients indicate that the drug has no substantial effect on
mortality or other critical patient outcomes like the need for artificial ventilation or the time it takes for clinical prog-
ress. Despite the fact that drug repurposing has the ability to reduce the time it takes for a drug to enter the market, it is
also a method riddled with difficulties, both regulatory and scientific. To investigate the efficacy and safety of medica-
tion for potential repurposing, close collaboration between numerous authorities is needed to exploit and critically
analyze the latest information and efficiently strategize the generation of new preclinical, clinical, and observational
evidence. One of the key goals of such a project should be to prevent the repetition of studies and to develop the best
possible strategy.

Acknowledgments
The authors are indebted to Dr. Buddolla’s Educational Society for their support and all the researchers cited in this chapter for their signifi-
cant and valuable research. No funding was received to perform this review.

Conflict of interest
The authors declare no relevant competing financial interests to disclose.
 Could repurposing existing vaccines and antibiotics help to control the COVID-19 pandemic? Chapter | 15 253

References
[1] Acter T, Uddin N, Das J, Akhter A, Choudhury TR, Kim S. Evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as
coronavirus disease 2019 (COVID-19) pandemic: a global health emergency. Sci Total Environ 2020;730:138996. Available from: https://doi.
org/10.1016/j.scitotenv.2020.138996.
[2] Han E, Tan MM, Turk E, Sridhar D, Leung GM, Shibuya K, et al. Lessons learnt from easing COVID-19 restrictions: an analysis of countries
and regions in Asia Pacific and Europe. Lancet 2020;396(10261):1525
34.
[3] Low ZY, Farouk IA, Lal SK. Drug repositioning: new approaches and future prospects for life-debilitating diseases and the COVID-19 pan-
demic outbreak. Viruses 2020;12(9):1058.
[4] Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, et al. Drug repurposing: progress, challenges and recommendations. Nat Rev
Drug Discov 2019;18(1):41
58.
[5] Zhou Y, Wang F, Tang J, Nussinov R, Cheng F. Artificial intelligence in COVID-19 drug repurposing. Lancet Digital Health 2020;2(12):
e667
76.
[6] LoRusso S, Johnson NE, McDermott MP, Eichinger K, Butterfield RJ, Carraro E, et al. Clinical trial readiness to solve barriers to drug develop-
ment in FSHD (ReSolve): protocol of a large, international, multi-center prospective study. BMC Neurol 2019;19(1):1
3.
[7] Singh TU, Parida S, Lingaraju MC, Kesavan M, Kumar D, Singh RK. Drug repurposing approach to fight COVID-19. Pharmacol Rep 2020;
72:1479
508.
[8] Kim TW. Drug repositioning approaches for the discovery of new therapeutics for Alzheimer’s disease. Neurotherapeutics 2015;12(1):132
42.
[9] Padhy BM, Gupta YK. Drug repositioning: re-investigating existing drugs for new therapeutic indications. J Postgrad Med 2011;57(2):153.
[10] WHO Solidarity Trial Consortium. Repurposed antiviral drugs for COVID-19—interim WHO solidarity trial results. N Engl J Med 2021;384
(6):497
511.
[11] Khan HJ, Rohondia SO, Ahmed ZS, Zalavadiya N, Dou QP. Increasing opportunities of drug repurposing for treating breast cancer by the inte-
gration of molecular, histological, and systemic approaches. Drug repurposing in cancer therapy. Academic Press; 2020. p. 121
72.
[12] Lee HM, Kim Y. Drug repurposing is a new opportunity for developing drugs against neuropsychiatric disorders. Schizophrenia Res Treat
2016;2016:6378137. Available from: https://doi.org/10.1155/2016/6378137.
[13] Cha Y, Erez T, Reynolds IJ, Kumar D, Ross J, Koytiger G, et al. Drug repurposing from the perspective of pharmaceutical companies. Br J
Pharmacol 2018;175(2):168
80.
[14] Saxena A. Drug targets for COVID-19 therapeutics: ongoing global efforts. J Biosci 2020;45(1):1
24.
[15] Mohan BS, Nambiar V. COVID-19: an insight into SARS-CoV-2 pandemic originated at Wuhan city in Hubei province of China. J Infect Dis
Epidemiol 2020;6:146. Available from: https://doi.org/10.23937/2474-3658/1510146.
[16] WHO. Official updates
coronavirus disease 2019. Geneva, Switzerland: World Health Organization; 2020.
[17] WHO. Naming the coronavirus disease (COVID-19) and the virus that causes it. Geneva: World Health Organization; 2020.
[18] WHO. COVID-19 Weekly Epidemiological Update, 13 April 2021; 2021.
[19] Pachetti M, Marini B, Benedetti F, Giudici F, Mauro E, Storici P, et al. Emerging SARS-CoV-2 mutation hot spots include a novel RNA-
dependent-RNA polymerase variant. J Transl Med 2020;18:179.
[20] Peck KM, Lauring AS. Complexities of viral mutation rates. J Virol 2018;92(14) e01031-17.
[21] Pathan RK, Biswas M, Khandaker MU. Time series prediction of COVID-19 by mutation rate analysis using recurrent neural network-based
LSTM model. Chaos Solitons Fractals 2020;138:110018.
[22] Rouse BT, Sehrawat S. Immunity and immunopathology to viruses: what decides the outcome? Nat Rev Immunol 2010;10(7):514
26.
[23] OECD. Organisation for economic co-operation and development. The territorial impact of COVID-19: managing the crisis across levels of
government; 2020.
[24] Pal M, Berhanu G, Desalegn C, Kandi V. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2): an update. Cureus 2020;12(3):
e7423.
[25] Naqvi AA, Fatima K, Mohammad T, Fatima U, Singh IK, Singh A, et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis
and therapies: Structural genomics approach. Biochim Biophys Acta (BBA)-Mol Basis Dis 2020;1866(10):165878.
[26] Boni MF, Lemey P, Jiang X, Lam TT, Perry BW, Castoe TA, et al. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible
for the COVID-19 pandemic. Nat Microbiol 2020;5(11):1408
17.
[27] Sharifkashani S, Bafrani MA, Khaboushan AS, Pirzadeh M, Kheirandish A, Yavarpour_Bali H, et al. Angiotensin-converting enzyme 2 (ACE2)
receptor and SARS-CoV-2: potential therapeutic targeting. Eur J Pharmacol 2020;884:173455.
[28] Lukassen S, Chua RL, Trefzer T, Kahn NC, Schneider MA, Muley T, et al. SARS-CoV-2 receptor ACE 2 and TMPRSS 2 are primarily
expressed in bronchial transient secretory cells. EMBO J 2020;39(10):e105114.
[29] Gheblawi M, Wang K, Viveiros A, Nguyen Q, Zhong JC, Turner AJ, et al. Angiotensin-converting enzyme 2: SARS-CoV-2 receptor and regu-
lator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ACE2. Circ Res 2020;126(10):1456
74.
[30] Tikellis C, Thomas MC. Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. Int
J Peptides 2012;2012:256294.
[31] Ni W, Yang X, Yang D, Bao J, Li R, Xiao Y, et al. Role of angiotensin-converting enzyme 2 (ACE2) in COVID-19. Crit Care 2020;24(1):422.
[32] Satarker S, Nampoothiri M. Structural proteins in severe acute respiratory syndrome coronavirus-2. Arch Med Res 2020;51(6):482
91.
[33] Samrat SK, Tharappel AM, Li Z, Li H. Prospect of SARS-CoV-2 spike protein: potential role in vaccine and therapeutic development. Virus
Res 2020;288:198141.
254 Pandemic Outbreaks in the 21st Century

[34] Tang T, Bidon M, Jaimes JA, Whittaker GR, Daniel S. Coronavirus membrane fusion mechanism offers a potential target for antiviral develop-
ment. Antivir Res 2020;178:104792.
[35] Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.
Nature 2020;581(7807):215
20.
[36] Song W, Gui M, Wang X, Xiang Y. Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor
ACE2. PLoS Pathogens 2018;14(8):e1007236.
[37] Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2
and is blocked by a clinically proven protease inhibitor. Cell 2020;181(2):271
80.
[38] Wu C, Liu Y, Yang Y, Zhang P, Zhong W, Wang Y, et al. Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by
computational methods. Acta Pharmaceutica Sin B 2020;10(5):766
88.
[39] Guy RK, DiPaola RS, Romanelli F, Dutch RE. Rapid repurposing of drugs for COVID-19. Science 2020;368(6493):829
30.
[40] Schlagenhauf P, Grobusch MP, Maier JD, Gautret P. Repurposing antimalarials and other drugs for COVID-19. Travel Med Infect Dis
2020;34:101658.
[41] Eastman RT, Roth JS, Brimacombe KR, Simeonov A, Shen M, Patnaik S, et al. Remdesivir: a review of its discovery and development leading
to emergency use authorization for treatment of COVID-19. ACS Cent Sci 2020;6(5):672
83.
[42] Gordon CJ, Tchesnokov EP, Woolner E, Perry JK, Feng JY, Porter DP, et al. Remdesivir is a direct-acting antiviral that inhibits RNA-
dependent RNA polymerase from severe acute respiratory syndrome coronavirus 2 with high potency. J Biol Chem 2020;295(20):6785
97.
[43] Gioia M, Ciaccio C, De Simone G, Fasciglione GF, di Masi A, Di Pierro D, et al. Role of proteolytic enzymes in the COVID-19 infection and
promising therapeutic approaches. Biochem Pharmacol 2020;182:114225.
[44] Uzunova K, Filipova E, Pavlova V, Vekov T. Insights into antiviral mechanisms of remdesivir, lopinavir/ritonavir and chloroquine/hydroxy-
chloroquine affecting the new SARS-CoV-2. Biomed Pharmacother 2020;131:110668.
[45] Ortega JT, Serrano ML, Pujol FH, Rangel HR. Unrevealing sequence and structural features of novel coronavirus using in silico approaches:
the main protease as molecular target. EXCLI J 2020;19:400.
[46] Cao B, Wang Y, Wen D, Liu W, Wang J, Fan G, et al. A trial of lopinavir
ritonavir in adults hospitalized with severe COVID-19. N Engl J
Med 2020;382:1787
99.
[47] Kumar P, Sobhanan J, Takano Y, Biju V. Molecular recognition in the infection, replication, and transmission of COVID-19-causing SARS-
CoV-2: an emerging interface of infectious disease, biological chemistry, and nanoscience. NPG Asia Mater 2021;13(1):1
4.
[48] Lee AJ, Ashkar AA. The dual nature of type I and type II interferons. Front Immunol 2018;9:2061.
[49] Sallard E, Lescure FX, Yazdanpanah Y, Mentre F, Peiffer-Smadja N. Type 1 interferons as a potential treatment against COVID-19. Antivir
Res 2020;178:104791.
[50] Chen X, Geiger JD. Janus sword actions of chloroquine and hydroxychloroquine against COVID-19. Cell Signal 2020;73:109706.
[51] Al-Bari MA. Targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases.
Pharmacol Res Perspect 2017;5(1):e00293.
[52] Fung TS, Liu DX. Human coronavirus: host-pathogen interaction. Annu Rev Microbiol 2019;73:529
57.
[53] Singh R, Vijayan V. Chloroquine: a potential drug in the COVID-19 scenario. Trans Indian Natl Acad Eng 2020;5:399
410.
[54] Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology. Biochim Biophys Acta
(BBA)-Gen Subj 2019;1863(10):1480
97.
[55] Zhang C, Wu Z, Li JW, Zhao H, Wang GQ. Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab
may be the key to reduce mortality. Int J Antimicrob Agents 2020;55(5):105954.
[56] Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science 2020;368(6490):473
4.
[57] Vázquez-Laslop N, Mankin AS. How macrolide antibiotics work. Trends Biochem Sci 2018;43(9):668
84.
[58] Furtado RH, Berwanger O, Fonseca HA, Corrêa TD, Ferraz LR, Lapa MG, et al. Azithromycin in addition to standard of care versus standard
of care alone in the treatment of patients admitted to the hospital with severe COVID-19 in Brazil (COALITION II): a randomised clinical trial.
Lancet 2020;396(10256):959
67.
[59] Sultana J, Cutroneo PM, Crisafulli S, Puglisi G, Caramori G, Trifirò G. Azithromycin in COVID-19 patients: pharmacological mechanism, clin-
ical evidence and prescribing guidelines. Drug Saf 2020;43(8):691
8.
[60] Petersen MW, Meyhoff TS, Helleberg M, Kjær MB, Granholm A, Hjortsø CJ, et al. Low-dose hydrocortisone in patients with COVID-19 and
severe hypoxia (COVID STEROID) trial—protocol and statistical analysis plan. Acta Anaesthesiol Scandinavic 2020;64(9):1365
75.
[61] Zhou N, Pan T, Zhang J, Li Q, Zhang X, Bai C, et al. Glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and
block the entry of ebola virus, middle east respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus
(SARS-CoV). J Biol Chem 2016;291(17):9218
32.
[62] Juárez-López D, Schcolnik-Cabrera A. Drug repurposing: considerations to surpass while re-directing old compounds for new treatments. Arch
Med Res 2020;52(3):243
51.
[63] Edwards KM, Orenstein WA. Anticipating severe acute respiratory syndrome coronavirus 2 vaccine testing, licensure, and recommendations for
use. J Pediatr 2020;224:124
8.
[64] Dai L, Gao GF. Viral targets for vaccines against COVID-19. Nat Rev Immunol 2021;21:73
82.
[65] Li F. Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol 2016;3:237
61.
[66] Kaur SP, Gupta V. COVID-19 Vaccine: a comprehensive status report. Virus Res 2020;288:198114.
 Could repurposing existing vaccines and antibiotics help to control the COVID-19 pandemic? Chapter | 15 255

[67] Pollard AJ, Finn A, Curtis N. Non-specific effects of vaccines: plausible and potentially important, but implications uncertain. Arch Dis Child
2017;102(11):1077
81.
[68] Saso A, Kampmann B. Maternal immunization: nature meets nurture. Front Microbiol 2020;11:1499.
[69] Italiani P, Boraschi D. Induction of innate immune memory by engineered nanoparticles: a hypothesis that may become true. Front Immunol
2017;8:734.
[70] Wouters OJ, Shadlen KC, Salcher-Konrad M, Pollard AJ, Larson HJ, Teerawattananon Y, et al. Challenges in ensuring global access to
COVID-19 vaccines: production, affordability, allocation, and deployment. Lancet 2021;397(10278):1023
34.
This page intentionally left blank
Chapter 16

Genetics of coronaviruses
Shanthala Mallikarjunaiah1, Basavaraja Metikurki2 and Hunasanahally Puttaswamygowda Gurushankara3
1
Center for Applied Genetics, Department of Zoology, Bangalore University, Bengaluru, India, 2Nitte College of Pharmaceutical Sciences, Bengaluru,
India, 3Department of Zoology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Kasaragod, India

16.1 History of coronaviruses

Coronaviruses (CoVs) have been described as novel respiratory tract viruses, which were observed in the samples col-
lected from the people who presented with respiratory tract infections. The first described CoV disease was in 1931; it
was known as Avian infectious bronchitis virus (IBV) of chickens [1]. In 1965 human coronavirus (HCoV) was first
isolated from a patient nasal discharge with symptoms of the common cold, which was named as B814 [2]. Then identi-
fied two viruses HCoV-229E (α-CoV) and HCoV-OC43 (β-CoV, lineage A) [3,4]. The name “coronavirus,” coined in
1968, is derived from their “crown-like” (corona in Latin means crown) spikes, which are 12
24 nm long on their sur-
face appearance by transmission electron microscopy [5].
SARS-CoV (β-CoV, lineage B) was identified from southern China in 2002, which causes severe acute respiratory
syndrome (SARS). Eventually, it spread to other countries in Asia, in addition to North America and Europe. SARS-
CoV was most likely originated in bats, then entered the human population through intermediate hosts, most likely the
“Himalayan palm civet” (Paguma larvata) and the raccoon dog (Nyctereutes procyonoides) [6]. More than 8000 people
were infected with SARS-CoV and 774 died by July 2003, with a case fatality rate (CFR) of 9%. The elderly were
more susceptible to SARS disease, with a mortality rate of over 50% [7].
NL63 (α-CoV) was detected in a baby suffering from bronchiolitis in the Netherlands in 2004. It is primarily associ-
ated with children, the elderly and immune-compromised patients with respiratory illnesses. An estimated 4.7% of com-
mon respiratory diseases are distributed globally [8]. A novel coronavirus HKU1 (β-CoV, lineage A), identified from a
71-year-old man with pneumonia, returned from Shenzhen, China, a previously SARS-endemic area, residing in Hong
Kong [9]. HKU1 cases outside Asia were detected in New Haven, USA, Australia, France, and Brazil, indicating a
global distribution of the virus [10].
MERS-CoV (β-CoV, lineage C) that causes Middle East Respiratory Syndrome (MERS) has started in Saudi Arabia
in 2012 [11]. MERS-CoV has a close relationship with two bat-CoVs (HKU4 and HKU5). The camelids (Camelus
dromedaries) serve as intermediaries between infected vespertilionid bats and humans, that is, camel-to-human trans-
mission and human-to-camel transmission [12]. MERS-CoV is less contagious than its SARS cousin since the human-
to-human transmission of MERS-CoV is minimal. There were two further MERS-CoV outbreaks in South Korea in
2015 and Saudi Arabia in 2018. There is an intermittent of MERS-CoV infections that continue to this day, but the epi-
sodes are usually well contained. Another serious CoV is the enteric bat CoV-HKU2, identified in China, which causes
severe piglet diarrhea and mortality and severely impacts the livestock industry [13].
The novel HCoV pandemic, known as COVID-19, was first reported in Wuhan, Hubei Province, China, in late
December 2019. The earliest date of symptom onset was December 1, 2019. These patients’ symptoms, including fever,
sickness, dry cough, and dyspnea, were diagnosed as viral pneumonia [14,15]. Initially, it was called “Wuhan pneumo-
nia” by the media because of the area and pneumonia-like symptoms. Whole-genome sequencing data confirmed that
the causative agent is a novel CoV; it is the seventh member of the coronaviridae family to infect humans [16]. The
World Health Organization (WHO) tentatively termed the new virus 2019 novel coronavirus (2019-nCoV) on January
12, 2020. Then officially called this as infectious coronavirus disease 2019 (COVID-19) on February 12, 2020. The
CoV formally was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International
Committee on Taxonomy of Viruses (ICTV) based on phylogenetic analysis, taxonomy, and established practice [17].

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00003-3

© 2021 Elsevier Inc. All rights reserved. 257
258 Pandemic Outbreaks in the 21st Century

On March 11, 2020, the WHO finally assessed that COVID-19 as a pandemic; it began spreading freely across the
World as a global threat. SARS-CoV-2 is believed to be a spillover of an animal CoV and later adapted the ability of
human-to-human transmission. Human-to-human transmission is through close contact of respiratory droplets, direct
contact with the infected individuals, or contact with contaminated objects and surfaces [7]. Because the CoV is highly
contagious, it rapidly spreads worldwide and continuously evolves in the human population [18]. Worldwide, 87.2 mil-
lion COVID-19 cases were confirmed, with 1.88 million deaths recorded as of January 7, 2021 [19] (Table 16.1).

16.2 Taxonomy of coronaviruses

The Nidovirales order contains Coronaviridae, Arteviridae, and Roniviridae families [20]. The variations in the
Nidovirales variety, kind, and sizes of the structural proteins cause considerable changes within the nucleocapsids and
virions’ structure and morphology. All viruses within the Nidovirales order have a vast genome having salient standard
features: gene expression through transcription of a set of multiple 30 -nested subgenomic RNAs; replicase polyprotein
expression via ribosomal frameshifting; unique enzymatic activities among the replicase proteins; a virion membrane
envelope; and a multispanning integral membrane protein in the virion. The first of these qualities provide the order’s
name, which was derived from the Latin Nido, meaning nest [21]. The Arteviridae family includes swine and equine
viruses, and the Roniviridae family comprises invertebrate viruses [22,23]. In 1975 the Coronaviridae family was estab-
lished by the ICTV. The Coronaviridae family was divided into Coronavirinae and Torovirinae, two subfamilies, at the
10th International Nidovirus Symposium in Colorado, in June 2005. The Toroviruses belong to the subfamily
Torovirinae that causes enteric diseases in cattle and possibly in humans. CoVs belong to the subfamily Coronavirinae
and the genus Coronavirus. Genus CoVs were classified into four genera that include alpha coronavirus (α-CoV), beta
coronavirus (β-CoV), gamma coronavirus (γ-CoV), and delta coronavirus (δ-CoV). Based on their serological cross-
reactivity, genomic structure, and phylogenetic clustering (habitat/genetic relatedness), CoVs are classified as above
[24,25].
In general, α-and β-CoVs primarily include CoVs from mammals, whereas γ- and δ-CoVs mostly include CoVs of
Avian origin. Phylogenetically, all currently known HCoVs belong to two genera α- or β-CoVs. The low-CFR strains
span both genera and the high-CFR strains belong solely to β-CoVs; these CoVs are mainly found in the bats. α-CoV
contains the porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, canine coronavirus, feline
infectious peritonitis virus, human coronaviruses 229E (HCoV-229E), and NL63 (HCoV-NL63) [26]. β-CoV contains
the murine hepatitis virus (MHV), bovine coronavirus, rat sialodacryoadenitis virus, porcine hemagglutinating encepha-
lomyelitis virus (PHEV), canine respiratory coronavirus, equine coronavirus, and human coronaviruses OC43 (HCoV-
OC43), SARS-CoV, HKU1, MERS-CoV, and SARS-CoV-2 [27,28]. γ-CoV contains the Avian IBV and turkey corona-
virus [29]. δ-CoV includes several newly evolved CoVs from the wild birds’ gene pools. Recombination frequently
occurs between the different viral lineages’ genome that encodes the host receptor-binding protein, contributing to new
viruses’ emergence—capable of interspecies transmission and adaptation to new animal hosts [28,30].
The β-CoV genus is divided into A, B, C, and D lineages. Lineage A includes OC43, HKU1, MHV, PHEV, equine,
rabbit, camel, bovine, and antelope CoVs. Lineage A CoVs encode a smaller protein called a hemagglutinin esterase,
which is functionally similar to the spike (S) protein [31]. SARS-CoV, SARS-like viruses of the bat and palm civet ori-
gin are under lineage B. Lineage C includes MERS-CoV and some bat-derived viruses. Lineage D contains only bat-
derived CoVs. In contrast to α- and β-CoVs, γ-CoVs consist mainly of Avian CoVs and CoVs isolated from aquatic
animals, whales, and dolphins. CoVs of wild-bird origin clustered into the δ-CoV, containing some swine-derived CoVs
[10]. Next-generation sequencing of SARS-CoV-2 shows 79% homology to SARS-CoV and 50% to MERS-CoV; hence
SARS-CoV-2 is placed under the subgenus Sarbecovirus of the genus β-CoV [32,33] (Table 16.2).

16.3 Naming of coronaviruses

The type of the disease, virus, and species have to be considered for naming a new viral disease. The WHO is responsi-
ble for the first (disease), expert virologists group for the second (virus), the ICTV for the third (species). The WHO
announced the disease name on February 11, 2020. WHO has named the disease COVID-19, short for “coronavirus dis-
ease 2019.” Co and Vi come from coronavirus, with D meaning disease and 19 standing for 2019, the year. The ICTV
is the global authority on the designation and naming of new viruses. The Coronavirus Study Group (CSG) of the ICTV
officially named it as the novel coronavirus SARS-CoV-2 on February 11, 2020. The viral genomes from several
patients were analyzed and assessed phylogenetic relationships between the new viruses (CoVs). The genome of viruses
isolated from patients was similar to SARS.
TABLE 16.1 History of human coronaviruses.

S. Coronavirus Location/ Natural host Intermediate Cellular receptor Type Year Respiratory References
no. strain origin host symptom
01 B814 UK Cultured a virus from a boy


1965
[2]
with cold
02 HCoV-229E Chicago, Bats Camelids Aminopeptidase N α 1966 Mild [3]
USA (CD13)
03 HCoV-OC43 Maryland, Rodents Cattle 9-O-Acetylated β-A 1967 Mild [4]
USA sialic acid
04 SARS-CoV China Bats Masked palm ACE2 β 2002 Severe acute [6]
civets
05 NL63 Netherlands Bats Unknown ACE2 α 2004 Mild [8]
06 HKU1 Hong Kong Rodents Unknown 9-O-Acetylated β-A 2005 Mild [9]
sialic acid
07 MERS-CoV Saudi Bats Dromedary DPP4 β-C 2012 Severe acute [11]
Arabia camels
08 SARS-CoV-2 China Bats Pangolin! ACE2 β-B 2009 Severe acute [16]
260 Pandemic Outbreaks in the 21st Century

TABLE 16.2 Typical taxonomic position of SARS-CoV-2.

Category Virus
Realm Riboviria
Order Nidovirales
Suborder Cornidovirineae
Family Coronaviridae
Subfamily Coronavirinae
Genus Betacoronavirus
Subgenus Sarbecovirus
Species Severe acute respiratory syndrome
related coronavirus
Individuum SARS-CoV-2

The clinical presentation, epidemiologic patterns, and host range of SARS-CoV-2 may differ from the original SARS-
CoV. The two viruses’ genetic similarity is confirming the same species. The CSG found fewer differences in the conserved
marker gene when comparing the SARS-CoV and SARS-CoV-2 genomes than between MERS and HCoV-OC43 viruses.
Hence, the CSG has named SARS-CoV (SARS-CoV-1) and SARS-CoV-2 as variants of the species known as SARS-related
CoVs [34]. The new virus’s provisional name stemmed from the year it was first seen (2019), the fact that it was new (n),
and a member of the CoV family. The functional naming convention provides a standard format to use for any future CoV
outbreaks. For example, if there is a new CoV outbreak in 2023, the disease’s name can be COVID-23.

16.4 Genome of coronaviruses

CoVs are pleomorphic, enveloped, and spherical particle that measure between 80 and 160 nm in diameter containing a
nonsegmented positive-sense, large single-stranded ribonucleic acid genomes. CoVs have the largest genomes among
RNA viruses, approximately 26.4
31.7 kilobases (kb), more than double the average RNA virus genome, and encode
for 22
29 proteins [35
37]. All CoVs shared their similar type of genome organization and gene expression. The typi-
cal genome of CoV contains polycistronic, a nested RNA, 50 -cap, 50 -untranslated region (UTR), 6
11 variable number
of open reading frames (ORFs), a 30 -UTR, and 30 -poly (A) tail [38
40].
The first two ORFs (ORF1a/b), representing approximately 67% of the entire genome, encode 16 nonstructural pro-
teins (nsp1 to nsp16) of .7000-amino-acid pp1a/pp1ab large polypeptide. The pp1a/pp1ab large polypeptide translated
from ORF1a/b is proteolytically processed in cis and trans viral proteases encoded by nsp3 and nsp5. Nsp3 contain one
or two papain-like proteases (PLpro1 and PLpro2), and nsp5 contains a chymotrypsin-like cysteine protease (3CLpro)
[41]. The 3CLpro, as the main protease, catalyzes the proteolytic cleavage of all nsps downstream of nsp4. The trans-
lated proteins include the putative RNA-dependent RNA polymerase (RdRp) (nsp12) and its cofactors (nsp7 and nsp8),
RNA helicase (HEL) and an adenosine triphosphatase (ATPase) (nsp13), exoribonuclease (nsp14) and co-factor
(nsp10), N7 methyltransferase (nsp14), endonuclease (nsp15), and 20 -O-methyltransferase (nsp16). These nsps are
involved in viral RNA replication/transcription, processing, capping and are directly translated from the genomic RNA
[10,42,43]. The protease activities are common to RNA viruses. The CoV replicase employs various RNA processing
enzymes that are not found in other RNA viruses and are unique to the coronaviridae. These include putative sequence-
specific endoribonuclease, 30 -to-50 exoribonuclease, 20 -O-ribose methyltransferase, ADP ribose 1v-phosphatase, and
cyclic phosphodiesterase [44]. The precise, unique strategy used by CoVs for genome replication is unknown, although
well established many features in the genome. Scientists are considered inhibitors of 3CLpro and PLpro as potential
drug targets, their cleavage recognition sequences are distinct from human proteases, and they are essential to viral rep-
lication [45,46]. PLpro is responsible for cleavage events in pp1a; it also functions as a deubiquitinase and
deISGylating enzyme, contributing to the avoidance of the initial antiviral response [47]. Therefore it is possible that
targeting PLpro would inhibit viral replication and prevent the dysregulation of cellular signaling pathways that could
lead to programmed cell death of surrounding cells [48].
 Genetics of coronaviruses Chapter | 16 261

The genome’s remaining ORFs encode structural proteins and several accessory proteins with unknown functions that do
not participate in viral replication [42]. Genes for the four major conserved structural proteins in all CoVs occur in the 50 to 30
order as the spike (S) protein, envelope (E) protein, transmembrane (M) protein, and nucleocapsid (N) protein. The spike (S),
the type I glycoprotein (B150 kDa) forms the peplomers on the virion surface, giving the virus its corona or crown-like mor-
phology that is characteristic of CoVs [49]. The S fusion glycoprotein plays an essential role in binding to receptors on the host
cell, determines host tropism and subsequent viral entry to host cells [50,51]. The CoV S protein binds to different host recep-
tors via receptor-binding domains (RBDs). SARS-CoV uses angiotensin-converting enzyme 2 (ACE2) as one of the primary
receptors with CD209L as an alternative receptor [52]. ACE2 is a cell-surface peptidase that hydrolyzes angiotensin II in most
organs, with exceptionally high in lung and small intestine [53]. After ACE2 receptor binding, S proteins are subsequently
cleaved and activated by the host cell
surface protease TMPRSS2 at the S1/S2 and S29 sites, leading to membrane fusion
[54]. Some S proteins are precleared at the S1/S2 site by the cellular protease furin during their biosynthesis in the producer
cell [55]. MERS-CoV uses dipeptidyl peptidase 4 (also known as CD26) as the primary receptor [16]. Multiple cellular pro-
teases, including TMPRSS2, endosomal cathepsins, and furin, have been involved in the cleavage [56,57]. The MHV S protein
uses the host CEACAM1a as its receptor and is subsequently cleaved at S29 by lysosomal proteases [58].
The smallest of the major structural proteins is the membrane-spanning envelope protein (E) (B8
12 kDa), a highly
hydrophobic protein part of the nucleocapsid of viral particles and participates in viral assembly and budding [59]. The
most abundant transmembrane (M) protein (B25
30 kDa) that spans the membrane three times and has a short N-
terminal ectodomain and a cytoplasmic tail defines the shape of the viral envelope [60]. The nucleocapsid (N) phospho-
protein with the RNA forms a helical capsid within the viral membrane involved in viral assembly and budding, result-
ing in complete virion formation [61]. Nonstructural proteins such as ORF3a, ORF7a, and ORF8 function as accessory
proteins playing a role in viral pathogenesis [62].
CoVs are the largest genomes among RNA viruses. They have an essential gene, RdRp, which is highly conserved; it
makes the gene useful as a stable genetic marker for measuring the evolutionary distance and relatedness of one RNA virus to
another [63]. The fully sequenced genomes of seven species of HCoVs are available in the National Center for Biotechnology
Information (NCBI) GenBank and Virus Pathogen Database and Analysis Resource (ViPR) (http://www.viprbrc.org/). As viral
genomes are made publicly available, we could track viral evolution, animal-to-human, human-to-human transmission, mutation
rate, and stable adaptation and pathogenicity. For instance, the SARS-CoV-2 genome sequence analysis across samples has
revealed the highest diversity in the structural genes: S protein, ORF3a, and ORF8. Five mutations have been identified, includ-
ing T8782C (in ORF1a, codons AGT to AGC, silent mutation), T9561C (in ORF1a, codons TTA to TCA, nonsilent mutation),
C15607T (in ORF1b, codons CTA to TTA, silent mutation), C28144T (in ORF8b, codons TCA to TTA, nonsilent mutation),
and T29095C (in nucleocapsid, codons TTT to TTC, silent mutation) [64]. Therefore SARS-CoV-2 is evolved from the SARS-
CoV virus and evolving new variants of such CoVs in the pandemic duration.

16.5 Coronavirus diversity

The diversity of CoVs results from the RdRp infidelity, high frequency of homologous RNA recombination, efficient adap-
tive evolution, and the large genomes of CoVs. The RdRp infidelity of CoVs makes their mutation rates (about
1.05 3 1023
1.26 3 1023 substitutions per site per year) in the order of one per 1000 to 10,000 nucleotides replication [65].
High mutation rates result in several slightly different versions of the CoVs genome made by replicating their genome every
time. The CoV population with diverse genomes is known as quasispecies. With each of its replication cycle, the differences
accumulate between the original parental and the progeny genomes that may contribute to clinical outcomes between
patients, as the viral populations that are infecting them are slightly different. The multiple versions of the CoVs also make it
challenging to categorize CoVs as distinct species. Hence, CSG assessed relative levels of relatedness between and within
SARS-CoV and SARS-CoV-2 and compared it to the level of relatedness between and within other CoV species.
Many CoVs of human and animal origin are currently circulating in the wild have a unique random template-
switching process of RNA replication, mediated by a “copy choice” mechanism. CoVs have a high frequency of homol-
ogous RNA recombination and undergo evolution resulting in novel viruses [65]. CoV’s recombination in camels has
resulted in a dominant recombinant CoVs MERS lineage that potentially caused human outbreaks in 2012 [10].
CoVs spread from animal hosts (bats, camels, dogs, and masked palm civets) to humans [6,42,66,67]. The signifi-
cant natural carrier of various CoVs that could transmit from animals to humans is bats. Seven CoVs are known to
infect humans: 229E, OC43, SARS-CoV, NL63, HKU1, MERS-CoV, and SARS-CoV-2. The 229E, OC43, NL63, and
HKU1 widely circulated in the human population generally cause mild upper respiratory illness associated with 10%

30% of common cold symptoms [68]. The remaining three highly pathogenic CoVs have emerged into the human popu-
lation from wildlife that can cause severe respiratory illness. SARS-CoV resulted in the outbreak of SARS in 2002
262 Pandemic Outbreaks in the 21st Century

and 2003 [69,70]. The CoVs responsible for the MERS-CoV, which emerged in 2012, remained in camels [11], and
SARS-CoV-2 appeared in December 2019 in Wuhan, China [15]. Among all hosts, the diversity of CoVs in bats and
birds may result from their species diversity, ability to fly, environmental pressures, and roosting and flocking habits.
The present evidence supports that bat CoVs are the gene pools of α- and β-CoVs, whereas bird CoVs are the gene
pool of γ-CoVs. With the increasing number of CoVs, more and more closely related CoVs observed from distantly
associated animals, resulting from recent interspecies jumping and maybe the cause of disastrous outbreaks of zoonotic
diseases. An estimated 100s to 1000s of CoVs may reside in bats alone [26], and the history of epidemics highlights the
potential for future CoVs zoonotic transmission. Furthermore, in light of CoVs’ ability to recombine, mutate, novel
strains of CoVs continue to evolve, emerge, and cause new zoonotic disease outbreaks in the future.

16.6 Genetics of coronavirus infection

Some young, healthy people experience mild, severe, very severe, or life-threatening illness when they get CoVs infection.
Why young people become so ill is one of the puzzles of the COVID-19. Extensive studies in geographically diverse popula-
tions have demonstrated substantial genetic variation in protein-coding regions, with widely varying allele frequencies
[71
73]. COVID-19 Host Genetics Initiative reveals genetic and nongenetic associations with COVID-19 susceptibility and
severity [74]. Severe responses to CoV infection, like those seen with other illnesses such as influenza, could be caused by
genetic differences between individuals [75]. The pathogenesis of COVID-19 associated respiratory failure is poorly under-
stood, but higher mortality is consistently associated with older age and male sex [76]. COVID-19 is associated with hyper-
tension, diabetes, obesity, and cardiovascular disease in individuals. The clinical risk factors in determining the severity of
COVID-19 are not clear [77,78]. The data on lymphocytic endotheliitis and vascular thromboembolic complications suggest
that COVID-19 is a systemic disease involving injury to the vascular endothelium resulting in pathogenesis [79
81]. In gen-
eral, an individual’s genetic landscape in particular, and a population seems to play a pivotal role in shaping the COVID-19
dynamics [82]. Geneticists have raced to see a person’s DNA explains why some get hit hard by the CoVs, and they have
uncovered provocative leads. Understanding the gene variants linked to the most severe disease cases and that point to exist-
ing drugs could be repurposed to help. It would provide a potential target for speedy treatment and even save many lives.
The human genome is the individual instruction manual containing all the information needed to make, maintain,
and repair DNA damage. Almost all human cells have DNA, which we inherit from our parents [83]. Genes can work
singly but much more commonly act together. They also interact with the environment. Any two people have roughly
99.9% of genomes the same; it is the remaining 0.1% that makes them different. This variation may be significant in
determining how other people respond to particular infections. When the immune system of CoV infected people, frag-
ments of the virus may differ from each other [84]. Genes involved in the immune response to virus infection are highly
diverse within the genome. Specific genes on the X chromosome could be why males (only one copy of X) are severely
affected than females (two copies). Variations in human genomes, both immune genes and others, could explain at least
some of the differences in coronavirus infection responses [85], exploration of human genetics explain differences in
how patients respond to treatments for COVID-19.
The mechanisms of host genetic resistance to CoVs infections at three sequential levels: (1) genetic control at the level
of cellular receptors, (2) genetic control at the macrophage level, and (3) genetic control at the acquired immunity level
[86]. The presence or absence of a specific cellular receptor controls viral entry. Once the virus has gained access, the host
cells’ factors restrict or permit viral growth and disease. Genetic factors exempt genetically resistant individuals from strict
social distancing restrictions and other suitable measures [87]. Those with genetic markers for susceptibility would face
more substantial limits. The host’s immune system’s humoral and cellular defenses determine whether the virus is elimi-
nated or disseminated and leads to chronic disease. Genetic markers raise even more significant concern in this regard, as
they are immutable. Those born with genetic susceptibility always have it, while those who lack immunity because they
have not been exposed to the CoVs can later acquire it. Moreover, those with known genetic susceptibility would be less
likely to gain antibody-building exposure because of their heightened need to avoid the disease [87].

16.7 Potential genes for pathogenesis of COVID-19

Genetic factors play a role in human susceptibility to infectious diseases. Genes play a major or minor part in determin-
ing the response to CoVs infection; tracking them is essential because they could provide clues about the biological
pathways involved in CoV disease. To finding genes that influence a COVID-19 disease, geneticists screen the DNA
marker sequences of many people looking for associations between specific markers and disease. The human genetics
community worldwide generates, shares, and analyzes data to determine the genetic determinants of COVID-19. There
 Genetics of coronaviruses Chapter | 16 263

is a considerable variation in disease behavior among patients infected with SARS-CoV-2. Genome-wide association
(GWAS) analysis may allow for identifying potential genetic factors involved in the development of COVID-19. The
available genetic data in the scientific literature could help to identify individuals at risk of disease, concepts for drug
repurposing, and contribute to global knowledge of SARS-CoV-2 infection. We do not know there is a link between
genes, immunity, and the severity of COVID-19. Understanding the illness and how people are affected is key to slow-
ing the spread of CoV diseases.

16.7.1 Chromosome 3P21.31 gene locus

The specific chromosome 3 genes are the most potent genetic factor in CoVs disease. The locus of B50 kb at chromo-
some 3p21.31 comprised six genes (SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6, and XCR1) that may have inherited
from Neanderthals reported in mildly and severely affected patients [74,88]. The risk allele GA of rs11385942 is associ-
ated with reduced expression of CXCR6 and increased expression of the sodium
imino acid (proline) transporter 1
(SIT1)-encoding SLC6A20 and LZTFL1 in human lung cells. There are functional interacts with the novel coronavirus’s
cell-surface receptor, ACE2 [89]. SLC6Z20 codes for a protein that interacts with the cell receptor used by SARS-CoV-
2 to enter cells. The remaining genes encode chemokine receptors of the C-C and CXC superfamily of G protein-
coupled receptors members. Chemokines control cell migration connected with immune surveillance by trafficking
effector cells to sites of infection and inflammation [90]. CXCR6 and its flanking genes (CCR1 and CCR2) regulate
lung-resident memory CD8 1 T cells’ specific location through sustained immune response to airway pathogens
[91,92]. The GWAS analysis highlights associations for a particular genetic variant between hospitalized patients with
severe disease, mildly symptomatic, or asymptomatic individuals [93]. The younger age of homozygous patients for the
risk allele than patients who were heterozygous or homozygous for the nonrisk allele suggests the chromosome’s
involvement 3p21.31 locus in COVID-19 disease severity [94]. Available variant database entries indicated that the fre-
quency of this risk allele varies among populations worldwide. Geneticists have identified gene variants that explain
why COVID-19 has hit African Americans. The chromosome 3 gene variant is absent in most of the African ancestry
people. Many gene link studies have found a genome region that may raise the risk of severe disease [95]. Identifying
more genetic risk factors in the future reveals the disease’s underlying biology and inspires better treatments.

16.8 ABO blood group genes

ABO blood group implicated in SARS-CoV-2 and SARS-CoV-1 infection [96]. Blood groups A and O have a reduced
risk of acquiring COVID-19 than other blood groups [97,98]. The biologic mechanisms involved with the ABO group
are developing neutralizing antibodies against protein-linked N-glycans or gene variants [99
101]. It has been evi-
denced for ABO and Rh blood groups’ association, depleting O and B blood group enrichment among SARS-CoV-2
patients. Rh(D)-positive blood types are associated with SARS-CoV-2 infection and death following infection, without
confounding demographics or other known risk factors [102]. A 3p21.31 genetic susceptibility locus in patients with
COVID-19 with respiratory failure confirmed the ABO blood group system’s potential involvement. The association
signal 9q34.2 is the ABO blood group locus; a blood group
specific analysis showed a higher risk in blood group A
than in other blood groups and a protective effect in blood group O compared with different blood groups [93].
Nonetheless, the observed associations with blood types differed from SARS-CoV-2, suggesting they may be incidental,
stemming from factors unrelated to COVID-19 [74].

16.8.1 Human leukocyte antigen genes

One right place to look for genetic factors controlling COVID-19 is within the HLA complex, which plays a crucial
role in regulating immunity. Multiple genes are controlling the different types of human leukocyte antigen (HLA) and
may involve severe response to CoVs infection. The potential associations between the genetic variability in major
histocompatibility complex (MHC) class I genes HLA A, B, and C with the susceptibility to SARS-CoV-2 infection and
severity of COVID-19 disease suggest that individuals harboring HLA-B*46:01 allele may be particularly vulnerable to
COVID-19 [103,104]. The highly conserved SARS-CoV-2 peptides shared among HCoVs detected HLA-B*15:03, sug-
gesting that this allelic variant could enable cross-protective T cell-based immunity [104]. A study from China showed
that the HLA-A*11:01, -B*51:01, and -C*14:02 alleles significantly predispose patients to the worst clinical outcome
[105]. Another report from Italy added HLA-DRB1*15:01, HLA -DQB1*06:02, and HLA-B*27:07 three HLA alleles to
the list that may predispose for a less favorable outcome by analyzing a group of patients affected by a severe or too
264 Pandemic Outbreaks in the 21st Century

severe form of COVID-19 compared to a reference group of individuals [106]. Analysis of the classical HLA loci (chro-
mosome 6, 25 through 34 Mb) showed no significant allele associations with the severity of COVID-19 disease. Further
research of heterozygote and divergent alleles of HLA-bound SARS-CoV-2 peptides did not show significant associa-
tions with COVID-19 [93].

16.8.2 X-chromosomal Toll-like receptor 7 gene

X-chromosome inactivation mechanisms contribute to sexual dimorphism and X-linked gene mosaicism. Males are hap-
loid for the X-chromosome that is inherited from their mothers. Any changes in X-chromosome genes are likely to be
expressed phenotypically and immunologically. Females have maternal and paternal X chromosome; the functional
mosaics of X-linked genes contribute to females’ immunological advantage in CoVs infections. Females tend to have
higher antibody responses and more adverse reactions to some vaccines and are more prone to developing autoimmu-
nity [107,108]. Testosterone is an immune suppressor through the upregulation of anti-inflammatory cytokines
interleukin-10. Estrogen hormone enhances the immune system by upregulating pro-inflammatory cytokines tumor
necrosis factor-alpha [109,110].
The X-chromosomal Toll-like receptor 7 (TLR7) gene’s loss is associated with impaired type I and II IFN in severe
COVID-19 patients [94]. Identified a maternally inherited 4-nucleotide deletion (c.2129_2132del; p.[Gln710Argfs*18])
in members of the first family that included a brother who died of the SARS-CoV-2 infection; the affected members of
the second family carried a missense variant (c.2383G . T; p.[Val795Phe]) in TLR7. Type I IFN signaling was tran-
scriptionally downregulated in primary peripheral blood mononuclear cells of patients. It has been observed that
decreased mRNA expression of IRF7, IFNB1, and ISG15 on stimulation with the TLR7 agonist imiquimod (an immune
response modifier) [111] seems to be an essential component of the innate immunity against SARS-CoV-2 [112,113]. It
is also reported that SARS-CoV-2 induces a lower antiviral transcriptional response, marked by low type I IFN levels
and elevated chemokine expression, compared to other respiratory viruses [114]. A new genetic link between the
X-chromosomal TLR7 gene and susceptibility and severity of COVID-19 could open a novel avenue to consider poten-
tial treatments. Numerous immune-related genes and regulatory elements (noncoding micro RNA: miRNA) involved in
immune responses are found in the X-chromosome and are associated with infectious diseases; it explains the trend of
higher fatalities in men than in women COVID-19 patients [115].

16.8.3 Apolipoprotein E
Preexisting dementia is a significant risk factor for COVID-19 severity in the older adult population [116]. The ApoE
gene has three major isoforms, ApoE2, ApoE3, and ApoE4, encoded by e2, e3, and e4 alleles, which are haplotypes of
the SNPs rs429358 and rs7412 on chromosome 19. The ApoE e4e4 homozygous genotype increased the risk of severe
COVID-19, independently of preexisting dementia, cardiovascular disease, and type-2 diabetes [117]. In addition to
affecting lipoprotein function and subsequent cardio-metabolic conditions, the ApoE e4 allele moderates macrophage
pro/anti-inflammatory phenotypes [118]. ApoE is one of the highly expressed genes in type II alveolar cells in the lungs,
where the ACE2 receptor that CoVs use for cell entry. Advance studies in this direction could help unravel the biologi-
cal mechanisms linking ApoE genotypes to COVID-19 severity.

16.8.4 Interferon-induced transmembrane protein 3-encoding gene

An SNP rs12252-C/C in the interferon-induced transmembrane protein 3-encoding gene (IFITM3) linked to severe
influenza was detected in a COVID-19 patient from Wuhan, China [119,120]. The prevalence of this SNP is 26.5% in
the Chinese population [121]. Further investigating the IFITM3-rs12252-C/C allele in people with COVID-19 may be
worth tracking.

16.8.5 Transmembrane protein 189-ubiquitin-conjugating enzyme E2 variant 1

The TMEM189-UEV mRNA is a rare but naturally occurring read-through transcript of the neighboring TMEM189 and
UBE2V1 genes involved in the IL-1 signaling pathway. Ubiquitin-conjugating E2 enzyme variant proteins have
sequence similarity to other ubiquitin-conjugating enzymes but lack the conserved cysteine residue critical for the cata-
lytic activity of E2s [122]. The overexpression of Uev1B [Transmembrane protein 189-ubiquitin-conjugating enzyme
E2 variant 1 (TMEM189-UBE2V1) isoform 2] has slowed the degradation of epidermal growth factor receptor
 Genetics of coronaviruses Chapter | 16 265

complexes. The first host genetic variant study in China confirms that the most significant gene loci TMEM189-
UBE2V1 are associated with COVID-19 disease severity [123].

16.8.6 ACE2 and TMPRSS2 receptor genes

Studies have explored ACE2 allele frequencies in various geographic/ancestral human populations. The differences in
allele frequencies and observed differences in gene expression may explain the differential impacts of COVID-19
[124]. SARS-CoV-2 infection depends on the host cell receptors: entry into cells (ACE2) and the host transmembrane
serine protease (TMPRSS2) for spike (S) protein priming [125]. The X-chromosome codes the ACE2 protein. ACE2
catalyzes angiotensin II to angiotensin-(1
7), which acts as a vasodilator and modulates the cardiovascular system. The
expression of ACE2 and TMPRSS2 are likely to command SARS-CoV-2 tissue tropism [126]. Clinical studies have
reported that occurrence and mortality rates are significantly different between male and female COVID-19 patients.
The COVID-19 is associated with preexisting conditions, such as cancer and cardiovascular disorders, particularly indi-
viduals with hypertension receiving antihypertensive medications [127]. The genetic analysis of human genomes across
different populations suggested possible associations of ACE2 and TMPRSS2 variants with COVID-19 susceptibility,
severity, and clinical outcomes [128]. Specifically, cardiovascular and pulmonary conditions by altering the
angiotensinogen
ACE2 interactions are associated with ACE2 polymorphisms, such as p.Arg514Gly in the human pop-
ulation [129]. The differential polymorphisms on susceptibility and the localization of ACE2 on the X chromosome
may enlighten the increased risk of COVD-19 in males compared to females. X-chromosome’s TLR7 gene may harm
the natural history and prognosis of COVID-19 males [130].
The differential genetic susceptibility in male COVID-19 patients is due to the unique and prevalent polymorphisms
of p.Val160Met (rs12329760) and expression of quantitative trait locus in TMPRSS2 [131]. TMPRSS2 linked to locali-
zation of the gene on 21q22.3 might place individuals with Down syndrome at high risk for COVID-19 infection
[128,132]. These studies suggested the ACE2 or TMPRSS2 DNA polymorphisms are likely associated with the genetic
susceptibility of COVID-19 [128], which calls for a human genetics initiative for fighting the COVID-19 pandemic.
Therefore a systematic investigation of the polymorphisms in ACE2 and TMPRSS2 among different populations paves
the way for precision and personalized treatment strategies for COVID-19. Beyond receptor studies, the site of viral
replication appears to vary among the CoV species, tissue-specific receptor expression, and age-specific differences
observed with COVID-19 in humans. One of the above potential factors that may explain why most children are mildly
affected by COVID-19 is age-related differences in ACE2 receptor expression [133].

16.8.7 Interferon-α and β receptor subunit 2

The immune system is complex, involves many genes, including those encoding cytokines known as interferons (IFNs).
Individuals who lack specific IFNs can be more susceptible to infectious diseases [134]. At the onset of the COVID-19
pandemic, researchers were involved in identifying the genetic and immunological factors that could explain the severe
forms of the disease. The researchers have highlighted genetic abnormalities in individual patients which reduce the
production of Type I IFN, which are potent antiviral molecules (3%
4% of severe forms). They have identified autoim-
mune diseases that block type I IFNs (10%
11% of harsh conditions) in other patients. All these discoveries would
explain 15% of the extreme forms of COVID-19 [135]. Interferon-α and β receptor subunit 2 (IFNAR2) gene codes for
a cell receptor of IFN, a powerful molecular messenger that assembles the immune system when a virus conquers a cell
[136]. Researchers identified a new significant, GWAS IFNAR2 gene at chromosome 21. Also found evidence support-
ing a causal link from the low expression of IFNAR2 and high expression of Tyrosine Kinase 2 (TYK2) in life-
threatening COVID-19 disease patients [137]. The data revealed that a variant of IFNAR2 found in one in four
Europeans raised the risk of severe COVID-19. These studies raise hopes for ongoing trials of IFNs for the COVID-19
treatment, at least early in the course of SARS-CoV-2 infection.

16.8.8 20 -50 oligoadenylate synthetase family

The OAS, an IFN-stimulated gene cluster on chromosome 12, includes OAS1, OAS2, and OAS3, which share significant
homology and differ only in their number of OAS units. These genes all encode a similar 20 ,50 oligoadenylate synthase
family of enzymes essential to host responses to viral infections [138]. These activated genes turn on an enzyme called
ribonuclease L, which is capable of degrading viral RNA and inhibiting viral replication [139]. Precisely, the OAS1
gene activates pathways in the body that directly attack viruses and stop them from spreading, but it might even
266 Pandemic Outbreaks in the 21st Century

contribute to longevity [140]. A change in one of these family genes might impair this activation, allowing the virus to
flourish. Specifically, the OAS1 gene helps our body destroy viruses’ genetic structure, including CoVs. OAS1 gene
polymorphisms in host immune response to several viral infection classes, including influenza, herpes simplex, hepatitis
C, Dengue, and SARS-CoV viruses [141
143]. The association between SARS-CoV-1 and OAS1 genetic variants
located in exon 3, exon 6, and in region 30 UTR was found [144,145]. A study found an increase in OAS1 levels associ-
ated with reduced COVID-19 death [146]. These studies suggest that an OAS1 variant is common and influential on
COVID-19 as the IFN genetic risk factor. Therefore increasing the activity of the OAS pathway may help fight against
CoV and prevent COVID-19.
CoVs first try to invade our cells by weakening our immune response; low OAS1 might further reduce our ability to
attack these CoVs early on. In severe cases, CoVs cause excessive inflammation. People are predisposed to low levels
of OAS1, further reducing the body’s chance of fighting off the infection. People who are genetically predisposed to
higher OAS levels may have enhanced antiviral defenses. Available medicines, such as IFN-β-1b, increase OAS1 and
could be explored for their effect on COVID-19 susceptibility and severity.

16.8.9 DPP9 and FOXP4 genes

DPP9 codes for an enzyme involved in lung disease and TYK2 encodes a signaling protein involved in inflammation.
Drugs that target these two genes’ proteins are already in use in DPP9 enzyme baricitinib for diabetes, which blocks
TYK2 arthritis product. Baricitinib is early clinical testing for COVID-19 [147]. The COVID-19 Host Genetics
Initiative has found a gene called FOXP4, implicated in lung cancer [74]. This gene previously connected to the flu’s
effects also boosts COVID-19 susceptibility in men more likely to die of the disease than women.

16.9 New SARS-CoV-2 variant

Many SARS-CoV-2 variants are circulating globally. SARS-CoV-2 VOC 202012/01 or B.1.1.7 a new variant strain was
discovered by Public Health England’s genomic surveillance. The agency notified the United Kingdom (UK) government
on December 18, 2020, of the new strain’s seriousness, and the UK submitted its findings to the WHO the same day [148].
SARS-CoV-2 mutates regularly, acquiring about one new mutation in its genome every 2 weeks. Many mutations are silent,
do not impact the protein’s function. The first VOC 202012/01 new variant contains a series of mutations (23 genetic
changes) in its genetic code. VOC 202012/01 variant has a mutation in the spike protein RBD at position 501, where amino
acid asparagine (N) is replaced with tyrosine (Y). This mutation is N501Y, sometimes distinguished as S: N501Y to specify
that in the spike protein. This variant carries various other mutations, including a double deletion that spontaneously and
likely leads to a change in the spike protein’s shape. P681H: close to the S1/S2 furin cleavage site, with high variability in
CoVs. The ORF8 stop codon (Q27stop): This mutation is not in the spike protein but in a different gene, the function of
which is unknown. Studies on variants with N501Y suggest that they may bind more tightly to the ACE2 receptor.
Notably, these changes can make the virus more rapidly transmissible than other circulating strains of SARS-CoV-2.
Reproductive number R-naught increasing from 1.1 to 1.3 compared to COVID-19 can significantly increase potential
infections. An increase in infections that grows exponentially (i.e., transmission) can have far more effect than the same
proportional increase in something that scales an outcome (i.e., severity) [149].
In one laboratory study, the transmission of the virus between ferrets (Mustela putorius furo), a mutation in the same
location (N501T), freely arose in the experiment’s ferrets. So far, VOC 202012/01 has no known association with animals
or animal contact. The rapid change from being a rare strain to becoming a common strain has concerned scientists working
on learning more about this variant to understand better how easily it might be transmitted and whether currently authorized
vaccines would protect people against it. Information regarding the variant’s virologic, epidemiologic, and clinical charac-
teristics is rapidly emerging [150]. Data indicate that the variant may be better spread among youngsters, highly prevalent
in London and southeast England. The latest data identified additional sequences in S:501 mutant variants from the UK,
Denmark, Australia, South Africa, and many other countries [151]. We know a lot about this new COVID-19 variant, but
scientists warn the World that it is significantly more contagious but not more lethal than other strains.

16.10 Impact of genetics on COVID-19 treatment

Currently, there are no approved effective medications against COVID-19, but several National and International
research groups have developed vaccines to prevent COVID-19. Several potentially repurposable drugs, including mela-
tonin, hydroxychloroquine, and chloroquine, are under investigation to treat COVID-19. Several drugs have been under
 Genetics of coronaviruses Chapter | 16 267

investigation to treat COVID-19 without well-established safety or data to support these. However, some of these
unproven therapies may have underlying genetic reasons for not being effective and resulting in fatal adverse effects.
The application of pharmacogenomic tests can help eliminate fatal hypersensitivity for patients prescribed certain drugs.
If selecting a COVID-19 medication or the dose using an individual’s genetic information could improve effectiveness
or safety.

16.11 Future perspectives

The CoV’s initial genome sequencing describes the emergence of SARS-CoV-2 and discusses the gaps in understanding
its origins. CoVs can jump species boundaries and adapt to new hosts, predicting that more can emerge in the future.
Detailed knowledge of how an animal virus jumped species boundaries to infect humans productively could prevent
future zoonotic events. Besides, identifying the closest viral relatives of SARS-CoV-2 circulating in animals helps to
understand the viral function. Studying ACE2, TMPRSS2, and other specific candidate host genes, epigenetic, and
GWAS across different racial and ethnic populations to identify genes and haplotypes associated with differential fac-
tors of infection, severity, and disease. Research on viral genetics over time and geography—in terms of the number
and frequency of viral mutations and recombination events and their connection to viral infection, transmission, disease
severity, and clinical phenotype—is essential. The large-scale GWAS is urgently needed to identify causal host genetic
risk factors for treating diseases using genetic data. Such knowledge would improve the risk stratification of individuals
exposed to or testing positive for SARS-CoV-2 and allow for precision medicine interventions. As a future recommen-
dation for preventing CoV diseases, the World’s respective countries’ governments should pay attention to avoid the
disease by promoting the laws concerning prevention strategies to combat the disease. Scientists, medical workers, and
pharmaceutical organizations should prepare a specific vaccine for prevention and control and discover a specific drug
to treat the disease.

Acknowledgment
The authors are thankful to their university authorities for providing facilities.

References
[1] Seifried O. Histopathology of infectious Laryngotracheitis in chickens. J Exp Med 1931;54(6):817
26.
[2] Tyrrell DA, Bynoe ML. Cultivation of a novel type of common-cold virus in organ cultures. Br Med J 1965;1(5448):1467
70.
[3] Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract. Proc Soc Exp Biol Med 1966;121(1):190
3.
[4] McIntosh K, Dees JH, Becker WB, Kapikian AZ, Chanock RM. Recovery in tracheal organ cultures of novel viruses from patients with respira-
tory disease. Proc Natl Acad Sci USA 1967;57:933
40.
[5] Tyrrel DAJ, Almedia JD, Berry DM, Cunningham CH, Hamre D, Hofstad MS, et al. Coronavirus. Nature 1968;220:650.
[6] Guan Y. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 2003;302
(5643):276
8.
[7] Peiris JS, Yuen KY, Osterhaus AD, Stohr K. The severe acute respiratory syndrome. New Engl J Med 2003;349(25):2431
41.
[8] van der Hoek L, Pyrc K, Jebbink MF, Vermeulen-Oost W, Berkhout RJ, Wolthers KC, et al. Identification of a new human coronavirus. Nat
Med 2004;10(4):368
73.
[9] Woo PC, Lau SK, Chu CM, Chan KH, Tsoi HW, Huang Y, et al. Characterization and complete genome sequence of a novel coronavirus, coro-
navirus HKU1, from patients with pneumonia. J Virol 2005;79(2):884
95.
[10] Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends
Microbiol 2016;24(6):490
502.
[11] Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in
Saudi Arabia. N Engl J Med 2012;367:1814
20.
[12] Corman VM. Rooting the phylogenetic tree of Middle East respiratory syndrome coronavirus by characterization of a conspecific virus from an
African bat. J Virol 2014;88(19):11297
303.
[13] Gong L, Li J, Zhou Q, Xu Z, Chen L, Zhang Y, et al. A new bat-HKU2-like coronavirus in swine, China, 2017. Emerg Infect Dis 2017;23(9).
[14] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet
2020;395:497
506.
[15] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med
2020;382(8):727
33.
[16] Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al. A new coronavirus associated with human respiratory disease in China. Nature
2020;579(7798):265
9.
268 Pandemic Outbreaks in the 21st Century

[17] ICTV. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 Coronaviridae
Study Group of the International Committee on Taxonomy of V Nat Microbiol 2020;(5):536
44.
[18] Liu YC, Kuo RL, Shih SR. COVID-19: the first documented coronavirus pandemic in history. Biomed J 2020;S2319
4170(20):30044
5.
[19] WHO. Coronavirus disease (COVID-19) pandemic. Numbers at glance. November 31, 2020.
[20] Cavanagh D. Nidovirales: a new order comprising Coronaviridae and Arteriviridae. Arch Virol 1997;142(3):629
33.
[21] Enjuanes L, Spaan W, Snijder E, Cavanagh D. Virus taxonomy. Seventh report of the International Committee on Taxonomy Of Viruses.
In: Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers MD, editors. Nidovirales. New York:
Academic Press; 2000. p. 827
34.
[22] Cowley JA, Dimmock CM, Spann KM, Walker PJ. Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and
ORF1b genes related to arteri- and coronaviruses. J Gen Virol 2000;81:1473
84.
[23] Enjuanes L, Cavanagh D, Holmes K, Lai MMC, Laude H, Masters P, et al. Virus taxonomy. Classification and nomemclature of viruses.
In: van Regenmortel MHV, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR,
Wickner RB, editors. Coronaviridae. San Diego, CA: Academic Press; 2000. p. 835
49.
[24] Woo PC, Lau SK, Lam CS, et al. Discovery of seven novel mammalian and Avian coronaviruses in the genus deltacoronavirus supports bat cor-
onaviruses as the gene source of alphacoronavirus and betacoronavirus and Avian coronaviruses as the gene source of gammacoronavirus and
deltacoronavirus. J Virol 2012;86(7):3995
4008.
[25] Rabi FA, Al Zoubi MS, Kasasbeh GA, Salameh DM, Al-Nasser AD. SARS-CoV-2 and coronavirus disease 2019: what we know so far.
Pathogens 2020;9(3):231.
[26] He B, Zhang Y, Xu L, Yang W, Yang F, Feng Y, et al. Identification of diverse alphacoronaviruses and genomic characterization of a novel
severe acute respiratory syndrome-like coronavirus from bats in China. J Virol 2014;88(12):7070
82.
[27] Tang Q, Song Y, Shi M, Cheng Y, Zhang W, Xia XQ. Inferring the hosts of coronavirus using dual statistical models based on nucleotide com-
position. Sci Rep 2015;5:17155.
[28] Wille M, Holmes EC. Wild birds as reservoirs for diverse and abundant gamma- and deltacoronaviruses. FEMS Microbiol Rev 2020;44(5):631
44.
[29] Woo PC, Lau SK, Huang Y, Yuen KY. Coronavirus diversity, phylogeny and interspecies jumping. Exp Biol Med (Maywood) 2009;234
(10):1117
27.
[30] de Groot RJ, et al. Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses, International Union of
Microbiological Societies, Virology Division. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Coronaviridae. London, United
Kingdom: Elsevier Academic Press; 2011.
[31] Langereis MA, van Vliet AL, Boot W, de Groot RJ. Attachment of mouse hepatitis virus to O-acetylated sialic acid is mediated by
hemagglutinin-esterase and not by the spike protein. J Virol 2010;84(17):8970
4.
[32] Forster P, Forster L, Renfrew C, Forster M. Phylogenetic network analysis of SARS-CoV-2 genomes. Proc Natl Acad Sci U S A 2020;117
(17):9241
3.
[33] Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus
origins and receptor binding. Lancet 2020;395(10224):565
74.
[34] Gorbalenya AE, Baker SC, et al. The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it
SARS-CoV-2. Nat Microbiol 2020;5:536
44.
[35] Gorbalenya AE, Enjuanes L, Ziebuhr J, Snijder EJ. Nidovirales: evolving the largest RNA virus genome. Virus Res 2006;117(1):17
37.
[36] Kim D, Lee JY, Yang JS, Kim JW, Kim VN, Chang H. The Architecture of SARS-CoV-2 transcriptome. Cell. 2020;181(4):914
21 e10.
[37] Masters PS. Coronavirus genomic RNA packaging. Virology 2019;537:198
207.
[38] Song Z, Xu Y, Bao L, Zhang L, Yu P, Qu Y, et al. From SARS to MERS, thrusting coronaviruses into the spotlight. Viruses 2019;11(1):59.
[39] Chan JF, Kok KH, Zhu Z, Chu H, To KK, Yuan S, et al. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated
from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 2020;9(1):221
36.
[40] Wu A, Peng Y, Huang B, Ding X, Wang X, Niu P, et al. Genome composition and divergence of the novel coronavirus (2019-nCoV) originat-
ing in China. Cell Host Microbe 2020;27(3):325
8.
[41] Brakier-Gingras L, Charbonneau J, Butcher SE. Targeting frameshifting in the human immunodeficiency virus. Expert Opin Ther Targets
2012;16(3):249
58.
[42] Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17(3):181
92.
[43] Gorbalenya AE, Koonin EV, Donchenko AP, Blinov VM. Coronavirus genome: prediction of putative functional domains in the non-structural
polyprotein by comparative amino acid sequence analysis. Nucleic Acids Res 1989;17(12):4847
61.
[44] Ziebuhr J. The coronavirus replicase. Curr Top Microbiol Immunol 2005;287:57
94.
[45] Zhang L, Lin D, Sun X, Curth U, Drosten C, Sauerhering L, et al. Crystal structure of SARS-CoV-2 main protease provides a basis for design
of improved α-ketoamide inhibitors. Science 2020;368(6489):409
12.
[46] Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS
drugs. Science 2003;300(5626):1763
7.
[47] Mielech AM, Kilianski A, Baez-Santos YM, Mesecar AD, Baker SC. MERS-CoV papain-like protease has deISGylating and deubiquitinating
activities. Virology 2014;450
451:64
70.
[48] Báez-Santos YM, St John SE, Mesecar AD. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral
compounds. Antiviral Res 2015;115:21
38.
 Genetics of coronaviruses Chapter | 16 269

[49] Kirchdoerfer RN, Cottrell CA, Wang N, et al. Pre-fusion structure of a human coronavirus spike protein. Nature 2016;531(7592):118
21.
[50] Bosch BJ, van der Zee R, de Haan CA, Rottier PJ. The coronavirus spike protein is a class I virus fusion protein: structural and functional char-
acterization of the fusion core complex. J Virol 2003;77(16):8801
11.
[51] Zhu Z, Zhang Z, Chen W, Cai Z, Ge X, Zhu H, et al. Predicting the receptor-binding domain usage of the coronavirus based on kmer frequency
on spike protein. Infect Genet Evol 2018;61:183
4.
[52] Li F. Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol 2016;3:237
61.
[53] Hamming I, Timens W, Bulthuis ML, Lely AT, Navis G, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS
coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004;203(2):631
7.
[54] Glowacka I, Bertram S, Müller MA, Allen P, Soilleux E, Pfefferle S, et al. Evidence that TMPRSS2 activates the severe acute respiratory syn-
drome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J Virol 2011;85(9):4122
34.
[55] Millet JK, Whittaker GR. Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike
protein. Proc Natl Acad Sci U S A 2014;111(42):15214
19.
[56] Shirato K, Kawase M, Matsuyama S. Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease
TMPRSS2. J Virol 2013;87(23):12552
61.
[57] Hartenian E, Nandakumar D, Lari A, Ly M, Tucker JM, Glaunsinger BA. The molecular virology of coronaviruses. J Biol Chem 2020;295
(37):12910
34.
[58] Tan K, Zelus BD, Meijers R, Liu JH, Bergelson JM, Duke N, et al. Crystal structure of murine sCEACAM1a[1,4]: a coronavirus receptor in the
CEA family. EMBO J 2002;21(9):2076
86.
[59] Venkatagopalan P, Daskalova SM, Lopez LA, Dolezal KA, Hogue BG. Coronavirus envelope protein remains at the site of assembly. Virology
2015;478:75
85.
[60] Neuman BW, Kiss G, Kunding AH, et al. A structural analysis of M protein in coronavirus assembly and morphology. J Struct Biol 2011;174
(1):11
22.
[61] Chang CK, Sue SC, Yu TH, et al. Modular organization of SARS coronavirus nucleocapsid protein. J Biomed Sci 2006;13(1):59
72.
[62] Zhu N, Zhang D, Wang, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 2020;382:727
33.
[63] Kasibhatla SM, Kinikar M, Limaye S, Kale MM, Kulkarni-Kale U. Understanding evolution of SARS-CoV-2: a perspective from analysis of
genetic diversity of RdRp gene. J Med Virol 2020;92(10):1932
7. Available from: https://doi.org/10.1002/jmv.25909.
[64] Sah R, Rodriguez-Morales AJ, Jha R, Chu DKW, Gu H, Peiris M, et al. Complete genome sequence of a 2019 novel coronavirus (SARS-CoV-
2) strain isolated in Nepal. Microbiol Resour Announcv 2020;9 e00169
20.
[65] Lai MM. RNA recombination in animal and plant viruses. Microbiol Rev 1992;56:61
79.
[66] Cavanagh D. Coronavirus avian infectious bronchitis virus. Vet Res 2007;38:281
97.
[67] Drosten C, Kellam P, Memish ZA. Evidence for camel-to-human transmission of MERS coronavirus. N Engl J Med 2014;371(14):1359
60.
[68] Paules CI, Marston HD, Fauci AS. Coronavirus infections-more than just the common cold. JAMA. 2020;323(8):707
8.
[69] Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute
respiratory syndrome. N Engl J Med 2003;348:1967
76.
[70] Zhong NS, Zheng BJ, Li YM, Poon, Xie ZH, Chan KH, et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in
Guangdong, People’s Republic of China, in February 2003. Lancet 2003;362(9393):1353
8.
[71] Lek M, Karczewski KJ, Minikel EV, et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature. 2016;536(7616):285
91.
[72] Di Maria E, Latini A, Borgiani P, Novelli G. Genetic variants of the human host influencing the coronavirus-associated phenotypes (SARS,
MERS, and COVID-19): rapid systematic review and field synopsis. Hum Genomics 2020;14(1):30.
[73] Ovsyannikova IG, Haralambieva IH, Crooke SN, Poland GA, Kennedy RB. The role of host genetics in the immune response to SARS-CoV-2
and COVID-19 susceptibility and severity. Immunol Rev 2020;296(1):205
19.
[74] COVID-19 Host Genetics Initiative. The COVID-19 Host Genetics Initiative, a global initiative to elucidate the role of host genetic factors in
susceptibility and severity of the SARS-CoV-2 virus pandemic. Eur J Hum Genet 2020;28(6):715
18.
[75] Shin DL, Hatesuer B, Bergmann S, Nedelko T, Schughart K. Protection from severe influenza virus infections in mice carrying the Mx1 influ-
enza virus resistance gene strongly depends on genetic background. J Virol 2015;89(19):9998
10009.
[76] Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective
cohort study. Lancet 2020;395:1054
62.
[77] Docherty AB, Harrison EM, Green CA, et al. Features of 20133 UK patients in hospital with covid-19 using the ISARIC WHO Clinical
Characterisation Protocol: prospective observational cohort study. BMJ 2020;369 m1985.
[78] Richardson S, Hirsch JS, Narasimhan M, et al. Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with
COVID-19 in the New York City area. JAMA 2020;323(20):2052
9.
[79] Ackermann M, Verleden SE, Kuehnel M, Haverich A, Welte T, Laenger F, et al. Pulmonary vascular endothelialitis, thrombosis, and angiogen-
esis in COVID-19. N Engl J Med 2020;383(2):120
8.
[80] Levi M, Thachil J, Iba T, Levy JH. Coagulation abnormalities and thrombosis in patients with COVID-19. Lancet Haematol 2020;7(6):
e438
40.
[81] Varga Z, Flammer AJ, Steiger P, et al. Endothelial cell infection and endotheliitis in COVID-19. Lancet 2020;395:1417
18.
[82] Debnath M, Banerjee M, Berk M. Genetic gateways to COVID-19 infection: implications for risk, severity, and outcomes. FASEB J. 2020;34
(7):8787
95.
270 Pandemic Outbreaks in the 21st Century

[83] International Human Genome Sequencing Consortium, Whitehead Institute for Biomedical Research, Center for Genome ResearchLander E,
et al. Initial sequencing and analysis of the human genome. Nature 2001;409:860
921.
[84] Tizaoui K, Zidi I, Lee KH, et al. Update of the current knowledge on genetics, evolution, immunopathogenesis, and transmission for coronavi-
rus disease 19 (COVID-19). Int J Biol Sci 2020;16(15):2906
23.
[85] Guo G, Ye L, Pan K, et al. New insights of emerging SARS-CoV-2: epidemiology, etiology, clinical features, clinical treatment, and preven-
tion. Front Cell Dev Biol 2020;8:410.
[86] Buschman E, Skamene E. Genetic resistance to coronavirus infection. In: Talbot PJ, Levy GA, editors. Corona- and related viruses. Advances
in experimental medicine and biology, 380. Boston, MA: Springer; 1995.
[87] Field RI, Orlando AW, Rosoff AJ. Genetics and COVID-19: how to protect the susceptible. Trends Genet 2020;37(2):106
8.
[88] Zeberg H, Pääbo S. The major genetic risk factor for severe COVID-19 is inherited from Neanderthals. Nature 2020;587:610
12. Available
from: https://doi.org/10.1038/s41586-020-2818-3.
[89] Vuille-dit-Bille RN, Camargo SM, Emmenegger L, Sasse T, Kummer E, Jando J, et al. Human intestine luminal ACE2 and amino acid trans-
porter expression increased by ACE-inhibitors. Amino Acids 2015;47(4):693
705.
[90] Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol 2007;25:787
820.
[91] Hickey MJ, Held KS, Baum E, Gao JL, Murphy PM, Lane TE. CCR1 deficiency increases susceptibility to fatal coronavirus infection of the
central nervous system. Viral Immunol 2007;20(4):599
608.
[92] Wein AN, McMaster SR, Takamura S, Dunbar PR, Cartwright EK, Hayward SL, et al. CXCR6 regulates localization of tissue-resident mem-
ory CD 8 T cells to the airways. J Exp Med 2019;216(12):2748
62.
[93] Severe Covid-19 GWAS GroupEllinghaus D, Degenhardt F, Bujanda L, Karlsen TH. Genomewide association study of severe COVID-19
with respiratory failure. N Engl J Med 2020;383(16):1522
34.
[94] van der Made CI, Simons A, Schuurs-Hoeijmakers J, et al. Presence of genetic variants among young men with severe COVID-19. JAMA
2020;324(7):1
11.
[95] Giudicessi JR, Roden DM, Wilde AAM, Ackerman MJ. Genetic susceptibility for COVID-19-associated sudden cardiac death in African
Americans. Heart Rhythm 2020;17(9):1487
92.
[96] Cheng Y, Cheng G, Chui CH, et al. ABO blood group and susceptibility to severe acute respiratory syndrome. JAMA 2005;293:1450
1.
[97] Wu Y, Feng Z, Li P, Yu Q. Relationship between ABO blood group distribution and clinical characteristics in patients with COVID-19. Clin
Chim Acta 2020;509:220
3.
[98] Zhao J, Yang Y, Huang H, Li D, Gu D, Lu X, et al. Relationship between the ABO blood group and the COVID-19 susceptibility. Clin Infect
Dis 2020; ciaa1150.
[99] Breiman A, Ruvën-Clouet N, Le Pendu J. Harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses
such as SARS-CoV-2. PLoS Pathog 2020;16(5) e1008556.
[100] Aziz M, Fatima R, Assaly R. Elevated interleukin-6 and severe COVID-19: a meta-analysis. J Med Virol 2020;92(11):2283
5. Available
from: https://doi.org/10.1002/jmv.25948.
[101] Murray GP, Post SR, Post GR. ABO blood group is a determinant of von Willebrand factor protein levels in human pulmonary endothelial
cells. J Clin Pathol 2020;73:347
9.
[102] Zietz M, Tatonetti NP. Testing the association between blood type and COVID-19 infection, intubation, and death. medRxiv 2020.
[103] Lin M, Tseng HK, Trejaut JA, Lee HL, Loo JH, Chu CC, et al. Association of HLA class I with severe acute respiratory syndrome coronavirus
infection. BMC Med Genet 2003;4:9.
[104] Nguyen A, David JK, Maden SK, Wood MA, Weeder BR, Nellore A, et al. Human leukocyte antigen susceptibility map for severe acute respi-
ratory syndrome coronavirus 2. J Virol 2020;94(13) e00510-20.
[105] Wang F, Huang S, Gao H, et al. Initial whole genome sequencing and analysis of the host genetic contribution to COVID-19 severity and sus-
ceptibility. medRxiv 2020.
[106] Novelli A, Andreani M, Biancolella M, Liberatoscioli L, Passarelli C, Colona VL, et al. HLA allele frequencies and susceptibility to COVID-
19 in a group of 99 Italian patients. HLA 2020;96(5):610
14.
[107] Jaillon S, Berthenet K, Garlanda C. Sexual dimorphism in innate immunity. Clin Rev Allergy Immunol 2019;56(3):308
21.
[108] Souyris M, Mejı́a JE, Chaumeil J, Guéry JC. Female predisposition to TLR7-driven autoimmunity: gene dosage and the escape from X chro-
mosome inactivation. Semin Immunopathol 2019;41(2):153
64.
[109] Klein SL, Marriott I, Fish EN. Sex-based differences in immune function and responses to vaccination. Trans R Soc Trop Med Hyg 2015;109(1):9
15.
[110] Cutolo M, Capellino S, Sulli A, Serioli B, Secchi ME, Villaggio B, et al. Estrogens and autoimmune diseases. Ann N Y Acad Sci
2006;1089:538
47.
[111] Casanova JL, Abel L, Quintana-Murci L. Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and
clinical genetics. Annu Rev Immunol 2011;29:447
91.
[112] Cervantes-Barragan L, Züst R, Weber F, Spiegel M, Lang KS, Akira S, et al. Control of coronavirus infection through plasmacytoid dendritic-
cell-derived type I interferon. Blood 2007;109(3):1131
7.
[113] Channappanavar R, Fehr AR, Zheng J, Wohlford-Lenane C, Abrahante JE, Mack M, et al. IFN-I response timing relative to virus replication
determines MERS coronavirus infection outcomes. J Clin Invest 2019;129(9):3625
39.
[114] Blanco-Melo D, Nilsson-Payant BE, Liu WC, Uhl S, Hoagland D, Møller R, et al. Imbalanced host response to SARS-CoV-2 drives develop-
ment of COVID-19. Cell 2020;181(5):1036
45 e9.
 Genetics of coronaviruses Chapter | 16 271

[115] Schurz H, Salie M, Tromp G, Hoal EG, Kinnear CJ, Möller M. The X chromosome and sex-specific effects in infectious disease susceptibility.
Hum Genomics 2019;13(1):2.
[116] Atkins JL, Masoli JAH, Delgado J, Pilling LC, Kuo CL, Kuchel GA, et al. Preexisting comorbidities predicting COVID-19 and mortality in
the UK Biobank Community Cohort. J Gerontol A Biol Sci Med Sci 2020;75(11):2224
30.
[117] Kuo CL, Pilling LC, Atkins JL, Masoli JAH, Delgado J, Kuchel GA, et al. APOE e4 genotype predicts severe COVID-19 the UK Biobank
Community Cohort. J Gerontol A Biol Sci Med Sci 2020;75(11):2231
2.
[118] Tudorache IF, Trusca VG, Gafencu AV. Apolipoprotein E
A Multifunctional protein with implications in various pathologies as a result of
its structural features. Comput Struct Biotechnol J. 2017;15:359
65.
[119] Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, et al. IFITM3 restricts the morbidity and mortality associated with influenza.
Nature 2012;484(7395):519
23.
[120] Thevarajan I, Nguyen THO, Koutsakos M, Druce J, Caly L, van de Sandt CE, et al. Breadth of concomitant immune responses prior to patient
recovery: a case report of non-severe COVID-19. Nat Med 2020;26(4):453
5.
[121] Wang Z, Zhang A, Wan Y, Liu X, Qiu C, Xi X, et al. Early hypercytokinemia is associated with interferon-induced transmembrane protein-3
dysfunction and predictive of fatal H7N9 infection. Proc Natl Acad Sci U S A 2014;111(2):769
74.
[122] Duex JE, Mullins MR, Sorkin A. Recruitment of Uev1B to Hrs-containing endosomes and its effect on endosomal trafficking. Exp Cell Res
2010;316(13):2136
51.
[123] Wang F, Huang S, Gao R, et al. Initial whole-genome sequencing and analysis of the host genetic contribution to COVID-19 severity and sus-
ceptibility. Cell Discov 2020;6:83.
[124] LoPresti M, Beck DB, Duggal P, Cummings DAT, Solomon BD. The role of host genetic factors in coronavirus susceptibility: review of ani-
mal and systematic review of human literature. Am J Hum Genet 2020;107(3):381
402.
[125] Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2
and is blocked by a clinically proven protease inhibitor. Cell 2020;181(2):271
80.
[126] Stopsack KH, Mucci LA, Antonarakis ES, Nelson PS, Kantoff PW. TMPRSS2 and COVID-19: serendipity or opportunity for intervention?
Cancer Discov 2020;10(6):779
82.
[127] Zou X, Chen K, Zou J, Han P, Hao J, Han Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of
different human organs vulnerable to 2019-nCoV infection. Front Med 2020;14(2):185
92.
[128] Hou Y, Zhao J, Martin W, Kallianpur A, Chung MK, Jehi L, et al. New insights into genetic susceptibility of COVID-19: an ACE2 and
TMPRSS2 polymorphism analysis. BMC Med 2020;18(1):216.
[129] Devaux CA, Rolain JM, Raoult D. ACE2 receptor polymorphism: susceptibility to SARS-CoV-2, hypertension, multi-organ failure, and
COVID-19 disease outcome. J Microbiol Immunol Infect 2020;53(3):425
35.
[130] Asselta R, Paraboschi EM, Mantovani A, Duga S. ACE2 and TMPRSS2 variants and expression as candidates to sex and country differences
in COVID-19 severity in Italy. Aging (Albany NY) 2020;12(11):10087
98.
[131] Vargas-Alarcón G, Posadas-Sánchez R, Ramı́rez-Bello J. Variability in genes related to SARS-CoV-2 entry into host cells (ACE2, TMPRSS2,
TMPRSS11A, ELANE, and CTSL) and its potential use in association studies. Life Sci 2020;260:118313.
[132] Yu J, Ouyang W, Chua MLK, Xie C. SARS-CoV-2 transmission in patients with cancer at a tertiary care hospital in Wuhan, China. JAMA
Oncol 2020;6(7):1108
10.
[133] Ortiz-Fernández L, Sawalha AH. Genetic variability in the expression of the SARS-CoV-2 host cell entry factors across populations. Genes
Immun 2020;21(4):269
72.
[134] Sallard E, Lescure FX, Yazdanpanah Y, Mentre F, Peiffer-Smadja N. Type 1 interferons as a potential treatment against COVID-19. Antiviral
Res 2020;178:104791.
[135] Zhang Q, Bastard P, Liu Z, Casanova JL. Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science 2020;370
(6515) eabd4570.
[136] Duncan CJ, Mohamad SM, Young DF, Skelton AJ, Leahy TR, Munday DC, et al. Human IFNAR2 deficiency: lessons for antiviral immunity.
Sci Transl Med 2015;7(307) 307ra154.
[137] Pairo-Castineira E, Clohisey S, Klaric L, Bretherick A, Rawlik K, Parkinson N, et al. Genetic mechanisms of critical illness in COVID-19.
medRxiv 2020.
[138] Hu J, Wang X, Xing Y, et al. Origin and development of oligoadenylate synthetase immune system. BMC Evol Biol 2018;18(1):201.
[139] Schwartz SL, Conn GL. RNA regulation of the antiviral protein 2’-5’-oligoadenylate synthetase. Wiley Interdiscip Rev RNA 2019;10(4):
e1534.
[140] Mullan PB, Hosey AM, Buckley NE, Quinn JE, Kennedy RD, Johnston PG, et al. The 2,5 oligoadenylate synthetase/RNaseL pathway is a
novel effector of BRCA1- and interferon-gamma-mediated apoptosis. Oncogene 2005;24(35):5492
501.
[141] Samuel CE. Antiviral actions of interferons. Clin Microbiol Rev 2001;14:778
809.
[142] Lim JK, Lisco A, McDermott DH, et al. Genetic variation in OAS1 is a risk factor for initial infection with West Nile virus in man. PLoS
Pathog 2009;5(2):e1000321.
[143] Simon-Loriere E, Lin RJ, Kalayanarooj SM, Sakuntabhai A. High anti-dengue virus activity of the OAS gene family is associated with
increased severity of dengue. J Infect Dis 2015;212(12):2011
20.
[144] Hamano E, Hijikata M, Itoyama S, Quy T, Phi NC, Long HT, et al. Polymorphisms of interferon-inducible genes OAS-1 and MxA associated
with SARS in the Vietnamese population. Biochem Biophys Res Commun 2005;329:1234
9.
272 Pandemic Outbreaks in the 21st Century

[145] He J, Feng D, de Vlas SJ, Wang H, Fontanet A, Zhang P, et al. Association of SARS susceptibility with single nucleic acid polymorphisms of
OAS1 and MxA genes: a case-control study. BMC Infect Dis 2006;6:106.
[146] Zhou S, Butler-Laporte G, Nakanishi T, et al. Circulating proteins influencing COVID-19 susceptibility and severity: a Mendelian randomiza-
tion study. medRxiv 2020. Available from: https://doi.org/10.1101/2020.10.13.20212092.
[147] Zhang X, Zhang Y, Qiao W, Zhang J, Qi Z. Baricitinib, a drug with potential effect to prevent SARS-COV-2 from entering target cells and
control cytokine storm induced by COVID-19. Int Immunopharmacol 2020;86:106749.
[148] Tang JW, Tambyah PA, Hui DS. Emergence of a new SARS-CoV-2 variant in the UK. J Infect 2020;82(4) e27
e28.
[149] Leung K, Shum MH, Leung GM, Lam TT, Wu JT. Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the
United Kingdom, October to November 2020. Eurosurveillance 2021;26(1):2002106.
[150] Conti P, Caraffa A, Gallenga CE, Kritas SK, Frydas I, Younes A, et al. The British variant of the new coronavirus-19 (SARS-CoV-2) should
not create a vaccine problem. J Biol Regul Homeost Agents 2021;35(1):1
4.
[151] Emerging SARS-CoV-2 Variants. National Center for Immunization and Respiratory Diseases (NCIRD), Division of Viral Diseases, January
3, 2021.
Chapter 17

Spike in electronic sports during the

coronavirus disease pandemic
Neha Singh
Department of Sports Biosciences, Central University of Rajasthan, Ajmer, India

17.1 COVID-19 pandemic, lockdown, and e-sports

The coronavirus COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2. It is a major
health and socioeconomic crisis of our time [1]. It created a devastating effect on the social, economic, and normal
everyday life of millions of people across the globe [1,2]. The virus was transmitted to humans from bats in Wuhan
City of Hubei Province of China in December 2019 and rapidly spread throughout the world [3]. The coronavirus
infected more than 100 million people worldwide and more than 2 million people died because of the pandemic [4]
COVID-19 pandemic had a negative impact on multiple sectors of the world economy and on every aspect of human
life due to social distancing, travel restrictions, closure of schools, and reduced workforce in almost all economic sec-
tors [1,5] The United Nations Development Program (2020) declared the virus as a major health and socioeconomic cri-
sis, which affected people on a global scale [6]. To prevent the spread of the virus, the lockdown was imposed in
several parts of the world with different durations in each of the countries affected by the deadly virus. The lockdown
was also an opportunity for the governments to prepare for the crisis, strengthen management, and strategize the
response and recovery for post-lockdown normalizing of lives. While the entire world was struggling with COVID-19,
the sports world also went through crises [7]. Sports events, Olympics, and other organized events require people to
come together, but this could have spread the virus uncontrollably in the pandemic situation. Therefore all the major
sports events, games, and tournaments were postponed or canceled [8,9]. This program implementation failure had the
most pronounced effect in the economic sector due to loss of match and advertisement revenues. Clubs have been
affected more negatively during the pandemic period because team sports and clubs have more financial obligations
than individual sports. And therefore financial assistance was given to clubs. For example, FIFA announced to provide
1.5 billion USD of aid to the clubs in three stages with the “COVID-19 aid plan” [10]. The economic setback forced
clubs to transfer some events from traditional sports to e-sports. The pandemic forced social and commercial life to be
digitalized to a great extent [7,11]. The lockdown due to pandemic significantly increased the number of participants of
online games [11]. While many areas were negatively affected in this period, the game industry was positively impacted
in terms of the economy due to a considerable increase in participants’ interest in e-sports and the gaming industry. The
video game industry giants, including Microsoft, Nintendo, Twitch, and Activision, thrived financially and increased
number of consumers in the situation created by the COVID-19 pandemic. Twitch, the most popular video game
streaming platform, recorded 1.49 billion gaming hours watched in April 2020 [12]. Interestingly, e-sports or video
game streaming platforms also helped some countries’ governments in conveying the stay home stay safe message to
the people.

17.2 E-sports: an introduction and research in different academic disciplines

Sports play a vital role in strengthening national identity and cultivating integrity and harmony. It promotes the devel-
opment and well-being of the residents of the country. Sports may include any type of serious and physical activity that
individuals play through sorted out or causal interest. It helps us all to improve and enhance aptitude skills and physical
capacity. Electronic sports (e-sports), Cyber sports, Gaming, Competitive computer gaming, and Virtual sports are

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00009-4

© 2021 Elsevier Inc. All rights reserved. 273
274 Pandemic Outbreaks in the 21st Century

synonyms for e-sports. Regardless of the term used, e-sports is getting social acceptance as a sport, and gamers are
being identified as athletes within society today [13
15]. In the near past, a new category of sports activity has
attracted a large part of the youth community: e-sports, which challenges hegemonic and modern sports. e-sports is the
opposite of involvement in organized/managed sports, not an accepted leisure activity by the society, including parents
and adults who do not support and encourage the activity in a similar manner. Over the years, the term e-sports was
considered to be different from sports, which is not the situation now. E-sports is a category of sports that is acted in a
virtual domain where an individual or a team competes against each other and includes physical and mental capacities
[13]. Four primary segments ought to be there in the computer game: Real-time system, Fighting, First-person shooter,
Multiplayer online battle arena (MOBA). E-sports is now recognized in 152 countries across the globe. The e-sports
audience is around 380 million and is predicted to increase to about 557 million by the end of 2021. In India the total
number of e-sports players is approximately 264 million, out of which 120 million are online e-sports players. This
number will also surge significantly in the near future understanding e-sports is complicated because of the relative nov-
elty of the industry and the convergence of culture, technology, sport, and business [14,16,17]. In contrast to traditional
sports such as hockey, baseball, and soccer, e-sports is an interconnection of multiple platforms. E-sports, which is also
synonymous with gaming, involves computing, media, and a sports event all wrapped up into one [17].
E-sports has been gaining interest since the beginning of the 21st century and is a subject of research for diverse
multiple academic disciplines, including business, cognitive science, law, sports science, media studies, sociology, and
informatics. As e-sports is becoming much more familiar now, the research focuses more on understanding the skills
and overall health requirements of e-sports players for better performance in competitive games. The research done by
business researchers using tools like interviews, surveys, and case studies indicates that the popularity of video games,
technology advancement, and social recognition of video game players attributed to its growth. These and some more
factors have helped businesses design effective marketing techniques by motivating e-sports consumption [18], explor-
ing the networks around e-sports players and the organization working with them [15,18
21]. The research is now
done at a global level studying the performance of e-sports players from different communities and how the diversity of
culture, tradition, and social acceptance in each country and continent impact the performance of e-sports players and
consumers [14,22
28].
In sports science, the research started with identifying and categorizing e-sports as a sport and understanding the
physical and mental skills employed in playing e-sports effectively [16,29
31]. After thorough research, some research-
ers established that e-sports involves physical activity, recreation, competitive elements, and organizational structure
[16]. Recent studies focused their research on e-sports player’s engagement in competitions using interviews and sur-
veys as a tool for investigating important factors like e-sports player’s actions during the game, the interaction between
players and teams, the influence of fans on the internet, and the effect of game interface on the performance of e-sports
players. Scientific research on e-sports has mainly focused on the e-sports players’ cognitive and behavioral capabilities
and how these attributes distinguish elite players in competitions [32]. The study of understanding and context of differ-
ent games by players is an important criterion adopted by researchers [32]. It was found that developing high skill level
in-game good habits with regular practice helps e-sports players to excel in their respective games [33
35]. Recently,
experimental studies have started focusing on complex human behavior that is a crucial factor in understanding the
skills required to improve e-sports’ performance [25,34,36]. Besides, players’ hormone levels were also analyzed in
some studies to understand the complexity of responses during the game [37]. E-sports research using informatics col-
lects data through different resources like game telemetry, user-generated play data, physiological data, and text-mining
to analyze performance, team dynamics, team formation, and interaction between players and teams [38
41]. Scientists
also developed a machine-learning algorithm to observe and develop high-skill strategies and player behavior models to
improve performance scale based on individual characteristics [42]. Similar research is also done to understand team
expertise where the player’s expertise during the game aligns with the team in contrast to their individual proficiency
when they are playing alone [43
45]. Also, research is done on social interaction between players and how they sup-
port each other irrespective of their performance in the game [45,46].

17.3 Effect of COVID-19 on economic growth of e-sports or gaming industry

The COVID-19 pandemic and the recurrent extended lockdown and quarantine periods led more and more people to be
involved in online gaming and other online means of entertainment like watching TV series and movies online. These
implications helped the gaming industry gain colossal popularity and economic progress. This phenomenon boosted the
financial growth of the e-sports industry and helped people cope with the stress of pandemic.
 Spike in electronic sports during the coronavirus disease pandemic Chapter | 17 275

According to sources for e-sports analytics and market research, the global gaming market generated USD 162.32
billion in 2020 and is predicted to generate USD 295.63 billion by 2026 [47,48]. The global online gaming industry is
expected to reach a compound annual growth rate of 12% during the period 2020
25. The increase in consumption of
video games increased by 20% in the COVID-19 pandemic compared to the USA’s previous year. More than 33.7 bil-
lion USD was spent by consumers in the United States on video games in the first few months of 2020 during the pan-
demic [49,50]. Game developers across the globe are striving to enhance gamer’s experience, rewriting codes for
diverse console/platforms, for example, PlayStation, Xbox, Windows PC, which are incorporated into one product pro-
vided to the gamers through the cloud platform. The number of gamers increased by 5.3% year-on-year in 2020 and the
estimated number was 2.7 billion online gamers around the world [51,52]. The money spent by players in e-sports or
online gaming increased on an average of 60% year over year in 2020 [53]. Some new consoles were also launched,
like the Microsoft Xbox Series X and S and PlayStation 5 (with Blu-ray Disk drive or disk drive-free digital version).
The number of gamers and time spent increased rapidly in the online gaming industry in every gaming platform, includ-
ing video games and e-sports. A holistic growth was seen across all age groups and urban as well as semiurban masses.
Technological advancements were incorporated in all gaming platforms during the COVID-19 pandemic and most
developments were seen in smartphone or mobile gaming. Since smartphones or mobiles are easily accessible to most
people across the globe, they are less software-intensive, portable, and more preferred by the younger generation; the
demand for mobile games increased with pandemic restrictions to stay home. Therefore a lot of investments were made
in the development of mobile games. The mobile game industry has witnessed the most prominent growth during the
COVID-19 pandemic. Around 13 billion mobile games were downloaded by users in the first quarter of 2020. The
global mobile game market generated 75.4 billion USD in 2020, which is 19.5% higher compared to 2019 [54] PUBG
mobile was the biggest revenue generator in mobile games available in the Google Play and App store in 2020 with a
value of 2.6 billion dollars, which was 64.3% higher compared to 2019 revenues. The top mobile games by worldwide
revenue in the year 2020 were PUBG mobile, Honor of Kings, Pokemon GO, Coin Master, Roblox, and Monster
Strike, which generated revenues of 2.6 billion USD, 2.5 billion USD, 1.2 billion USD, 1.1 billion USD, 1.1 billion
USD, and 958 million USD, respectively. These games were already global hits and got an advantage of lockdown dur-
ing the pandemic with more people staying at home and using mobile games as an alternate entertainment resource
[54]. The worldwide PC gaming market generated revenue of almost 37 billion USD in 2020. Tencent, Sony, and
Apple, the major players in the video gaming industry, generate billion-dollar revenues every year. The bestseller
among current-generation consoles is PlayStation4 from Sony, and more than 112 million units of PlayStation4 were
sold in 2020 during the pandemic. The video game market revenue worldwide in 2020 by the device was 63.6 billion
USD for smartphone games, 33.9 billion USD for boxed/downloaded PC games, 13.7 billion USD for tablet games, and
3 billion USD for browser PC games [55]. Compared to the previous year, the growth rate of e-sports on different
devices and segments were as follows: 13.3% growth in mobile games, 2.7% growth in tablet games, 15.8% growth in
smartphone games, 4.8% growth in PC games, 6.7% growth in boxed/downloaded PC games, and 6.8% growth in con-
sole games [56]. Overall the awareness and interest in e-sports are increased during the COVID-19 pandemic, which
will foster e-sports growth even in the future in postpandemic times.
The COVID-19 pandemic lockdown was taken as a measure to slow down the spread of the virus and therefore people
were forced to stay at home. E-sports provided a platform for people to stay connected, engaged, and enjoy while being
safe at home during the pandemic. And thus player numbers in e-sports soared as more and more youngsters got involved
in video gaming and different kind of e-sports. Some of the popular games, such as Call of Duty (CoD) and FIFA saw a
demand jump during the pandemic. The company Activision Blizzard reported an average of 407 million people playing its
games each month in the first quarter of the year [57]. The latest game in the CoD series “Warzone” racked up more than
60 million players since its launch in March 2020 [58]. Some other games from the same company such as Overwatch,
World of Warcraft, and Candy Crush also held the highest popularity and helped the company push its net revenues to 1.44
billion dollars. Electronic arts saw an increase of more than 25 million players via its latest edition in the FIFA football
franchise. Similarly, the American football game “Madden NFL 20” engaged the highest number of players in the fran-
chise’s history [58]. And its newly released “Star Wars Jedi: Fallen Order” attracted more than 10 million users. Electronic
arts revenues also rose to 1.4 billion dollars during the initial months of the lockdown [58,59]. Besides, many gaming indus-
try giants ranging from Microsoft to Steam reported a significant increase in user numbers. The United States video game
sales saw the highest growth in sales that was not even seen in a decade. E-sports is one of the emerging entertainment ser-
vices, and lockdown promoted people’s involvement even more within few months of the lockdown. This situation also
resulted in record sales in the gaming industry because of the spike in the number of e-sports players. The gaming industry
analysts expect the demand and surge in e-sports players to remain strong with repeated lockdowns in different countries
around the world and stay at home for safety during the COVID-19 pandemic.
276 Pandemic Outbreaks in the 21st Century

17.4 Advances in e-sports during the COVID-19 pandemic

Cloud gaming is a new trend in the gaming industry that came into existence due to the development of improved cloud
technology [60]. Cloud gaming provides huge storage space and a server that stores all the games and performs compu-
tations like game logic processing, game rendering, video encoding and streaming [60]. This technology has helped
increase the number of players by accommodating smartphone users with limited storage space for online games. It is
also a big advantage for all the gamers for ease of playing online without the restraint of available storage space with
their devices. There has been a rapid rise in growth in e-sports and number of e-sports players because of the increased
market demand during the pandemic lockdown. The increased investment in 5G internet plans and unlimited 4G inter-
net plans has also helped the gaming industry’s growth. This infrastructure and technology help gamers worldwide use
cloud gaming along with their preferred portable devices. Therefore more players can join and e-sports consumption is
increased further. Smartphone gaming is another development in recent years that has pushed the growth of the gaming
industry further. And it was also a major factor in increasing e-sports consumption across many countries because of
the ease of access to mobile games developed using recent technological advancements such as artificial intelligence,
augmented reality, virtual reality, machine learning and cloud gaming, and so on. Many e-sports like e-football, chess,
Ludo King have attracted a vast number of players and are also adopted on the professional front as a full-time or part-
time career path [61]. Strangely, COVID-19 was an advantage for the online gaming industry, as it gained immense
popularity for home entertainment during the pandemic.
The e-sports growth data showed a significant increase in number of players during the pandemic in different
e-sports games: Shooters (40% growth), gambling games (36% growth), deck-building games (34% growth), arcade
games (28% growth), platformers (25% growth), and battle royale (17% growth). The games in the MOBA group like
League of Legends, Heroes of the storm, smite, and so on saw the most significant increase in number of players [62].
Many e-sports events are organized offline, but because of the uncertainties and travel restrictions caused by the pan-
demic, the events were postponed, conducted online, or canceled. E-sports events such as Arena of Valor World Cup
2020, ESL One Birmingham 2020 Online Leagues, PUBG 2020 Global Series, Combo Breaker 2020, The OGA Dota
PIT Minor 2020, Taipei Major 2020, Rainbow Six Siege Pro League, NorCal Regionals 2020, League of Legends Mid-
Season Invitational, Tekken World Tour, WESG APAC Finals, Rocket League World Championship, LEC Spring
Finals, Pokemon World Championships 2020, Combo Breaker 2020, The Evolution Championships Series, Call of
Duty League, and Mortal Kombat 11 were canceled. E-sports events that were postponed included Street Fighter
League Season 3, ESL One Los Angeles Major, NBA 2K League, Fighter’s Spirit 2020, Asia Pacific Predator League
2020, China’s League of Legends Pro League, SNK World Championship Japan Tour Final, ESL One Los Angeles
Major Chinese Qualifiers, Overwatch League, League of Legends Championship Korea, Apex Legend Global Series,
and The ESL One Rio 2020 CS [63]. Interestingly, many traditional sports organizations organized e-sports events; for
example, the ePremier League Invitational Tournament was organized by the English Premier League and Premier
League players of the English Premier League FIFA 20 game participated in the event [64]. Similarly, the “NBA2K20
Players Tournament” was organized and NBA players joined the tournament online. Likewise, “The Checkdown NFL
Madden 20 Tournament” was organized by NFL in which 10 NFL players participated [50,65]. Another new trend seen
in e-sports during the pandemic was the popularity of Sim racing games [66]. Simulation racing or Sim racing games
attracted a lot of players in the COVID-19 pandemic. Sim racing games are the e-sports games that offer the most real-
istic experience to the player with the help of simulation [65]. These features are incorporated and updated in the simu-
lation racing game by scanning real tracks with laser scanning technology and transferring the information to digital
media. Additionally, the physics of the vehicles are kept as close to real as possible. Also, the weather conditions, tire
and brake wear, and other actual effects are included in the games. Players are allowed to play it online and they also
get feedback [40]. iRacing, F1, rFactor, Assetto Corsa, and Dirt Rally game series are preferred PC Sim racing games
and Gran Turismo, Project Cars, and Forza are preferred console game series [41]. The “24 Hours of Le Mans Virtual
2020” event was organized on 13
14 June and the race was played on the rFactor2 game [67]. The race was followed
by a total of 20 million people on TV and social media. Similarly, “Formula 1 Virtual Grand Prix series,2020” was
organized, followed by more than 30 million viewers on TV and social media in more than 100 countries [68].

17.5 E-sports: tool for social connectedness and psychological healing in the
COVID-19 pandemic
The sports events, tournaments, and overall outdoor field training were canceled all over the world due to the COVID-
19 pandemic. Therefore not only common people but also sportspersons were attracted toward e-sports as a means of
 Spike in electronic sports during the coronavirus disease pandemic Chapter | 17 277

alternative entertainment and involvement. E-sports and gaming became very popular among youngsters and different
age groups during the lockdown as it filled the social gap and connected people while staying at home for the safety of
all [69]. Some people enjoyed playing online, while some just enjoyed watching the gaming content. Besides, the main-
stream organizations like The Grand National, Formula1, Moto GP, the Spanish La Liga Football league and NASCAR
also invested in organizing virtual competitions and online events, broadcasted them on TV and streaming services to
millions of people as a substitute for entertainment to compensate for the lost revenues due to cancellation of events in
lockdown [70]. For example, in collaboration with Gfinity, the Premier League launched an inaugural ePremier League
Invitational Tournament in the lockdown [64]. The COVID-19 pandemic and lockdown had a huge impact on college-
going students’ gaming behavior. The uncertainty caused by the pandemic induced stress in students who are concerned
about their careers and future. To cope with such stress, many students increased their online gaming participation
based on the belief that gaming helps relieve stress [71].
With the spread of the coronavirus, lockdown and social distancing were implemented. Restaurants, bars, leisure
centers, cinema halls, gyms, outdoor games, and all other public places and activities were closed around the world. In
addition to passing the time by watching TV, people were craving for social context and found creative ways to engage
in social activities online and to some people, e-sports turned out to be more than just a lockdown distraction [72]. In
some video games, such as Animal Crossing: New Horizons, players can meet colorful characters and friends, chat with
one another, and explore the virtual world in a safe, enjoyable environment avoiding exposure to the deadly coronavirus
[73]. It has been an excellent way for people to hang with each other and spend quality time socializing in a virtual
world and staying connected. Some players have also used this game for dating. Games like this are creative ways to
stay connected socially and have fun meetings and activities even while being at home. Some of the other examples of
such social games are CoD: Warzone, Fortnite, Honor of Kings, which allow people to meet up in the game and interact
in groups to meet certain challenges [74]. In addition to being an alternative to stay connected to friends, online games
also provide a safe virtual environment to people helping them relieve their panic and stress due to the sudden coronavi-
rus pandemic by momentarily forgetting the harsh reality of the situation. There are hundreds of online games featuring
gigantic virtual worlds where people or e-sports players can meet online [74]. For example, Eve online is a famous
social game where participants can cooperate or battle against one another. It also encourages people to make teams
and work together to face and overcome many social groups’ challenges, which results in forging friendships among
players. CCP games own this game and they recorded nearly 11,000 new accounts per day in March 2020 when the
lockdown was in place in almost all countries around the world [74]. According to some scientists, such games help
people fulfill their psychological needs while real-world daily life is restricted considering the safety of all during the
pandemic [69]. The psychological needs being satisfied through such social online games to include the need for social-
izing, feelings of being in control, a sense of efficacy, and a choice over what one does [72]. The sales of games rose
by 40%
60% every week in lockdown. The game Honor of King became very popular in China and its demands
spiked in February 2020, resulting in a 20% increase in its revenues. The surge in online gaming also increased the data
usage by 100% compared to pre-COVID-19 use. UK government also found e-sports as an innovative way to pass on
the crucial information for people’s safety during the pandemic. The government’s games industry supported such an
initiative and helped reach out to people by sharing the Stay Home Save Lives message in some popular games like
FIFA and Fortnite. Moreover, many big gaming companies like EA, Konami, Xbox, and Sega granted free access to
download more than 85,000 video games to the NHS staff as a token of appreciation for their work during the coronavi-
rus pandemic as a part of the Games for Carers initiative [75].

17.6 E-sports: role in health and well-being during the pandemic

In uncertain times like the one now with the fatal COVID-19 pandemic, some online games can help people calm down
in this corona crisis [69,72]. An example of one such game is Everything, where one can choose to be a solar system or
a single-celled organism and many more infinite possibilities. One can occasionally communicate with others or click
on a thought bubble. Another sister game of Everything is Mountain and both of the games can be attributed to being
absorbing, deep, beautiful with a chorus of satisfying sounds. These games have calming effects and help people forget
the stress and insanity of a restricted lifestyle in lockdown [76]. Most of the online gaming industry is dominated by
goal-driven games and has fight or flight content. Such games activate the sympathetic nervous system, release adrena-
line followed by dopamine and thus make the player feel excited, powerful, and happy. However, such games might
not be relevant to some people, and there are exceptions to the fight or flight imperative in gaming. Considering the
need for joyfully expansive online games, one of the new games company Tru Luv launched games like self-care with
features designed to de-stress the player with satisfying and straightforward tasks to complete [76,77]. People engage in
278 Pandemic Outbreaks in the 21st Century

regular games, social media, and many other apps to escape the tensions and stress of daily life; however, the stress
response is triggered more by online activities. Therefore games designed to provide a calm space and instill a deeper
quality of stillness can create nourishing and compelling experiences for the players [78]. Another example is
Andromeda Entertainment’s SoundSelf; it is a game that tracks the experience of religious ceremonies, psychedelics,
chanting, meditation, and hypnosis. It takes you on an “inward journey” to instill a deeper quality of stillness [79].
The e-sports popularity came out as an unexpected outcome of the COVID crisis. And with this popularity came
some positive effects of e-sports to the society. Some companies in the e-sports industry were doing philanthropic work
with real positive impact and far-reaching benefits even before lockdown; for example, League of Legends launched
the initiative Riot Games Social Impact Fund in October 2019 10th-anniversary celebrations. The campaign raised
more than 10 million dollars supporting 50 nonprofit organizations in 15 countries [80]. The potential substantial posi-
tive impact of e-sports is not just limited to the fundraisers, but it goes beyond that. The e-sports players and audience
are getting involved in addressing the most salient and least discussed issues to which people mostly shy away, such as
mental health, gender inequality, bullying, depression, health and environmental responsibility, and so on. However, the
current generation does not shy away from these issues and encourages bringing out these discussions in the open and
doing something about them. And that is where the online gaming and e-sports community and the gaming industry
come forward to take the responsibility to create a positive change in society in the long run. The e-sports community,
audience, and industry evolve with time and take measures to empower the next generation with educational and inspi-
rational content. Since the pandemic had a significant impact on the mental health of all people, it affected the mental
health of e-sports players and further affected their games and participation in various competitions. Good mental health
is essential for an e-sports player [69,81]. E-sports has been a growing industry and the growth multiplied fast during
the pandemic. Disruption is an inevitable part of this sector and it was most evident during the COVID-19 lockdown.
The pandemic had a positive impact on the e-sports and digital game industry. Sim racing and development of mobile
and smartphone games attracted a lot of attention and investment. Technological advancement/upgradation of many
e-sports attracted more players and viewers in the pandemic. Many new platforms offered online games and live broad-
casting to people, making e-sports and digital games more popular and increased awareness and acceptance of these
games.

References
[1] Nicola M, et al. The socio-economic implications of the coronavirus pandemic (COVID-19): A review. Int J Surg (London, England)
2020;78:185
93.
[2] Sohrabi C, et al. World Health Organization declares global emergency: a review of the 2019 novel coronavirus (COVID-19). Int J Surg
2020;76:71
6.
[3] Singhal T. A review of coronavirus disease-2019 (COVID-19). Indian J Pediatr 2020;87(4):281
6.
[4] WHO. WHO Coronavirus Disease (COVID-19) Dashboard; 2020. Available from: https://covid19.who.int/. [Accessed 2 February 2021].
[5] Laborde D, et al. COVID-19 risks to global food security. Science 2020;369(6503):500
2.
[6] UNDP. COVID-19 socio-economic impact; 2020. Available from: https://www.undp.org/content/undp/en/home/coronavirus/socio-economic-
impact-of-covid-19.html.
[7] Türkmen M., Özsari A. COVID-19 salgını ve spor sektörüne etkileri. Int J Sport Cult Sci 8(2): 55
67.
[8] Parnell D, et al. COVID-19, networks and sport. Manag Sport Leis 2020;1
7.
[9] Hull JH, Loosemore M, Schwellnus M. Respiratory health in athletes: facing the COVID-19 challenge. Lancet Respir Med 2020;8(6):557
8.
[10] FIFA. FIFA Council|COVID-19 Relief Plan; 2020. Available from: https://www.fifa.com/who-we-are/news/fifa-council-unanimously-approves-
covid-19-relief-plan. [Accessed 3 February 2021].
[11] Daniel LK, et al. Problematic online gaming and the COVID-19 pandemic. J Behav Addict 2020;9(2):184
6.
[12] Clement J. Numbers of hours watched on Streamlabs worldwide in 3rd quarter 2020, by platform; 2020. Available from: https://www.statista.
com/statistics/1030795/hours-watched-streamlabs-platform/. [Accessed 3 February 2021].
[13] Wagner M. On the scientific relevance of eSports, In: International conference on internet computing. CSREA Press; 2006. p. 437
442.
[14] Funk DC, Pizzo AD, Baker BJ. eSport management: embracing eSport education and research opportunities. Sport Manag Rev 2018;21
(1):7
13.
[15] Hallmann K, Giel T. eSports
competitive sports or recreational activity? Sport Manag Rev 2018;21(1):14
20.
[16] Hallmann K, Giel T. eSports
competitive sports or recreational activity? Sport Manag Rev 2018;21(1):14
20.
[17] Smithies TD, et al. Life after esports: a grand field challenge. Front Psychol 2020;11 883.
[18] Hamari J, Sjöblom M. What is eSports and why do people watch it? Internet Res 2017;27(2):211
32.
[19] Lee D, Schoenstedt LJ. Comparison of eSports and traditional sports consumption motives. ICHPER-SD J Res 2011;6(2):39
44.
[20] Weiss, T. Fulfilling the Needs of eSports Consumers: A Uses and Gratifications Perspective. Bled eConference; 2011.
[21] Seo Y, Jung S-U. Beyond solitary play in computer games: the social practices of eSports. J Consum Cult 2016;16(3):635
55.
 Spike in electronic sports during the coronavirus disease pandemic Chapter | 17 279

[22] Seo Y. Electronic sports: a new marketing landscape of the experience economy. J Mark Manag 2013;29(13
14):1542
60.
[23] Karhulahti V-M. Reconsidering esport: economics and executive ownership. Phys Cult Sport Stud Res 2017;74(1):43
53.
[24] Parshakov P, Zavertiaeva M. Success in eSports: does country matter?; 2015 10.2139/ssrn.2662343.
[25] Reitman JG, et al. Esports research: a literature review. Games Cult 2020;15(1):32
50.
[26] Szablewicz M. From addicts to athletes: participation in the discursive construction of digital games in urban China. AoIR Sel Pap Internet Res
2011.
[27] Menasce RM. From casual to professional: How Brazilians achieved esports success in counter-strike: global offensive. Northeastern
University; 2017.
[28] Natalia L, Karashchuk O, Kornilova O. Analysis of eSports as a commercial activity. Probl Perspect Manag 2018;16:207
13.
[29] Hemphill D. Cybersport. J Philos Sport 2005;32(2):195
207.
[30] Jonasson K, Thiborg J. Electronic sport and its impact on future sport. Sport Soc 2010;13(2):287
99.
[31] Guttmann A. From ritual to record: The nature of modern sports. Columbia University Press.; 1978.
[32] Ash J. Technology, technicity, and emerging practices of temporal sensitivity in videogames. Environ Plan A 2012;44(1):187
203.
[33] Huang J, et al. Master maker: understanding gaming skill through practice and habit from gameplay behavior. Top Cognit Sci 2017;9
(2):437
66.
[34] Gray WD. Game-XP: action games as experimental paradigms for cognitive science. Wiley Online Library; 2017.
[35] Brock T, Fraser E. Is computer gaming a craft? prehension, practice, and puzzle-solving in gaming labour. Inf Commun Soc 2018;21
(9):1219
33.
[36] Fatehi B. Gamifying psychological testing: insights from Gamifying the TAT. Northeastern University Boston; 2017.
[37] Gray P, et al. Testing men’s hormone responses to playing League of Legends: no changes in testosterone, cortisol, DHEA or androstenedione
but decreases in aldosterone. Comput Hum Behav 2018;83.
[38] El-Nasr, M.S., A. Drachen, and A. Canossa, Game analytics. Springer.
[39] El-Nasr, M.S., A. Drachen, and A. Canossa, Game Analytics: Maximizing Value Player Data Springer-Verlag London.
[40] Nagel GL. Use of eye tracking for esports analytics in a MOBA game. The University of Bergen; 2017.
[41] Olshefski E. Game-changing event definition and detection in an esports corpus. In Proceedings of the the third workshop on EVENTS: defini-
tion, detection, coreference, and representation; 2015.
[42] Low-Kam C, et al. Mining statistically significant sequential patterns. In: 2013 IEEE 13th international conference on data mining. IEEE; 2013.
[43] Kim J, et al. The proficiency-congruency dilemma: virtual team design and performance in multiplayer online games. In: Proceedings of the
2016 CHI conference on human factors in computing systems; 2016.
[44] Chen Z, et al. The art of drafting: a team-oriented hero recommendation system for multiplayer online battle arena games. In: Proceedings of
the twelfth ACM conference on recommender systems; 2018.
[45] Freeman G, Wohn DY. Understanding eSports team formation and coordination. CSCW 2019;28(1):95
126.
[46] Freeman G, Wohn D. Social support in eSports: building emotional and esteem support from instrumental support interactions in a highly com-
petitive environment. In: CHI PLAY ’17: Proceedings of the annual symposium on computer-human interaction in play; 2017. p. 435
447.
[47] Mordor Intelligence. Gaming market - growth, trends, COVID-19 impact, and forecasts (2021
2026); 2020. Available from: https://www.mor-
dorintelligence.com/industry-reports/global-games-market [Accessed 3 February 2021].
[48] ReportLinker. Gaming market - growth, trends, forecasts (2020
2025); 2020. Available from: https://www.globenewswire.com/news-release/
2020/11/12/2125769/0/en/Gaming-Market-Growth-Trends-Forecasts-2020-2025.html [Accessed 3 February 2021].
[49] The NPD Group. The NPD Group: United States consumer spend on video game products continues to break records; 2020. Available from: https://
www.npd.com/wps/portal/npd/us/news/press-releases/2020/the-npd-group-us-consumer-spend-on-video-game-products-continues-to-break-records/.
[50] Fakazlı A. The effect of COVID-19 pandemic on digital games and eSports. International Journal of Sport Culture and Science 2020;335
44.
[51] Newzoo. Global games market report; 2020. Available from: https://newzoo.com/products/reports/global-games-market-report/. [Accessed 3
February 2021].
[52] Wijiman T. The World’s 2.7 billion gamers will spend $159.3 billion on games in 2020; The market will surpass $200 billion by 2023; 2020.
Available from: https://newzoo.com/insights/articles/newzoo-games-market-numbers-revenues-and-audience-2020-2023/. [3 February 2021].
[53] Facteus. Facteus insight report on consumer spending and transactions (FIRST); 2020. Available from: https://www.facteus.com/reports/first-
report-10-14-2020/. [Accessed 3 February 2021].
[54] Tower S. What 5 billion-dollar games tell us about mobile in 2020; 2020. Available from: https://venturebeat.com/2020/12/17/what-5-billion-
dollar-games-tell-us-about-mobile-in-2020/. [Accessed 3 February 2021].
[55] Clement J. Video game market revenue worldwide in 2020, by device; 2020; Available from: https://www.statista.com/statistics/292751/
mobile-gaming-revenue-worldwide-device/. [Accessed 3 February 2021].
[56] Newzoo. The world’s 2.7 billion gamers will spend $159.3 billion on games in 2020; The market will surpass $200 billion by 2023; 2020.
Available from: https://newzoo.com/insights/articles/newzoo-games-market-numbers-revenues-and-audience-2020-2023/. [Accessed 3 February
2021].
[57] Santa Monica. Activision blizzard announces strong first-quarter 2020 financial results; 2020. Available from: https://www.businesswire.com/
news/home/20200505006004/en/Activision-Blizzard-Announces-Strong-First-quarter-2020-Financial-Results. [4 February 2021].
[58] BBC. Lockdown and loaded: coronavirus triggers video game boost. 2020. Available from: https://www.bbc.com/news/business-52555277?
intlink_from_url 5 https://www.bbc.com/news/business/companies&link_location 5 live-reporting-story. [4 February 2021].
280 Pandemic Outbreaks in the 21st Century

[59] Clement J. Electronic Arts (EA) net income 2005
2020; 2021. Available from: https://www.statista.com/statistics/698262/income-loss-elec-
tronic-arts/. [Accessed 4 February 2021].
[60] Clement J. COVID-19 Has increased interest in cloud gaming services; 2020. Available from: https://in.pcmag.com/game-streaming-services/
139833/covid-19-has-increased-interest-in-cloud-gaming-services. [Accessed 4 February 2021].
[61] Yannakakis, G.N. and J. Togelius, Artificial intelligence and games. 2. Springer.
[62] Jackson J. What gamers are playing & watching during the coronavirus lockdown: player share & viewership spikes for games & genres; 2020.
Available from: https://newzoo.com/insights/articles/games-gamers-are-playing-watching-during-coronavirus-covid19-lockdown-quarantine/.
[Accessed 4 February 2021].
[63] Hayward A. The esports leagues and events affected by coronavirus; 2020. Available from: https://esportsinsider.com/2020/03/esports-events-
affected-by-coronavirus/. [Accessed 4 February 2021].
[64] League P. ePremier league invitational; 2020. Available from: https://www.premierleague.com/epl-invitational. [Accessed 4 February 2021].
[65] Krustofski N. Esports racing year 2020 in facts and figures; 2020. Available from: https://www.overtake.gg/news/2903-2020-review-on-the-
sim-racing-and-racing-games-boom. [Accessed 4 February 2021].
[66] Post TW. The giants of the video game industry have thrived in the pandemic. Can the success continue?; 2020. Available from: https://www.
washingtonpost.com/video-games/2020/05/12/video-game-industry-coronavirus/. [Accessed 4 February 2021].
[67] Mans H. The ultimate guide to 24 hours of Le Mans virtual; 2020. Available from: https://www.24h-lemans.com/en/news/the-ultimate-guide-to-
24-hours-of-le-mans-virtual-53875. [Accessed 4 February 2021].
[68] Formula1. Formula 1 Virtual Grand Prix series achieves record-breaking viewership; 2020. Available from: https://www.formula1.com/en/latest/arti-
cle.formula-1-virtual-grand-prix-series-achieves-record-breaking-viewership.7bv94UJPCtxW0L5mwTxBHk.html. [Accessed 4 February 2021].
[69] Jones C, et al. Gaming well: links between videogames and flourishing mental health. Front Psychol 2014;5:260.
[70] McCarthy J. Is esports prepared for the coming influx of freed-up marketing spend?; 2020. Available from: https://www.thedrum.com/news/
2020/05/04/esports-prepared-the-coming-influx-freed-up-marketing-spend. [Accessed 4 February 2021].
[71] Balhara YPS, et al. Impact of lockdown following COVID-19 on the gaming behavior of college students. Indian J Public Health 2020;64
(Supplement):S172
6.
[72] Baraniuk C. Computer games: More than a lockdown distraction; 2020. Available from: https://www.bbc.com/news/business-52210938.
[Accessed 4 February 2021].
[73] Griffin M.S. Animal crossing: new horizons
the video game where we can still be together; 2020. Available from: https://www.theguardian.
com/games/2020/mar/20/animal-crossing-new-horizons-video-games-nintendo. [Accessed 4 February 2021].
[74] Stuart K., Stuart D., Webber J.E. 25 best video games to help you socialise while self-isolating; 2020. Available from: https://www.theguardian.
com/games/2020/mar/17/25-best-online-video-games-coronavirus-self-isolating.
[75] Pattle A. Games for carers: website offering 85,000 free games to NHS workers overcomes early technical issues; 2020. Available from:
https://www.independent.co.uk/arts-entertainment/games/news/games-carers-nhs-free-website-not-working-loading-a9489646.html. [Accessed 4
February 2021].
[76] Spicer K. How gaming became a form of meditation; 2020. Available from: https://www.bbc.com/culture/article/20200409-how-gaming-
became-a-form-of-meditation. [Accessed 4 February 2021].
[77] Etherington D. Mobile developer Tru Luv enlists investors to help build a more inclusive alternative to gaming; 2020. Available from: https://tech-
crunch.com/2020/06/29/mobile-developer-tru-luv-enlists-investors-to-help-build-a-more-inclusive-alternative-to-gaming/. [Accessed 4 February 2021].
[78] Marston HR, Kowert R. What role can videogames play in the COVID-19 pandemic? Emerald Open Res 2020;2:34.
[79] Cronin AM. SoundSelf: A Technodelic - a new way to cope with confinement stress - digital meditation is about to be revolutionized; 2020.
Available from: https://www.globenewswire.com/news-release/2020/04/07/2012758/0/en/SoundSelf-A-Technodelic-A-New-Way-to-Cope-with-
Confinement-Stress-Digital-Meditation-is-About-to-be-Revolutionized.html. [Accessed 4 February 2021].
[80] Partleton K., Riot Games has raised $10 million for its social impact fund so far; 2020. Available from: https://www.pocketgamer.biz/news/
73357/riot-games-raises-10-million-usd-social-impact-fund/#:B:text 5 As%20announced%20in%20its%202019,celebrations%20for%20League
%20of%20Legends. [Accessed 4 February 2021].
[81] DiFrancisco-Donoghue J, et al. Managing the health of the eSport athlete: an integrated health management model. BMJ Open Sport Exerc
Med 2019;5(1):e000467.
Chapter 18

How digital health and pandemic

preparedness proved a game changer?
A case of Singapore in COVID-19
management
Sibasis Hense1, Pratik Mukherjee2 and Hunasanahally Puttaswamygowda Gurushankara3
1
Department of Public Health and Community Medicine, Central University of Kerala, Kasaragod, India, 2Woodlands Health Campus, Singapore,
3
Department of Zoology, School of Biological Sciences, Central University of Kerala, Tejaswini Hills, Kasaragod, India

18.1 The context

Infectious diseases have existed since hunter-gatherer days, but they are known to take the shape of pandemics in the
last 10,000 years. Some of the earlier pandemics experienced by human civilization included Athens plague of 430
BCE, Antonine Plague of CE 165, Cyprian Plague of CE 250, Black death of 1350, Spanish Flu of 1918, and so on [1].
The recent pandemic of COVID-19 has a stark contrast to these pandemics as it has hit the world in an era that is digi-
tally connected. Besides, it has affected over 200 countries, causing significant morbidities and mortalities, at a time
when medical sciences and public health disciplines are well developed [2]. Despite well-established public health mea-
sures (such as testing, tracing, masking, hand hygiene, and social distancing) and treatment modalities the number of
infections and deaths has continued to grow worldwide. Countries like the U.K., Germany appeared to handle the first
wave well but witnessed rising cases and deaths during the second wave. Globally, over 103 million cases and 2 million
COVID-19 related deaths are recorded as of January 31, 2021 (Table 18.1) [3].
The United States continues to rank number 1 in terms of COVID-19-related cases and deaths. Brazil, Italy, U.K.,
Spain, and France witnessed over 1000 deaths per million population [3]. On a positive note, given the test, trace, and
isolate strategy [4], Singapore conducted the highest number of tests (1,074,538 per 1 million population as of January
31, 2021) and attained remarkable success in controlling the virus [3,5]. However, in the absence of an established
effective antiviral therapy/vaccination, the cornerstone of COVID-19 management, the strategies were largely contain-
ment and mitigation [6]. But it is increasingly felt that health technology particularly digital health can be widely used
to facilitate COVID-19 pandemic management effectively, which is otherwise difficult to attain manually [7]. Therefore
it is important to understand the concepts of digital health, its scope, and how its usage can facilitate and supplement
preparedness and address the health challenges posed by the virus (Fig. 18.1).
What is digital health? A sizeable amount of literature has attempted to define digital health, but a universally
agreed definition is elusive. In the past, digital health was defined using several terms such as e-Health, mHealth, virtual
care, remote health, and telehealth [8]. A rigorous examination of the extant literature focuses on three levels of infor-
mation used to define digital health. They are type/use of digital technology, improvement of health delivery, and a
strategy for healthcare system transformation (Fig. 18.2) [8
11].
However, for ease of understanding, in this chapter we adhere to the definition given by the Healthcare Information
Management System and Society (HIMSS) for digital health. The HIMSS defines digital health “as a system that con-
nects and empowers people and population to manage health and wellness, augmented by accessible and supportive
provider teams working within flexible, integrated, interoperable, and digitally-enabled care environments that strategi-
cally leverage digital tools, technologies and services to transform care delivery” [8]. Some of the tools and technology

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00007-0

© 2021 Elsevier Inc. All rights reserved. 281
282 Pandemic Outbreaks in the 21st Century

TABLE 18.1 Top 10 countries with largest number of COVID-19 cases as of January 31, 2021.

Rank Country Total cases Total Total cases/1M Deaths/1M Tests/1M

deaths population population population
World 103,528,810 2,237,790 13,282 287.1 NA
1 USA 26,767,229 452,279 80,590 1,362 939,773
2 India 10,758,619 154,428 7,752 111 142,004
3 Brazil 9,204,731 224,534 43,125 1052 133,993
4 Russia 3,850,439 73,182 26,378 501 698,788
5 U.K. 3,817,176 106,158 56,057 1,559 1,052,607
6 France 3,197,114 76,057 48,917 1,164 674,262
7 Spain 2,830,478 58,319 60,525 1,247 684,859
8 Italy 2,553,032 88,516 42,262 1,465 540,755
9 Turkey 2,477,463 25,993 29,191 306 349,141
10 Germany 2,225,65 57,777 26,514 688 459,056
93 Singapore 59,536 29 10,130 05 1,074,538

Source: Worldometer. COVID-19 Coronavirus pandemic. Available from: https://www.worldometers.info/coronavirus/. [Accessed 31 January 2021].

Type/use of digital
technology

Improvements of health
delivery

Strategy for healthcare system

informaon

FIGURE 18.1 Levels of information used to define digital health.

•Facilitate capture, • Research and

access and sharing of development
data using internet • Insight development
• Aid in early and decision making
diagnosis, quaranne •Inform public health
and aer recovery Internet Big data policies
measures
of things and
(IoT) analycs

Tele mHealth
• Remote treatment
medicine
& consultaon • Contact tracing
•Prevent hospital • Health advisories
crowding • Virtual consulaon
• Infecon prevenon • Health educaon
FIGURE 18.2 Role of digital health in COVID-19 pandemic management. COVID-19, Coronavirus disease.
 How digital health and pandemic preparedness proved a game changer Chapter | 18 283

that are used in digital health includes mHealth, internet of things (IoT), telemedicine, smart wearable devices, big-data
analytics, artificial intelligence (AI), blockchain technology, and techniques enabling remote data capture, storage, and
exchange across the healthcare ecosystem. Generally, digital health technologies use computing platforms, connectivity,
software, and sensors that are proven to improve efficiency, access, quality, reduce cost, and make medicine more per-
sonalized. How digital health tools and technologies are better utilized in the management of pandemics and particu-
larly in COVID-19 responses are discussed in the following section.

18.2 Digital health and COVID-19 pandemic

The World Health Organization (WHO) defines pandemic as “an epidemic occurring worldwide, or over a very wide
area, crossing international boundaries and affecting a large number of people” [12]. It is a health emergency spread
over a larger geographical area demanding greater mobilization of health resources including its management and effec-
tive coordination within and across countries/or even continents. Given every possibility that a pandemic could inflict
heavy casualties of human lives and disrupt social and economic processes, the use of digital health technologies can be
vital in pandemic management that are often difficult to attain manually. How the use of different digital tools and tech-
nologies, such as IoT, big-data analytics, telemedicine, mHealth, facilitated early surveillance, testing, contact tracing,
and quarantine strategies in pandemic management are discussed (Fig. 18.2).
1. IoT: IoT include objects that can be connected to the internet. These objects are embedded with sensors, software,
and other technologies for the purpose of exchanging data over the internet. It can provide platforms that allow pub-
lic health agencies to access data for the management of pandemics. IoT has been widely used in response to
COVID-19 for early diagnosis, quarantine, and after recovery [7,13,14]. IoT devices are known to speed up the
detection process by capturing information from patients thus facilitating early diagnosis of the cases. These are
implemented for capturing body temperatures using a smart thermometer from suspicious individuals. Similarly, to
monitor the quarantine time, once the patient is diagnosed with COVID-19, IoT can monitor patients remotely and
authorities can send advisory messages to the smartphone users connected the internet. Some of the IoT enabled
device commonly used in the early diagnosis, quarantine, and after recovery of COVID-19 includes wearables,
smartphone applications, smart thermometers, robots, and drones [15].
2. Big data and analytics: Big data analytics refers to the application of advanced analytics techniques in analyzing
very large and diverse data sets of sizes ranging from terabytes to zettabytes [16]. The COVID-19 pandemic has
resulted in the production of a staggering amount of data related to patients, healthcare providers, and insurance
companies. The analysis of these data using techniques like predictive analytics, data visualization techniques, data
mining, AI, machine learning, and natural language processing has been used to inform public health policies and
the implementation of preventive measures. For example, data scientists in the Institute of Data Science, Columbia
University, United States launched a start-up called WVQLV that created a computer algorithm capable of generat-
ing, screening, and optimizing millions of therapeutics of antibodies [16]. Researchers are also using AI techniques
to identify individuals who are vulnerable to severe complications from COVID-19 [17].
3. Telemedicine: Telemedicine uses information communication and technology (ICT) technology to improve patient
outcomes by increasing access to care and clinical information. The application of telemedicine services was pre-
dominantly offered to patients where distance was a critical factor and specialist consultation was unavailable.
However, the COVID-19 pandemic changed this mindset of health providers and regulators. Authorities globally are
now urging the public and clinicians to use telemedicine for nonurgent cases so as to reduce the burden of the hospi-
tals and prevent cross infections in hospital settings [18]. The use of telemedicine during the pandemic also provided
a valuable platform in bridging the gap between clinicians, patients, and healthcare systems. It has proved a valuable
tool for symptomatic patients and rendered care to them by establishing communication with physicians and other
health professionals through the virtual platform. The provision of remote consultation helped reduce the spread of
the virus to mass populations and the clinicians and staff working in healthcare settings.
4. m-Health: While there is no universally accepted definition of mHealth, the WHO defines mHealth as “medical and
public health practice supported by mobile devices, such as mobile phones, patient monitoring devices, personal dig-
ital assistants, and other wireless devices” [19]. mHealth apps are important in disease containment during pan-
demics. It can support health systems in risk assessment, case identification, contact tracing, surveillance, and
situation monitoring [20]. In the context of COVID-19 pandemic many countries developed their tracking app that
facilitated users in risk assessment and sending relevant advisories [15]. These apps are useful in identifying indivi-
duals who might have come in contact with someone who tested positive for COVID-19. Subsequently, research has
284 Pandemic Outbreaks in the 21st Century

also found that mHealth technologies, such as wearable sensors and electronic patient-reported data obtained
through smartphone or other IoT devices, can monitor COVID-19 patients at home and predict COVID-19 virus
exposure in individuals presumed to be free of infection [21,22], thus providing relevant information that could help
prioritize testing and treatment planning.

18.3 Preparedness and leveraging digital health in COVID-19 management:

a case of Singapore
In response to the COVID-19 pandemic, many countries were seen adopting a number of containment and mitigation
strategies, which varied in terms of implementation, scale, and tenacity. These strategies primarily focused on delaying
the surge of cases, protecting the most vulnerable (elderly and patients with comorbid conditions) from getting the
infection, and limiting the deaths. Notwithstanding, countries striving for a common goal, that is, to contain the virus
and keeping the fatalities low, a mixed outcome was experienced, with some countries limiting the infection to the low-
est level and observing the minimum loss of human lives, compared to others. One such country was Singapore, which
has been successful in keeping the cases low and mortalities to bare minimum. This was made possible despite the chal-
lenges faced in terms of importation of cases, higher population density, and geographical and cultural proximity to the
epicenter of the virus.
Singapore is a small country in the South-East Asian region with a population of 5.87 million and a density of 7866
per sq. km [23]. It was one of the first countries affected by COVID-19 (along with Hong Kong and Taiwan) and
reported the highest cases outside of China, during the month of February 2020 [24]. As of January 31, 2021, 279 active
cases, 59,228 recoveries, and 29 deaths were reported, representing one of the top-performing countries in terms of low-
est fatality (0.07%), and highest testing capacity (757,835 per million population) [3]. These impressive
figures reflected Singapore’s stringent implementation of measures as part of its multipronged approach to contain the
virus. Some of its measures included: preparedness of its health systems, leveraging technology and conducting compre-
hensive surveillance, tracking and testing, emphasizing on community and social responsibilities, lockdown of affected
areas, unified public communication, and fiscal stimulus [25]. However, instilling such measures raises million-dollar
questions. Why similar measures did not prove effective in other countries? and what distinguishes Singapore from
other countries in tackling COVID-19? Thus it can be argued that much of Singapore’s success story is attributed to its
existing outbreak response mechanism which the country has put in place since its Severe Acute Respiratory Syndrome
(SARS). Therefore it may not be naive to mention that the mechanism in place helped the country in terms of prepared-
ness, technology adoption, timely decision making, clear command and direction, effective communication, better inter-
sectoral coordination, and stringent implementation of different measures at the ground level, which could be seen
missing in other countries.

18.4 What was different in Singapore’s response and what lessons

can other countries learn?
Early in its fight against COVID-19, the Singapore government was able to contain the virus using preemptive mea-
sures. However, most of these measures adopted were the legacy lessons from SARS and H1NI outbreaks of 2003 and
2009, respectively. The inclusion of this short case study in this chapter has analyzed and presented Singapore’s
response in its fight against COVID-19 and argued how preparedness coupled with digital technology has proved a
game changer for Singapore in keeping the virus under control and limiting deaths to bare minimum.
Experience and preparedness matters: Singapore was one of the worst affected countries in the 2003 SARS out-
break and this had unveiled limitations in terms of infrastructure, coordination, and systemic deficiencies. Post-SARS,
Singapore established state-of-the-art centers like the “National Center for Infectious Disease,” National Public Health
Laboratory (NPHL), and expanded its isolation capacities in public hospitals. Additionally, it built primary care public
health preparedness centers, enhanced staff competencies in respiratory infectious crisis response, developed infectious
disease guidelines, strengthened communication channels for cross-hospital sharing and Health Department, and devel-
oped procedures to ensure medical supplies. This level of preparedness indicated the presence of a well-defined out-
break response mechanism in place to address COVID-19 without wasting valuable time.
Stringency of alert system and moral responsibilities of Singaporeans counted: The Ministry of Health, Government
of Singapore, adopted the Disease Outbreak Response System Condition—the DORSCON, developed during SARS
outbreak, a color-coded framework (Green, Yellow, Orange, and Red) for pandemic response. It guides the general
 How digital health and pandemic preparedness proved a game changer Chapter | 18 285

public and authorities on what needs to be done, and how to prevent and reduce the impact of infections. DORSCON
takes into account the nature of the disease, its R(0), impact on daily life and shares advisories to the public on required
measures. Although a framework similar to DORSCON was used in other countries for COVID-19, the difference lies
with its stringency in implementation, prior experience, seriousness, and moral responsibilities among the general public
to not evade the DORSCON alert.
Leveraging digital health technology: In Singapore digital health technologies such as m-Health apps, EMR, smart-
phones, GPS tracking, CCTV cameras, robots, drones, and wearables were used effectively to prevent, identify, manage
and monitor COVID-19 cases and minimize the spread of infection in the community. The Singapore Government had
set up a dedicated WhatsApp channel for transparency which could be used by anyone in the country. This instilled a
sense of confidence in the public and informed them about any new measures and advisories being rolled out by the
authorities. The general public was updated on a daily basis about the number of new infections, recoveries, reminders
for safety tips, new measures to be implemented (with dates), and shared the necessary information for the locals which
helped them face the challenges during the “Circuit breaker-CB”/lockdown phase.
Time was crucial: Singapore was the first country outside mainland China to be hit by coronavirus. Acting on the
situation promptly and proactively was the key to contain the virus and prevent community transmission. Unlike other
countries such as, the United States, Italy, Spain, and so on, which were passive in their approach, Singapore was swift
and well-prepared to instill border control measures in Airports and all other ports of entry. The DORSCON alert sys-
tem proves crucial in this regard.
Testing, tracing, and isolating were the key drivers: The model “test, trace, and isolate” that emerged out of the 2003
SARS outbreak were arguably the key drivers to flatten the curve in the early stage of the epidemic. Singapore adopted a
comprehensive surveillance system to detect as many cases as possible and contained them quickly at the individual level.
This was made possible with widespread testing, extensive contact follow-up, phone surveillance, and strict quarantine mea-
sures which were done free of cost and with incentives. The aggressive testing was done in a coordinated and integrated
manner, and later they used locally manufactured testing kits that were low priced and readily available.
Straight forward and unified channel of communication eased the panic: What makes a pandemic like COVID-19
more dangerous? It is the spread of rumors and misinformation, primarily through the internet that spreads even faster
than the virus itself. While most of the countries focused on restricting rumors and misinformation through ICT mea-
sures and legal frameworks, Singapore went one step ahead and unified the source of information that was meant for
public education and dissemination. Unlike the United States where messages related to COVID-19 contradicted among
the policymakers and scientific community, this did not happen in Singapore. All along, Singapore’s public communi-
cation had been consistent with no division seen among different stakeholders which helped keep confusion and public
panic at bay and addressed any stigma associated with the virus.
Reactivation of public health preparedness clinics proves effective: The public health preparedness clinics (PHPCs)
in Singapore were an important line of defense during public health outbreaks during SARS and H1NI and these were
reactivated as the first COVID-19 case was detected in January 2020. The aim of this clinic was to detect the virus early
and reduce the risk of further transmission. Unlike some developed health systems (such as the U.K. and Australia)
where primary care physicians were instructed to refrain from any face-to-face assessment of suspected cases and refer
them to COVID-19 dedicated health centers, Singapore reactivated its well-resourced 800 PHPCs to manage any respi-
ratory infection at the grass-root level. The well-trained medical practitioners of these health centers were instructed to
provide treatment at subsidized rates [as low as (SG$ 10)] and patients with respiratory symptoms were offered medical
leave up to 5 days, and subsequently referred for COVID-19 testing if flu-like symptoms persisted.

18.5 Conclusion
The world is currently facing a global health emergency of tremendous proportions due to the COVID-19 pandemic. Its
consequences are not only confined to the health and wellness of the human population but extended to the disruption of
social and economic processes. Specifically, the pandemic has resulted in food insecurity, economic crisis, loss of employ-
ment, drop-out of students from schools/colleges, increased domestic violence, and staggering mental health problems glob-
ally. The trajectory and tipping points of many countries are yet to be known as they are in the midst of second and third
waves. Technology is widely used as an alternate measure to mitigate and address the challenges posed by the virus and
deliver the social and economic processes. Governments, private, and not-for-profit agencies are trying their level best to
leverage technology in offering the essential services, be it healthcare, education, or financial services in this tormenting sit-
uation. In context to Singapore the response to managing the COVID-19 pandemic has been born out of its important les-
sons and past experiences of handling the SARS and H1N1 outbreaks of 2003 and 2009. The structure and response
286 Pandemic Outbreaks in the 21st Century

mechanisms that are put in place demonstrate Singapore’s timely and effective control measures of the pandemic in a well-
co-ordinated and integrated manner including leveraging digital health technology which supplemented and proved crucial
in the pandemic management. However, no two countries or outbreaks are similar. Therefore it is crucial to better under-
stand the disease-causing agent, its environment, and the socio-cultural, political, economic, and geographic context of the
region, prior to instilling the best practices and replicable lessons of another country.

References
[1] Huremovic D. Brief history of pandemics (pandemics throughout history). Psychiatr Pandemics. Cham: Springer; 2019. p. 7
35.
[2] David M. COVID-19 (coronavirus): a global emergency outbreak and its implications in India. Int J Zool Appl Biosci 2020;5(2):89
98.
[3] Worldometer. COVID-19 Coronavirus pandemic. Available from: https://www.worldometers.info/coronavirus/. [Accessed 31 January 2021].
[4] Contreras S, Dehning J, Loidolt M, Zierenberg J, Spitzner FP, Urrea-Quintero JH, et al. The challenges of containing SARS-CoV-2 via test-
trace-and-isolate. Nat Commun 2021;12(1):1
3.
[5] Lee WC, Ong CY. Overview of rapid mitigating strategies in Singapore during the COVID-19 pandemic. Public Health 2020;185:15
17.
[6] Vijayvargiya P, Garrigos ZE, Almeida NE, Gurram PR, Stevens RW, Razonable RR. Treatment considerations for COVID-19: a critical review
of the evidence (or lack thereof). Mayo Clin Proc 2020;95(7):1454
66.
[7] Ting DS, Carin L, Dzau V, Wong TY. Digital technology and COVID-19. Nat Med 2020;26(4):459
61.
[8] Snowdon A. Digital health: a framework for healthcare transformation. Healthcare Information Management System and Society (HIMSS);
2020. Available from: https://www.himss.org/resources/digital-health-framework-healthcare-transformation-white-paper. [Accessed 21 October
2020].
[9] Lupton D. Critical perspectives on digital health technologies. Sociol Compass 2014;8(12):1344
59.
[10] Rowlands D. What is digital health and why does it matter? Digital Health Workforce Academy; 2019.Available from: https://www.hisa.org.au/
wp-content/uploads/2019/12/What_is_Digital_Health.pdf?x97063. [Accessed 03 January 2021].
[11] Rich E, Miah A. Understanding digital health as public pedagogy: a critical framework. Societies 2014;4(2):296
315.
[12] Doshi P. The elusive definition of pandemic influenza. Bull World Health Organ 2011;89:532
8.
[13] Singh RP, Javaid M, Haleem A, Suman R. Internet of things (IoT) applications to fight against COVID-19 pandemic. Diabetes Metab
Syndrome Clin Res Rev 2020;14(4):521
4.
[14] Rahman MS, Peeri NC, Shrestha N, Zaki R, Haque U, Ab, et al. Defending against the novel coronavirus (COVID-19) outbreak: how can the
internet of things (IoT) help to save the world? Health Policy Technol 2020;9(2):136
68.
[15] Nasajpour M, Pouriyeh S, Parizi RM, Dorodchi M, Valero M, Arabnia HR. Internet of Things for current COVID-19 and future pandemics: An
exploratory study. J Healthc Inform Res 2020;1
40.
[16] What is Big data Analytics? International Business Machine (IBM). Available from: https://www.ibm.com/in-en/analytics/hadoop/big-data-ana-
lytics. [Accessed 27 December 2020].
[17] Kent J. Data scientists use machine learning to discover COVID-19 treatments. Health IT analytics. Available from: https://healthitanalytics.
com/news/data-scientists-use-machine-learning-to-discover-covid-19-treatments#:B:text 5 Andrew%20Satz%20and%20Brett%20Averso,of%
20millions%20of%20therapeutic%20antibodies. [Accessed 21 December 2020].
[18] Siwicki B. Telemedicine during COVID-19: benefits, limitations, burdens, adaptation. Healthcare IT News. March 19; 2020. Available from:
https://www.healthcareitnews.com/news/telemedicine-during-covid-19-benefits-limitations-burdens-adaptation. [Accessed 18 December 2020].
[19] Kay M, Santos J, Takane M. mHealth: new horizons for health through mobile technologies, 64. World Health Organization; 2011. p. 66
71. 7.
[20] Kodali PB, Hense S, Kopparty S, Kalapala GR, Haloi B. How Indians responded to the Arogya Setu app? Indian J Public Health 2020;64
(6):228.
[21] Adans-Dester CP, Bamberg S, Bertacchi FP, Caulfield B, Chappie K, Demarchi D, et al. Can mHealth technology help mitigate the effects of
the COVID-19 pandemic? IEEE Open J Eng Med Biol 2020;1:243
8.
[22] Giansanti D. The role of the mHealth in the fight against the Covid-19: successes and failures. Healthcare 2021;9(1):58. Available from: https://
www.mdpi.com/2227-9032/9/1/58#cite.
[23] Singapore population. World popul review; 2021. Available from: https://worldpopulationreview.com/countries/singapore-population.
[Accessed 03 February 2021].
[24] Lee VJ, Chiew CJ, Khong WX. Interrupting transmission of COVID-19: lessons from containment efforts in Singapore. J Travel Med 2020;27
(3):039 Apr.
[25] Legido-Quigley H, Asgari N, Teo YY, Leung GM, Oshitani H, Fukuda K, et al. Are high-performing health systems resilient against the
COVID-19 epidemic? Lancet 2020;395(10227):848
50.
Chapter 19

COVID-19 and its effects on neurological

expressions
Roopkumar Sangubotla and Jongsung Kim
Department of Chemical and Biological Engineering, Gachon University, Seongnam-Si, South Korea

19.1 Introduction
The novel coronavirus disease (COVID-19) is a vigorously transmissible and cluster kind of viral infection caused by
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. This virus belongs to the family of Coronaviridae
being the largest type of virus with a genomic size between 26 and 32 kilobases (kb) of single-stranded RNA [2]. Since
March 11, 2020, when the WHO has declared the COVID-19 as the global pandemic until now there are massive publica-
tions have been reporting related to the COVID-19 [3]. Till now, seven coronaviruses were found including the COVID-19
virus that infected humans. Among these, four viruses such as OC43, 229E, HKU1, and NL63 can cause mild symptoms
like the common cold. Unfortunately, the remaining three viruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2,
are responsible for causing severe disease with high morbidity and mortality rate [4,5]. The researchers have suggested that
the mammalian species, including bats and pangolins, as the leading zoonotic hosts for COVID-19 and reasonable for
human to human transmission. Thereby it generates potentially life-threatening complications in humans [6]. Initially,
COVID-19 was responsible for the mild symptoms related to the respiratory system such as cough, fever, and shortness of
breath. Furthermore, it has shown it’s widespread to multiple organs such as the heart, pancreas, liver, kidneys, and brain
[7]. At the beginning of the COVID-19 pandemic, highly substantial mortality rates were identified in older people com-
pared to children. Most of the adults aged over 65 years have shown symptomatic features starting from a mild cough to
severe pneumonia infections. Unfortunately, children also have responded with COVID-19 in an asymptomatic fashion and
in very limited proportions [8]. In May 2020 different authors from England identified few clinical manifestations including
hyperinflammatory shocks in children. Interestingly, all of these children have already reported positive for COVID-19 [9].
They have extremely suffered from toxic shock syndrome and Kawasaki disease shock syndrome, where symptoms include
but not limited to shock, rash, fever, conjunctivitis, edema in extremities, and other gastrointestinal symptoms. Again,
authors from the New York City Department of Health also issued health alerts related to similar cases [10]. Next, in the
middle of May 2020, the Centers for Disease Control and Prevention also defined the hyperinflammatory syndrome and
represented it as the multisystem inflammatory syndrome in children by issuing the public health advisory [11,12]. In this
regard, COVID-19 is adequately contributing clinical symptoms from minor headaches, seizures, paralysis, and disabilities
in taste and smell (i.e., hypogeusia and hyposmia) to the extreme levels of neurological complications such as stroke,
encephalopathy, hemiplegia, encephalomyelitis, cerebral hemorrhages, and other neuromuscular disorders [13,14]. Focused
attention has intended to unlock the link between COVID-19 and its neurotropic behaviors [15]. A plethora of respiratory
viruses was found as the neurotropic viruses such as influenza, echovirus, the genus of enterovirus, and other extremely
pathogenic zoonotic-originated viruses including Nipah virus, Hendra virus, Henipavirus, and so on [16,17]. Among differ-
ent human coronaviruses mainly, 229E and OC43 (circulating viral strains), SARS-CoV, and Middle East respiratory syn-
drome coronaviruses have been responsible for neurological complications [18]. Severe respiratory complications and
encephalitis have been attributed to the viral entry to the respiratory centers of the brain or directly the brain itself [19].
Moriguchi et al. reported the first case regarding meningitis/encephalitis related to the COVID-19. For the first time, in this
report, COVID-19 was detected in the cerebrospinal fluid instead of nasopharyngeal fluid [20]. The autopsy studies con-
ducted in COVID-19 patients have demonstrated a variety of neurological complications along with numerous brain lesions
[21]. More importantly, severe neurological symptoms such as convulsions, ataxia, meningitis, encephalitis, acute

Pandemic Outbreaks in the 21st Century. DOI: https://doi.org/10.1016/B978-0-323-85662-1.00014-8

© 2021 Elsevier Inc. All rights reserved. 287
288 Pandemic Outbreaks in the 21st Century

disseminated encephalomyelitis (ADEM), and in some cases, Guillain-Barre syndrome and other psychotic symptoms also
have been reported [22
25]. In this chapter typical criteria such as the morphology of spike protein, possible entry routes,
and associated severe neurological complications for COVID-19 infection are discussed. Fig. 19.1 illustrates the schematic
representation of all typical criteria related to COVID-19.

19.1.1 Significance of “S” proteins in COVID-19

The structural and physiological objectives of the spike proteins (S proteins) should be examined thoroughly. Due to
the surface anchoring capacity of these glycoproteins, they play an immense and significant role in cell infection and
invasive potential in the brain. Furthermore, they are involved in the extensive contribution of COVID-19 transmission
in the brains of the fetus during pregnancy. Again, these are highly attributed to the smell and tastes related disorders
which are typical features of the COVID-19 [26]. These proteins are highly responsible for enhancing inflammatory
responses in the endothelial cells of the brain. Thereby, they displayed an excellent influence on the physiological func-
tions of the blood
brain barrier (BBB), which easily enabled viral entry [19,27].
Interestingly, morphological characteristics of S proteins and their mode of interactions with the host are highly
advantageous for the successful improvement of vaccines and antiviral medications [28]. Noteworthy, S protein is a
kind of homotrimer surrounding the virus and their monomers contain receptor binding domains (RBDs) that assist in
the binding of angiotensin-converting enzyme 2 (ACE2) receptors. Cryo-electron microscopy has been employed for
investigating the morphology of the trimeric form of S protein. This suggested that the RBDs of the S protein have
been existing in both opened and closed modes [29,30]. More importantly, S protein has been in the inactive form prior
to infection but once the viral infection has been started, cell proteases of the host cells will cleave S protein into two
subunits, namely, S1 and S2. The serine protease TMPRSS2 can be employed as a protein primer and this process is
important for stimulating the fusion domains of the membrane, immediately after the invasion of the virus into host
cells [31,32]. ACE2 is an enzyme bonded to the exterior surfaces of the epithelial cells covering the brain, lungs, kid-
neys, heart, arteries, and intestines [33,34]. Especially in the brain, endothelial, glial, and neuronal cells also capable of
expressing ACE2. In this manner, the S protein has been providing outstanding potentials through receptor binding
(ACE2) and efficient viral fusion during the viral infection to the host cells [35,36]. Hassanzadeh et al. reported that S
proteins of COVID-19 have a similar sequence identity (77%) and greater binding energy (30%) toward ACE2 recep-
tors of the host cells when compared with the SARS-CoV [30,37].

19.2 Routes of entry for COVID-19 into the brain

Human coronaviruses can be entering into the brain by means of different routes such as epithelial cells of the BBB, synap-
tic connections, blood circulation, and neuronal dissemination [13]. Mondolfi et al. first reported the direct presence of the

FIGURE 19.1 Schematic representation of the structure of spike protein, viral routes, and major neurological complications in COVID-19.
 COVID-19 and its effects on neurological expressions Chapter | 19 289

COVID-19 virus in the brain tissues of the human. Furthermore, their studies have suggested that capillary endothelial cells
of the brain and hematogenous routes are the favorable pathways for the COVID-19 into the brain. Again, their investiga-
tions were supported by the expression of the ACE receptor in the endothelium of the brain as the binding target for the S
protein of COVID-19 [38,39]. Mao et al. conducted a study on 214 COVID-19 patients, which revealed 78 patients
(36.4%) have shown neurological manifestations [40]. It is worth to not that ACE2 co-receptors have excellently enabled
the viral entry into the host cells, and few co-receptors also have been reported for COVID-19 including CD147, which is a
transmembranous glycoprotein. The transmembrane serine proteases like TMRSS2 and TMPRSS4 have been promoting
the fusion of S protein to the plasma membrane of the host cells [41,42]. More importantly, mucosae including oral and
nasal mucosae have played an important role in the dissemination of primary and secondary viral infections of COVID-19
by acting as viral reservoirs. Here, the surface area also has played a vital role in COVID-19 infections due to the viral
spreading and binging capacity of host-cell ACE2 receptors. For instance, the surface area of nasal mucosae and oral cavi-
ties are around 150
160 and 215 cm2, respectively [43
45]. On contrary, the surface area of the intestinal mucosae and
the pulmonary alveolar region is approximately between 250 and 118 6 22 m2, respectively. The surface area of oral and
nasal mucosae is less when compared with the intestinal mucosae and alveolar region. However, both nasal and oral muco-
sae are “the major entry points” for the viral infection by having the following factors. On the one hand, the anatomical
location of the nasal mucosae plays a significant role, where the olfactory nerve (first cranial nerve) is connected to the
olfactory bulb of the forebrain that contributes to the sensation of smell. On the other hand, the tongue in the oral mucosa
displays much higher expressions of ACE2 receptors [46
49]. In the brain, viral entry is mainly responsible for the occur-
rence of several neuro-related pathogenic mechanisms that account for neurological disturbances. Currently available inves-
tigations on the animal models recommend that the reason for the COVID-19 infection might be the cytopathic effects of
the virus directly. The other reasons could be the indirect influence of the cytokine-mediated neuroinflammation and other
immune cell-triggered responses on the neurons and endothelial cells of the cerebrum; this further enhances cell apoptosis,
edema, and even permeability of vasculature [50].

19.3 Neuroinflammation and immune responses in COVID-19

COVID-19 involves the mediation of several inflammatory reactions that leads to the myriad of lung infections and CNS-
related complications. In fact, patients who suffered from COVID-19 along with associated neurological disorders like altered
mental status and stroke possess an extreme threat of death compared to patients with other complications [51]. Different types
of cells such as endothelial cells, macrophages, dendritic cells, and neutrophils have been infected by COVID-19.
Interestingly, interferons (IFNs) have substantially increased as a result of immune reaction through the immediate viral attack
to the host cells [52]. The continuous immune response from IFNs is associated with the triggering of the CD4 1 helper and
CD8 1 cytotoxic T cells [53]. The immune response triggered by these cells results in the signaling of B cells, which devel-
ops the antiviral reaction by producing antibodies against COVID-19 [52]. On the other hand, natural killer cells have initiated
the immune response immediately after the entry of COVID-19 [54,55]. The first line of defense in the immune system was
initiated from the macrophages, dendritic cells, and neutrophils. Several studies have postulated the abundant availability of
infiltration of macrophages in the COVID-19 patients who suffered from bronchopneumonia [56]. Next, nucleoprotein anti-
gens of COVID-19 patients also demonstrated the macrophages consisting of ACE2 in the spleen and lymph nodes. The over-
expressions of interleukin-6 were found in these macrophages. This evidenced that the macrophages might be associated with
the massive inflammation process in COVID-19 [57]. Furthermore, excessive production of neutrophils and maximum
neutrophil-to-lymphocyte ratios has also been related to the prognosis of the COVID-19 disease [58]. All these observations
postulated that the severity and significance of inflammatory reactions during COVID-19 are highly detrimental when com-
pared to the sole response of the COVID-19 [59]. Generally, antibodies were developed after a week in the patients admitted
with the COVID-19 virus. During this time, the detection capability of COVID-19 antibodies was found to be ,40% in
patients. Hence, serological-based examinations have minimal scope for the diagnosis of COVID-19 [60]. For instance,
COVID-19-specific neutralizing antibodies have been reported in the COVID-19 patients. These are synthesized from B cells
as a consequence of viral infection and highly capable enough to resist the viral entry into the host cells. In this manner, these
antibodies encouraging virus clearance [59]. Also, some experimental studies have recommended that the possible link exists
between the active triggering of NLRP3 inflammasome during COVID-19 and the pathology of Alzheimer’s disease [61].

19.4 Limitations for clinical performances during COVID-19

Viral outbreaks like COVID-19 have many limitations in the clinical perspectives for analyzing neurological manifesta-
tions. First, clinical observations are based on the number of patients to clarify biases in terms of the accuracy of
290 Pandemic Outbreaks in the 21st Century

diagnosis. Next, mild or minimal clinical manifestations such as hypogeusia and hyposmia cannot be thoroughly inves-
tigated due to the complexity of underlying mechanisms. There are also possibilities of cross infections in patients dur-
ing COVID-19. Hence, performing the neuroimaging techniques on every COVID-19 patient was also not practical
[40]. Unfortunately, a massive delay has occurred for conducting clinical trials due to the restricted healthcare research
professionals and human subjects in clinical trials during global pandemic COVID-19. For instance, the leading network
named the National Institute of Health (NIH) StrokeNet conducts clinical trials for assessing stroke [62]. The disruption
of clinical trials has adversely restricted the progress in the therapeutic developments in neurological disorders.
Moreover, regulatory agencies were also severely struggled to cope with the losses that occurred from these conse-
quences [63].

19.5 Conclusion and future prospects

The pathology of COVID-19 is not limited to respiratory organ systems but also affected nervous systems like CNS
and peripheral nervous system. Furthermore, COVID-19 raises severe neurological complications without showing the
difference in the age barriers. They start with mild symptoms like headache, dizziness, and disturbed consciousness and
end with severe complications such as meningitis, ADEM, Guillain
Barre syndrome, and even psycho-related pro-
blems. The neurotropic potential of COVID-19 leads to the overexpressed cytokines in CNS that cause devastation of
the whole immune system [14,15,64]. Further investigations suggest the significance of viral entry, neuroinflammation,
and the typical structural complexity. They are associated with the therapeutic development of COVID-19 [13,54].
There is an urgent need to form alliances with promising researchers and clinical therapists in combating COVID-19.
To ensure better results against COVID-19 in 2021, we require proper safety guidelines and validation protocols in con-
ducting human and animal clinical trials [63,65,66].

Acknowledgments
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education (NRF2017R1D1A1B04035070) and the Ministry of Trade, Industry and Energy (MOTIE) of the Republic of
Korea (20174030201530).

Conflict of interest
The authors declare that there is no conflict of interest in the publication of this chapter.

References
[1] Liu, et al. Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.
EBioMedicine. 2020;55 102763.
[2] Su S, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 2016;24:490
502.
[3] Korsukewitz C, Reddel SW, Or AB, Wiendl H. Neurological immunotherapy in the era of COVID-19-looking for consensus in the literature.
Nat Rev Neurol 2020;16:493
505.
[4] Huang Y, Yang C, Xu X, Xu W, Liu S. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development
for COVID-19. Acta Pharmacol Sin 2020;41:1141
9.
[5] Hoek LVD. Human coronaviruses: what do they cause? Antivir Ther 2015;12:651
8.
[6] Anderson KG, et al. The proximal origin of SARS-CoV-2. Nat Med 2020;26(2020):450
2.
[7] John ALS, Rathore APS. Early insights into immune responses during COVID-19. J Immunol 2020;205:555
64.
[8] Lindan CE, et al. Neuroimaging manifestations in children with SARS-CoV-2 infection: a multinational, multicentre collaborative study.
Lancet Child Adolesc Health 2020;5:167
77.
[9] Riphagen S, Gomez X, Martinez CG, Wilkinson N, Theocharis P. Hyperinflammatory shock in children during COVID-19 pandemic. Lancet
2020;395:1607
8.
[10] New York City Health Department. 2020 health alert #13:pediatric multi-system inflammatory syndrome potentially associated with COVID-
19; 2020. Available from: https://www1.nyc.gov/assets/doh/downloads/pdf/han/alert/2020/covid-19-pediatric-multi-system-inflammatory-syn-
drome.pdf.
[11] Centers for Disease Control and Prevention. Multisystem Inflammatory Syndrome in Child (MIS-C) Associated Coronavirus Dis 2019
(COVID-19); 2020. Available from: https://emergency.cdc.gov/han/2020/han00432.asp.
[12] Chiotos K, Bassiri H, Behrens EM, Blatz AM, Chang J, Diorio C, et al. Multisystem inflammatory syndrome in children during the coronavirus
2019 pandemic: a case series. J Pediatr Infect Dis Soc 2020;9:393
8.
 COVID-19 and its effects on neurological expressions Chapter | 19 291

[13] Li YC, Bai WZ, Hashikawa T. The neuroinvasive potential of SARS-CoV2 may play a role in the respiratory failure of COVID-19 patients. J
Med Virol 2020;92:552
5.
[14] Berlit, et al. “Neurological manifestations of COVID-19”-guideline of the German society of neurology. Neurol Res Pract 2020;2:51.
[15] Baig AM, Khaleeq A, Ali U, Syeda H. Evidence of the COVID-19 virus targeting the CNS: tissue distribution, host 2 virus interaction, and pro-
posed neurotropic mechanisms. ACS Chem Neurosci 2020;11:995
8.
[16] Dawes BE, Freiberg AN. Henipavirus infection of the central nervous system. Pathog Dis 2019;77(2).
[17] Bohmwald K, Gálvez NMS, Rı́os M, Kalergis AM. Neurologic alterations due to respiratory virus infections. Front Cell Neurosci 2018;12:386.
[18] Desforges M, Coupanec AL, Dubeau P, Bourgouin A, Lajoie L, Dubé M, et al. Human coronaviruses and other respiratory viruses: underesti-
mated opportunistic pathogens of the central nervous system? Viruses. 2019;12(1):14.
[19] Rhea EM, Logsdon AF, Hansen KM, Williams LM, Reed MJ, Baumann KK, et al. The S1 protein of SARS-CoV-2 crosses the blood
brain
barrier in mice. Nat Neurosci 2020;24:368
78. Available from: https://doi.org/10.1038/s41593-020-00771-8.
[20] Moriguchi T, et al. A first case of meningitis/encephalitis associated with SARS-Coronavirus-2. Int J Infect Dis 2020;94:55
8.
[21] Solomon I, et al. Neuropathological features of COVID-19. N Eng J Med 2020;383:989
92.
[22] Gutiérrez-Ortiz, et al. Miller Fisher syndrome and polyneuritis cranialis in COVID-19. Neurology 2020;95:e601
5.
[23] Paterson R, et al. The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings. Brain 2020;143:3104
20.
[24] Poyiadji N, et al. COVID-19-associated acute hemorrhagic necrotizing encephalopathy: imaging features. Radiology 2020;296:201187.
[25] Varatharaj A, et al. Neurological and neuropsychiatric complications of COVID-19 in 153 patients: a UK-wide surveillance study. Lancet
Psychiat 2020;7:875
82.
[26] Varma P, Lybrand ZR, Antopia MC, Hsieh J. Novel targets of SARS-CoV-2 spike protein in human fetal brain development suggest early preg-
nancy vulnerability. Front Neurosci 2021;14:614680.
[27] Buzhdygan TP, et al. The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human
blood-brain barrier. Neurobiol Dis 2020;146:105131.
[28] Papageorgiou AC, Mohsin I. The SARS-CoV-2 spike glycoprotein as a drug and vaccine target: structural insights into its complexes with
ACE2 and antibodies. Cells 2020;9:2343.
[29] Walls AC. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181:281
92 e6.
[30] Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion confor-
mation. Science. 2020;367:1260
3.
[31] Hoffmann M, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell.
2020;181:271
80 e8.
[32] Bertram S, et al. TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells
in the respiratory epithelium. J Virol 2013;87:6150
60.
[33] Doobay MF, Talman LS, Obr TD, Tian X, Davisson RL, Lazartigues E. Differential expression of neuronal ACE2 in transgenic mice with over-
expression of the brain renin-angiotensin system. Am J Physiol Regul Integr Comp Physiol 2007;292:R373
81.
[34] Sun C, Chen L, Yang J, Luo C, Zhang Y, Li J, et al. SARS-CoV-2 and SARS-CoV spike-RBD structure and receptor binding comparison and
potential implications on neutralizing antibody and vaccine development. bioRxiv 2020. Available from: https://doi.org/10.1101/
2020.02.16.951723.
[35] Tortorici MA, et al. Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol 2019;26:481
9.
[36] Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science
2020;367:1444
8.
[37] Hassanzadeh K, Pena HP, Dragotto J, Buccarello L, Iorio F, Pieraccini S, et al. Considerations around the SARS-CoV-2 spike protein with par-
ticular attention to covid-19 brain infection and neurological symptoms. ACS Chem Neurosci 2020;11:2361
9.
[38] Helms, et al. Neurologic features in severe SARS-CoV-2 infection. N Engl J Med 2020;382:2268
70.
[39] Mondolfi, et al. Central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). J Med Virol
2020;92:699
702.
[40] Mao, et al. Neurological manifestations of hospitalized patients with COVID-19 in Wuhan, China: a retrospective case series study. JAMA
Neurol 2020;77:683
90.
[41] Wang K, et al. SARS-CoV-2 invades host Cell via a Nov route: CD147-spike protein. biorXiv 2020; https://www.biorxiv.org/content/biorxiv/
early/2020/03/14/2020.03.14.
[42] Zang R, et al. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol 2020;5 eabc3582.
[43] Lochhead JJ, Thorne RG. Intranasal delivery of biologics to the central nervous system. Adv Drug Deliv Rev 2012;64:614
28.
[44] Mygind N, Anggård A. Anatomy and physiology of the nose
pathophysiologic alterations in allergic rhinitis. Clin Rev Allergy
1984;2:173
88.
[45] Collins LM, Dawes C. The surface area of the adult human mouth and thickness of the salivary film covering the teeth and oral mucosa. J Dent
Res 1987;66:1300
2.
[46] Ladecola C, Anrather J, Kamel H. Effects of COVID-19 on the nervous system. Cell 2020;183:16
27 e1.
[47] Meinhardt J, et al. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19. Nat
Neurosci 2021;24:168
75.
[48] Xu H, et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 2020;12:8.
292 Pandemic Outbreaks in the 21st Century

[49] Lima M. Unraveling the possible routes of SARS-COV-2 invasion into the central nervous system. Curr Treat Options Neurol 2020;22:37.
[50] Kumar A, et al. Possible routes of SARS-CoV-2 invasion in brain: in context of neurological symptoms in COVID-19 patients. J Neurosci Res
2020;98:2376
83.
[51] Amruta N, et al. SARS-CoV-2 mediated neuroinflammation and the impact of COVID-19 in neurological disorders. Cytokine Growth Factor
Rev 2021;58:1
15. Available from: https://doi.org/10.1016/j.cytogfr.2021.02.002.
[52] Shah VK, Firmal P, Alam A, Ganguly D, Chattopadhyay S. Overview of immune response during SARS-CoV-2 infection: lessons from the
past. Front Immunol 2020;11:1949.
[53] Mahmoudi S, Rezaei M, Mansouri N, Marjani M, Mansouri D. Immunologic features in coronavirus disease 2019: functional exhaustion of t
cells and cytokine storm. J Clin Immunol 2020;40:974
6.
[54] Kandikattu HK, Venkateshaiah SU, Kumar S, Mishra A. IL-15 immunotherapy is a viable strategy for COVID-19. Cytokine Growth Factor Rev
2020;54:24
31.
[55] Masselli E, Vaccarezza M, Carubbi C, Pozzi G, Presta V, Mirandola P, et al. NK cells: a double edge sword against SARS-CoV-2. Adv Biol
Regul 2020;77:100737.
[56] Barton LM, Duval EJ, Stroberg E, Ghosh S, Mukhopadhyay S. COVID-19 autopsies, Oklahoma, USA. Am J Clin Pathol 2020;153:725
33.
[57] Park MD. Macrophages: a Trojan horse in COVID-19? Nat Rev Immunol 2020;20:351.
[58] Liu T, Gong D, Xiao J, Hu J, He G, Rong Z, et al. Cluster infections play important roles in the rapid evolution of COVID-19 transmission: a
systematic review. Int J Infect Dis 2020;99:374
80.
[59] Paces J, Strizova Z, Smrz D, Cerny J. COVID-19 and the immune system. Physiol Res 2020;69:379
88.
[60] Zhao, et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin Infect Dis 2020;71:2027
34.
[61] Ising, et al. NLRP3 inflammasome activation drives tau pathology. Nature. 2019;575:669
73.
[62] Broderick JP, et al. National institutes of health StrokeNet during the time of COVID-19 and beyond. Stroke 2020;51:2580
6.
[63] Jicha GA, Abner EL. COVID-19 and the neurological disease therapeutic pipeline. Nat Rev Neurol 2021;17:71
2.
[64] Correia AO, et al. Neurological manifestations of COVID-19 and other coronaviruses: a systematic review. Neurol Psychiatry Brain Res
2020;37:27
32.
[65] Schindler SE, et al. Maximizing safety in the conduct of Alzheimer’s disease fluid biomarker research in the era of COVID-19. J Alzheimers
Dis 2020;76:27
31.
[66] Cro S, et al. A four-step strategy for handling missing outcome data in randomised trials affected by a pandemic. BMC Med Res Methodol
2020;20:208.
Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Avian flu, 219 Chrysin, 219

A(H1N1) p09 virus, 57
58, 61
62, 65
66, 76 Avian influenza, 213 Clinical management
ABO blood group genes, 263
266 Avian influenza A virus. See Influenza A virus of Middle East respiratory syndrome
ACE2 and TMPRSS2 receptor genes, 265 (IAV) (MERS)-CoV virus, 118
119
Apolipoprotein E, 264 AXL protein, 92
93 of swine-origin influenza A (H1N1) virus,
DPP9 and FOXP4 genes, 266 Azithromycin, 18, 249 72
75
human leukocyte antigen genes, 263
264 alternative approach, 74
75
interferon-induced transmembrane protein 3- finding an ultimate cure for the disease,
encoding gene, 264 B 74
interferon-α and β receptor subunit 2, 265 Bamlanivimab, 25
26 M2 inhibitors, 73
2’-5’ oligoadenylate synthetase family, Baricitinib, 36
38 rimantadine, 73
265
266 Betacoronaviridae, 1
5 shikimic acid, 74
transmembrane protein 189-ubiquitin- Betacoronavirus, 11 Clinical manifestations of Zika virus (ZIKV),
conjugating enzyme E2 variant 1, Big data and analytics, 283 91
264
265 BioNTech/Pfizer, 129 Clio-epidemiology, 140
X-chromosomal Toll-like receptor 7 gene, Biosensors, 51 Cloud gaming, 276
264 Bird flu. See Avian influenza Clustered regularly interspaced short
ACE2 and TMPRSS2 receptor genes, 265 Black Death (1347
51), 141
142 palindromic repeats (CRISPR), 194,
Acquired immunodeficiency syndrome (AIDS), Blood-borne transmission of COVID-19, 195f, 196
124, 147 35
36 Comirnaty, 241
Acute kidney injury (AKI), in COVID-19 Bronchoalveolar lavage fluid (BALF), 197 Communicable diseases, origin of, 139
155
patients, 33
34 Bundibugyo ebolavirus (BDBV), 159 cholera, 145
146
Acute respiratory distress syndrome (ARDS), clio-epidemiology, 140
30, 63, 187 coronaviruses, 149
150
Adaptor-associated kinase 1 and cyclin G- C COVID-19, 150
associated kinase, 203 Candida auris, 13 Ebola virus (EBOV), 146
147
Adenovirus vectors, 129
134, 238 Cap snatching, 61 lessons learned from EBOV outbreaks,
Adjunctive therapy, for COVID-19, 39 Cardiovascular system, in COVID-19 patients, 147
Aerosol-generating procedures (AGPs), 32
33 future perspectives, 155
118
119 Casirivimab/Imdevimab combination, 38
39 historic pandemics, 141
Airborne transmission of COVID-19, 35 Caspase activation and recruitment domains HIV/AIDS, 147
α-mangostin, 219 (CARDs), 48
49 influenza, 143
145
Amantadine, 73 Catechin, 219 leprosy, 142
143
1-Amino-adamantane, 73 Cathepsin L, 203 lessons, 150
154
Amperometric sensors, 70 CD41 T cell, 169 neo-epidemiology, 140
Andrographis paniculata, 219 CD8 1 cells, 93 plague, 141
142
Angiotensin-converting enzyme 2 (ACE2) CD81 T-cells, 47, 49, 169 prehistoric pandemic, 141
receptors, 3
4, 188
190, 202, 246, CD26, 114 swine flu, 148
261 CD147, 288
289 worst diseases outbreaks in history,
Antiviral activity of plant-derived compounds Chagas disease, 12 140
against coronaviruses, 215t Chemical compounds virtual screening, Zika virus, 149
against Ebola and Zika viruses, 216 198
199 Computer-aided drug discovery (CADD),
Antiviral therapy, for COVID-19, 36
38 Chemokines, 46
49 198
199
Antonine plague, 123, 141 Chikungunya virus (CHIKV), 13, 125 Congenital Zika syndrome (CZS), 91, 96
102
Apolipoprotein E, 264 Chimpanzee adenovirus serotype 3
vectored hearing implications related to ZIKV
Arbidol, 18 vaccine (ChAd3-EBO-Z), 173 infection, 102
Asian avian influenza A (H5N1) virus, 144 Chloroquine, 18, 36
38, 249 ZIKV-associated congenital ocular
Asian flu, 144 Chloroquine phosphate, 214 anomalies, 101
102
Asiatic flu, 143 Cholera, 145
146 ZIKV-mediated autophagy and cell stress
Aspirin, 72 Cholera Pandemic, 123 responses, 99
101

293
294 Index

Congenital Zika syndrome (CZS) (Continued) genomic and molecular epidemiology, D

ZIKV-mediated cell cycle disruption, cell 15
17 Dengue virus, 124
death, and altered gene expression, health care workers infections due to Deoxyribonucleic acid biosensors. See Geno
97
99 SARS-CoV-2, 17
18 sensors
Contact and droplet transmission of COVID- pharmacoepidemiology of therapeutic Dexamethasone, 18
19, 34 approaches, 18 Diagnosis, of Ebola virus disease (EVD), 172
Convalescent plasma, 153 previous epidemiological situation of Digital health in COVID-19 management,
Convalescent plasma therapy (CPT), 153
154 major infectious diseases, 12
13 283
284
Coronanomics, 152
153 vaccinations against COVID-19, 18
19 levels of information used to define digital
Coronaviridae, taxonomical location of, 12f future prospects, 290 health, 282f
Coronavirus disease 2019. See COVID-19 history and epidemiology, 186
187 preparedness and leveraging (case of
Coronaviruses (CoVs), 1
3, 36, 127, 149
150 influenza and, 145t Singapore), 284
ABO blood group genes, 263
266 largest number of COVID-19 cases, 282t Singapore government in containing the
ACE2 and TMPRSS2 receptor genes, 265 limitations for clinical performances during, virus, 284
285
Apolipoprotein E, 264 289
290 Dipeptidyl peptidase 4 (DPP-4), 113
114
DPP9 and FOXP4 genes, 266 lockdown, and e-sports, 273 Direct RNA sequencing (dRNAseq),
human leukocyte antigen genes, 263
264 neuroinflammation and immune responses 194
195
interferon-induced transmembrane protein in, 289 Disseminated intravascular coagulation, 171
3-encoding gene, 264 outbreaks across the world, 2f DNA-stabilized silver nanoclusters (DNA-
interferon-α and β receptor subunit 2, 265 pathogenesis, 187
188 AgNCS), 227
2’-5’ oligoadenylate synthetase family, repurposing existing vaccines and antibiotics DNA vaccine, 240
265
266 to control, 245
246 development and mechanism of, 240f
transmembrane protein 189-ubiquitin- azithromycin, 249 Double immunodiffusion test (DI), 67
conjugating enzyme E2 variant 1, chloroquine and hydroxychloroquine, 249 Double-membrane vesicles (DMVs), 29
264
265 corticosteroid, 249
250 DPP4, 114
115
X-chromosomal Toll-like receptor 7 gene, COVID-19 vaccination programs and DPP9 and FOXP4 genes, 266
264 repurposing of existing vaccines, 250 Droplet transmission of COVID-19, 34
antiviral activity of plant-derived compounds drug repurposing, 247
against, 215t evolving strains of coronavirus genome
classification of, 186f and ineffectiveness of the vaccines, E
diversity, 261
262 251
252 Ebola outbreaks, lessons learned from, 147
future perspectives, 267 genome of coronavirus, 246
247 Ebola pathogenesis, molecular mechanisms of,
genetics of coronavirus infection, 262 interferons (pegylated Ifnα-2a and 162
167
genome of, 246
247, 260
261 pegylated Ifnα-2b), 248
249 degradation of IRF3 and IRF7 by VP35-
history of, 257
258, 259t limitations to drug repurposing approach, mediated SUMOylation, 165
166
impact of genetics on COVID-19 treatment, 250 dysregulation of innate immune response
266
267 lopinavir-ritonavir combination, 248 during Ebola infection, 162
naming of, 258
260 remdesivir, 248 subversion of IFN-induced signaling by
new SARS-CoV-2 variant, 266 SARS CoV-2 EBOV, 163
165
potential genes for pathogenesis of COVID- upsurge of, 246 VP24, 167
19, 262
263 teicoplanin, 249 Ebola virus (EBOV), 146
147, 159, 167
chromosome 3P21.31 gene locus, 263 therapeutic options for COVID-19 life cycle, 163f
replication cycle of, 189f management, 247
250 pathogenesis, 168f
taxonomy of, 258 therapeutic targets, 247 structure and its genomic organization, 161f
Corticosteroids, 38
39, 249
250 tocilizumab, 249 transmission, 160f
COVID-19, 1
3, 5, 150, 186
188, 214, 220, routes of entry for COVID-19 into the brain, Ebola virus disease (EVD), 125
126, 159,
287
288. See also Severe acute 288
289 214, 219
respiratory syndrome coronavirus 2 SARS-CoV-2 association with other adaptive immune response during EBOV
(SARS-CoV-2) comorbidities, 188 infection, 167
169
cytokine storm and, 198 “S” proteins in COVID-19, 288 dysregulation of adaptive immune
diagnosis, with micro- and nanosystems, therapeutic options for COVID-19 response, 169
225
226 management, 247
250 diagnosis, 172
limitations and future prospectus, transmission and course of infection, 187 epidemiology, 159
161
230
231 2009 swine flu and, 148t ecology and spreading of Ebola virus,
micro- and nanosystems for, 226
230 vaccines for, 237t 160
161
SARS CoV-19 structure, 226 vector-based vaccines in, 130
134 life cycle, 162
on economic growth of e-sports or gaming COVID-19 vaccination programs and phase I
III clinical trials, EVD candidate
industry, 274
275 repurposing of existing vaccines, 250 vaccines in, 173t
epidemiology, in Latin America, 11 COVID-19 vaccine race, frontrunners in, 129 postsymptomatic complications of, 171f
COVID-19 arrival at Latin America, Cranial neural crest cells (CNCCs), 94
96 prevention and control of, 175
13
15 Cyprian plague, 141 therapeutics, 173
174, 174t
daily new confirmed COVID-19 cases per Cytokines, 46
48 vaccines, 172
173
million people, 13f, 14f Cytokine storm and COVID-19, 198 vascular permeability and coagulation
emerging situations, 17 Cytokine storm syndrome (CSS), 188, 198 defects, 170
172
 Index 295

Ebola-postinfection manifestation, H Geno sensors, 52

171
172 H5N1 viruses, 44
45, 47
48 immunosensors, 51
virus structure, 161
162 H7N9 virus, 44
46 whole-cell biosensors, 52
Eicosanoids, 49 H9N2 virus, 45, 48 diagnosis, 50
Electrical biosensors, 51 Hansen’s disease, 142
143 epidemiology, 44
45
ELISA, 67
68 HA protein, 59
60 exposure risk factors to humans, 45
46
Emerging situations during COVID-19 Health care workers infections due to SARS- H5N1 infection, clinical findings in, 50
pandemic in Latin America, 17 CoV-2, 17
18 innate immunity and adaptive immunity,
End of pandemics, 154 Helicase (nsp13), 202 48
50
Endoplasmic reticulum
Golgi intermediate Hemagglutination inhibition assay, 67 major lineages, 43
44
compartment (ERGIC), 191 Highly active antiretroviral therapy (HAART), pathophysiology, 46
48
Enfuvirtide, 118 124 chemokines and cytokines, role of, 46
48
Envelope (E) protein, 193 Highly pathogenic avian influenza (HPAI) viral replication in host, 46
48
Enzymatic biosensors, 51
52 viruses, 44 Influenza viruses, 76
EOTAXIN, 47 Hispidulin, 219 antiviral activity of plant-derived compounds
Epidemic and pandemic, legacy of, 139 Historic pandemics, 141 against, 217t
Escherichia coli, 74 History of notable world disease outbreaks, Inoculated vaccinia, 153
E-sports 151t Inoculation, 153
advances in e-sports during the COVID-19 Hofbauer cells (HCs), 90
91 Interferon-alpha, 126
127
pandemic, 276 Hong Kong strain of influenza virus A2 H3N2, Interferon-induced transmembrane protein 3-
COVID-19 effect on economic growth of, 144 encoding gene, 264
274
275 Host-based druggable targets, 202
204 Interferons (pegylated Ifnα-2a and pegylated
COVID-19 pandemic, lockdown, and e- adaptor-associated kinase 1 and cyclin G- Ifnα-2b), 248
249
sports, 273 associated kinase, 203 Interferon-α and β receptor subunit 2, 265
research in different academic disciplines, angiotensin I converting enzyme 2 receptor Interferon β promoter stimulator protein-1
273
274 (ACE2), 202 (IPS-1), 163
165
role in health and well-being during the Cathepsin L, 203 Internet of things (IoT), 283
pandemic, 277
278 furin, 202 Intra-uterine transmission of COVID-19,
tool for social connectedness and immune response, 204 35
36
psychological healing in COVID-19 phosphatidylinositol 3-phosphate 5-kinase IP-10, 47
pandemic, 276
277 (PIKfyve), 204 Ivermectin, 18, 36
38
transmembrane serine protease 2, 202
two-pore channel (TPC2), 204
F Human coronavirus (HCoV). J
Favipiravir, 36
38, 214 See Coronaviruses (CoVs) Janus Kinase (JAK) inhibitor, 36
38
Fecal
oral transmission of COVID-19, 35 Human Coronavirus 229E (HCoV-229E), 11 Justinian Plague, 141
Flavivirus, 87 Human Coronavirus 229E (HCoV-OC43), 11
Fomite transmission of COVID-19, 35 Human Coronavirus HKU1 (HCoV-HKU1), 11
FOXP4 gene, 266 Human Coronavirus NL63 (HCoV-NL63), 11 K
Furin, 202 Human immunodeficiency virus (HIV), 124, Kaempferol, 219
147
Human leukocyte antigen genes, 263
264
G Human papillomavirus (HPV), 229 L
Gam-COVID-Vac, 238 Hydroxychloroquine, 18, 36
38, 214, 249 Leprosy, 142
143
Gaming industry, COVID-19 on economic Hypercytokinemia, 198 Letovirinae, 11
growth of, 274
275 Life cycle, of Ebola virus disease (EVD), 162
γ d T-cells, 47 Lockdown, COVID-19 pandemic and, 5
6,
Garcinone C, 219 I 273
Gastrointestinal system, in COVID-19 patients, IFN-stimulated genes (ISGs), 46
47 Lopinavir, 18, 214
31 I-Kappa-B kinase epsilon (IKKe), 163
165 Lopinavir/ritonavir, 36
38, 248
Genetic drift, 76 Immune-based therapy, for COVID-19, 38
39 Low pathogenic avian influenza (LPAI)
Genetic shift, 76 Immune response, 204 viruses, 44
Genome of SARS-CoV-2, 191f Immunoevasion mechanisms, 163 Luteolin, 219
Genomic and molecular epidemiology of Immunofluorescence (IF), 67 Lymphopenia, 169
COVID-19 in Latin America, Immunosensors, 51
15
17 Impedimetric biosensor, 69
70
Geno sensors, 52 Inflammasomes, 46
47 M
Ginkgo biloba, 74 Influenza, 43, 143
145 M2 inhibitors, 73
Global lockdowns, 5
6 Influenza (1918
19) pandemic, 124 Magnetic biosensors, 70
Glycoprotein (GP), 161
162 Influenza A virus (IAV), 43, 57, 75, 77 Magnets-plasmonic nanoparticles (MPNPs),
GOARN mission, 5 detection of, 50
52 230
Graphene-field-effect transistors (Gr-FET), biosensors, 51 Membrane protein, 192
230 electrical biosensors, 51 MERS-CoV. See Middle East respiratory
Guillain
Barre syndrome (GBS), 13, 91 enzymatic biosensors, 51
52 syndrome coronavirus (MERS-CoV)
296 Index

M-Health, 283
284 COVID-19, 220 of Ebola virus disease (EVD), 175

Microcephaly, 87
88 Ebola virus disease, 219 of Middle East respiratory syndrome
Middle East respiratory syndrome coronavirus in silico screening of plant-derived antiviral (MERS)-CoV virus, 118
119
(MERS-CoV), 1
3, 11, 25
26, compounds against pandemics of 21st of swine-origin influenza A (H1N1) virus,
111
112, 126
127, 149
150, 186, century, 218 71
72
213, 218
219, 257 SARS and MERS, 218
219 Pro-inflammatory cytokines, 49
clinical management approaches, 118
119 swine flu and avian flu, 219 Pulmonary system, in COVID-19 patients,
prevention and control of MERS, Zika fever, 219
220 30
31
118
119 N polypeptide, 192
diagnostics, 117 N protein, 30
ecology and spreading of, 113, 114f Nuclear export protein (NEP), 59 Q
epidemiology, 112
113 Nucleic acid vaccine, 240
241 Quarantine, 142, 150
future prospective, 119 DNA vaccine, 240 Quercetin, 219
immune responses to MERS infection, 116 RNA vaccine, 241
initial and postinfection manifestations, Nucleocapsid, 192
116
117 Nucleocapsid protein, 201 R
molecular mechanisms of pathogenesis, Nucleotide sequencing and phylogenetic Rapid antigen tests, 67
115
116 analysis, 68
69 Rapid influenza detection tests, 68
outbreak response priorities, 117 Real-time polymerase chain reaction (RT-
structure and life cycle, 113
115, 115f PCR), 69, 117
treatment strategies, 118 O Reassortment, 76
vaccines, 117
118 OAS1 gene, 265
266 Receptor-binding domains (RBDs), 114
115,
Ministry of Family Welfare of the Government 2’-5’ Oligoadenylate synthetase family, 261
of India, 6
7 265
266 Recombinant adenovirus type 5 (rAd5) vector,
Moderna, 129 “Omics” technologies, 195f 130
Molecular epidemiology, defined, 15 Optical biosensors, 69 Recombinant adenovirus type 26 (rAd26)
Monocytes, 49 Orthocoronavirinae, 11 vector, 130
M polypeptide, 192 Oseltamivir, 72
73, 144
145, 213 Remdesivir, 18, 36
38, 214, 248
Müller cells, 101
102 Renal system, in COVID-19 patients,
Mycobacterium indicus pranii (MIP) vaccine, 33
34
142
143 P Replicase protein expression, 190
Mycobacterium leprae, 142
143 Pathogen-associated molecular patterns Respiratory illnesses, influenza pandemics
Mycobacterium tuberculosis (MTB), 229 (PAMPs), 46
47 with, 78
Myocardial injury, 32
33 Pathogen recognition receptors (PRRs), 48 Reston ebolavirus (RESTV), 146
147, 159
Pathogen transmission, prediction of, 153 Reverse transcription-polymerase chain
Pattern recognition receptors (PRRs), 46
47 reaction (RT-PCR), 172, 225, 230
231
N Peripheral blood mononuclear cells (PBMC), Ribavirin, 18, 126
127
Naming new viruses, 154 197 Ribonucleoprotein (RNP) complex, 59
NA protein, 59
60 Personal protective equipment (PPE), 35, 150 Rig-like receptors (RLRs), 163
165
Naringenin, 219 Phosphatidylinositol 3-phosphate 5-kinase Rimantadine, 73
Neo-epidemiology, 140 (PIKfyve), 204 RNA-dependent RNA polymerase, 202
Nervous system, in COVID-19 patients, 31
32 Piezoelectric biosensors, 70 RNA vaccine, 241
Neural progenitor cells (NPCs), 87
88, 92, Pisoniviricetes, 11 Rubraxanthone, 219
94
96 Pisuviricota, 11 Russian flu, 143
Neuraminidase inhibitors (NAIs), 61, 72 Plague, 141
142
Neuroinflammation and immune responses in Plague of Justinian, 123
COVID-19, 289 Plant-derived compounds S
Neutrophils, 49 antiviral activity against Ebola and Zika Sarbecovirus, 11
Nidovirales, 3
4 viruses, 216 SARS-CoV. See Severe acute respiratory
NK T-cells, 47 as antiviral drugs for influenza viruses, 216 syndrome coronavirus (SARS-CoV)
NL63 (α-CoV), 257 as therapeutics for coronaviruses, 214
215 Sci-B-Vac, 239
Non-polymerase chain reaction-based RNA Polymerase chain reaction-based detection Seasonal influenzas, 63
detection methods, 68 methods, 68 Secretory glycoprotein (sGP), 161
162
Nonstructural protein 2 (NS2). See Nuclear Polyproteins, 27 Sentrin-specific proteases (SENPs),
export protein (NEP) Porcine epidemic diarrhea virus (PEDV), 230 165
166
Nonstructural proteins, 192, 201
202 Porcine transmissible gastroenteritis virus Serology, 172
3CLpro and PLpro, 201 (TGEV), 230 Severe acute respiratory syndrome (SARS),
Helicase (nsp13), 202 Potentiometric biosensors, 70 125, 149, 213, 218
219
RNA-dependent RNA polymerase, 202 Poxviruses as vaccine vector, 129 Severe acute respiratory syndrome coronavirus
Nonvector transmission of Zika virus (ZIKV), Prehistoric pandemic, 141 (SARS-CoV), 1
3, 25
26
90
91 Prenatal transmission of Zika virus (ZIKV), future directions, 6
7
Novavax, 118 90
91 global experiences, 5
6
Novel drugs, computational approaches in Prevention mechanism of action, 3
5
identifying, 216
220 of COVID-19, 36
39 structure, 3, 3f, 226, 226f
 Index 297

Severe acute respiratory syndrome coronavirus transmission, 34
36 clinical management, 72
75
2 (SARS-CoV-2), 1
3, 11, 25, airborne transmission, 35 alternative approach, 74
75
185
186, 236. See also COVID-19 blood-borne transmission, 35
36 finding an ultimate cure for the disease,
cardiovascular system, 32
33 contact and droplet transmission, 34 74
chemical compounds virtual screening, fecal
oral transmission, 35 M2 inhibitors, 73
198
199 fomite transmission, 35 rimantadine, 73
COVID-19 disease, 186
188 intra-uterine transmission, 35
36 shikimic acid, 74
history and epidemiology, 186
187 sexual transmission, 35
36 shikimic acid, importance and uses of, 74
pathogenesis, 187
188 upsurge of, 246 shikimic acid production, limitations of,
SARS-CoV-2 association with other vaccine candidates specific to, 131t 74
comorbidities, 188 viral life cycle as drug targets, 200
201 diagnostics, 66
69
transmission and course of infection, 187 viral transcriptome of, 194
195 H1N1 swine flu (pdm09), diagnostics of,
cytokine storm and COVID-19, 198 virus-based targets, 201
202 66
68
evolution and genomic analyses, 26
27 nonstructural proteins, 201
202 non-polymerase chain reaction-based
functional aspects of SARS-CoV-2 structural proteins, 201 RNA detection methods, 68
nonstructural proteins (Nsp’s), 28t virus
host dependency factors, nucleotide sequencing and phylogenetic
gastrointestinal system, 31 identification of, 196
197 analysis, 68
69
health care workers infections due to, 17
18 virus
host proteome and interactome, rapid influenza detection tests, 68
host-based druggable targets, 202
204 identification of, 196 surveillance methods, 68
adaptor-associated kinase 1 and cyclin G- Sexual transmission of COVID-19, 35
36 epidemiology, 61
63
associated kinase, 203 S gene of SARS-CoV-2, 27 incidence and mortality, 62
63
angiotensin I converting enzyme 2 S glycoprotein, 114 “2009 swine flu”/“A(H1N1) p09”, origin
receptor (ACE2), 202 Shikimate-3-phosphate (S3P), 74 of, 61
62
Cathepsin L, 203 Shikimic acid, 74 fusion and entry, 60
furin, 202 importance and uses of, 74 gene functions, 59
immune response, 204 limitations of production of, 74 host cell nucleus, trafficking to, 60
phosphatidylinositol 3-phosphate 5-kinase Sick, blaming people who get, 154 packaging of RNA and assembly of virus, 61
(PIKfyve), 204 Singapore government in containing the virus, prevention and control, 71
72
transmembrane serine protease 2, 202 284
285 vaccination, 71
72
two-pore channel (TPC2), 204 Single radial-immunodiffusion test, 67 replication and transcription, 60
61
host factors identification in COVID-19 Smallpox, 153 threats and challenges, 75
79
patient samples, 197
198 Small secretory glycoprotein (ssGP), 161
162 viral attachment, 59
60
and its structural proteins, 201f Small ubiquitin-related modifiers (SUMOs), virion structure, 59
molecular biology of, 27
30 165
166 virus budding and release, 61
mutation rate of, 26 Social distancing, 150 vmRNAs, host-cell translation of, 61
nervous system, 31
32 Spanish flu, 57, 143
origin of, 25
26 Specific age group, pandemics to, 154
prevention and treatment, 36
39 Spike glycoprotein, 113
114 T
adjunctive therapy, 39 Spike protein, 188
190, 192, 201, 288 Tai Forest ebolavirus (TAFV), 159
antiviral therapy, 36
38 Sporadic autopsy, 32
33 Tamiflu drug, 74
immune-based therapy, 38
39 Structural proteins, 192, 201 Tank binding kinase 1 (TBK1), 163
165
vaccines, 36, 37t nucleocapsid protein, 201 Technology and innovation, 153
pulmonary system, 30
31 spike protein, 201 Teicoplanin, 249
receptor-binding motif (RBD motif), 25
26 Subgenomic flavivirus RNAs (sfRNAs), 89 Telemedicine, 283
renal system, 33
34 Sudan ebolavirus (SUDV), 146, 159 Terminalia arjuna, 219
220
replication cycle, 188
193 SUMOylation, 165
166 Th17 lymphocytes, 47
assembly and release, 191
192 Survivors’ blood, 153
154 Therapeutics, of Ebola virus disease (EVD),
attachment and entry, 188
190 Swine flu, 148, 213, 219 173
174
elements of SARS-CoV-2 genome, 192 Swine-origin influenza A (H1N1) virus, 57
58 3CLpro and PLpro, 201
envelope protein, 193 biosensors, 69
71 TLRs, 30
31
membrane protein, 192 amperometric sensors, 70 TMPRSS2 gene, 265
nonstructural proteins, 192 impedimetric biosensor, 69
70 Tocilizumab, 214, 249
nucleocapsid, 192 magnetic biosensors, 70 Toll-like receptor 7 (TLR7) gene, 264
replicase protein expression, 190 optical biosensors, 69 Toll-like receptors (TLRs), 48
replication and transcription, 191 piezoelectric biosensors, 70 Transcription regulatory sequences (TRS), 191
spike protein, 192 potentiometric biosensors, 70 Transmembrane protein 189-ubiquitin-
structural proteins, 192 case fatality rate (CFR) of “2009 swine flu”, conjugating enzyme E2 variant 1,
structural proteins, translation of, 191 63 264
265
requirement of host factors for life cycle of, clinical features, 64
66 Transmembrane Protein 41B (TMEM41B), 197
194 complications, 66 Transmembrane serine protease 2 (TMPRSS2),
S gene of, 27 infectious versus incubation period, 200, 202
structure of SARS-CoV-2 virion, 28f 64
65 Transmission
tissue tropism and molecular pathogenesis symptoms, 65
66 of COVID-19, 34
36, 187
of, 30 transmission, 64 airborne transmission, 35
298 Index

Transmission (Continued) severe acute respiratory syndrome (SARS), Virus culture in mammalian cells, 67
blood-borne transmission, 35
36 125 Virus
host dependency factors, identification
contact and droplet transmission, 34 for swine-origin influenza A (H1N1) virus, of, 196
197
fecal
oral transmission, 35 71
72 Virus
host proteome and interactome,
fomite transmission, 35 vector-based vaccines in COVID-19 identification of, 196
intra-uterine transmission, 35
36 pandemic, 130
134 Virus-like proteins (VLPs), 191
192
sexual transmission, 35
36 Zika virus (ZIKV), 125 Vitexin, 219
of swine-origin influenza A (H1N1) virus, Vaccine VP24, 167
64 for COVID-19, 36, 37t, 237t VP30, 162
of Zika virus (ZIKV), 89
91, 90f frontrunners, 129 VP35, 162
165, 165f, 166f
nonvector transmission, 90
91 for Ebola virus disease (EVD), 172
173 VP40, 162
prenatal transmission, 90
91 ideal characteristics of, 128
vector-borne transmission, 89
90 major global infections prevented by, 128t
Treatment for Middle East respiratory syndrome W
of COVID-19, 36
39 (MERS)-CoV virus, 117
118 Whole-cell biosensors, 52
adjunctive therapy, 39 poxviruses as vaccine vector, 129
antiviral therapy, 36
38 strategies for the development of, 128
azithromycin, 249 viral vector-based, 129 X
corticosteroid, 250 Vaccine candidates specific to SARS-CoV-2, X-chromosomal Toll-like receptor 7 gene, 264
genetics on, 266
267 131t
immune-based therapy, 38
39 Vaccine technologies, 235
teicoplanin, 250 challenges in the success of vaccination, 241 Y
of Middle East respiratory syndrome future perspectives, 241
242 Yersinia pestis, 123
(MERS)-CoV virus, 118 nucleic acid vaccine, 240
241
Troponin, 33 DNA vaccine, 240
Two-pore channel (TPC2), 204 RNA vaccine, 241 Z
“2009 swine flu”/“A(H1N1) p09”, origin of, pandemic outbreaks and challenges in Zaire ebolavirus, 146, 159
61
62 vaccine development, 235
237 Zaire EBOV (ZEBOV), 160, 171
172
Tyrosine Kinase 2 (TYK2), 265 viral-like particles (VLP), 239 Zanamivir, 72
73, 144
145, 213
viral vector vaccines, 238
239 Zika fever, 91, 214, 219
220
Vector-based vaccines in COVID-19 pandemic, Zika virus (ZIKV), 13, 87
88, 125, 149.
U 130
134 See also Congenital Zika syndrome
Ubiquitin-conjugating E2 enzyme variant Vector-borne transmission of Zika virus, (CZS)
proteins, 264
265 89
90 cellular targets and entry of, 92
93
Vero cells, 3
4 clinical manifestations, 91
Vibrio cholerae, 123
124 genome organization and polyprotein
V Viral life cycle as drug targets, 200
201 processing, 88f
Vaccination, 123
124 Viral-like particles (VLP), 239 induction and suppression of innate immune
adenovirus vectors, 129 development and mechanism of, 239f mechanisms mediated by, 93
96
against COVID-19 in Latin America, 18
19 Viral transcriptome of SARS-CoV-2, 194
195 -mediated mechanisms to induce congenital
chikungunya virus (CHIKV), 125 Viral vector-based vaccines, 129 Zika syndrome, 96
102
COVID-19 vaccine race, frontrunners in, 129 Viral vector vaccines, 238
239 molecular biology of, 88
89
dengue virus, 124 development and mechanism of, 238f genome organization, 88
89
Ebola virus (EBOV), 125
126 Virtual world, 277 replication, 89
evolution of vaccine technologies, 127
128 Virus-based targets, 201
202 prenatal transmission, 90
91
human coronavirus, 127 nonstructural proteins, 201
202 transmission, 89
91
human immunodeficiency virus/acquired 3CLpro and PLpro, 201 nonvector transmission, 90
91
immunodeficiency syndrome, 124 Helicase (nsp13), 202 vector-borne transmission, 89
90
ideal characteristics of a vaccine, 128 RNA-dependent RNA polymerase, 202 transmission cycle of, 90f
Middle East respiratory syndrome (MERS) structural proteins, 201 Zoonosis, 153
coronavirus, 126
127 nucleocapsid protein, 201 Zoonotic disease, 44
45, 261
262
spike protein, 201