Review

A Comprehensive Review of the Immunological

Response against Foot-and-Mouth Disease Virus
Infection and Its Evasion Mechanisms
Ibett Rodríguez-Habibe 1,2,† , Carmen Celis-Giraldo 1,† , Manuel Elkin Patarroyo 3,4 ,
Catalina Avendaño 1, * and Manuel Alfonso Patarroyo 3,5, *
1 Animal Science Faculty, Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A),
Bogotá 111166, Colombia; ibetrodriguez@udca.edu.co (I.R.-H.); ccelis@udca.edu.co (C.C.-G.)
2 Masters Programme in Veterinary Science, Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A),
Bogotá 111166, Colombia
3 Fundación Instituto de Inmunología de Colombia (FIDIC), Bogotá 111321, Colombia; mepatarr@gmail.com
4 Faculty of Medicine, Universidad Nacional de Colombia, Bogotá 111321, Colombia
5 School of Medicine and Health Sciences, Universidad del Rosario, Bogotá 112111, Colombia
* Correspondence: cavendano@udca.edu.co (C.A.); mapatarr.fidic@gmail.com (M.A.P.);
Tel.: +57-6684-700 (C.A.); +57-1324-4672 (M.A.P.)
† Equally contributed as first author.




Received: 7 October 2020; Accepted: 27 November 2020; Published: 14 December 2020


Abstract: Foot-and-mouth disease (FMD) is a highly contagious viral disease, which has been reported
for over 100 years, and against which the struggle has lasted for the same amount of time. It affects
individuals from the order Artiodactyla, such as cattle, swine, sheep, wild animals from this order,
and a few non-cloven hoofed species, such as mice and elephants. FMD causes large-scale economic
losses for agricultural production systems; morbidity is almost 100% in an affected population,
accompanied by a high mortality rate in young animals due to myocarditis or an inability to suckle if
a mother is ill. The aetiological agent is an Aphthovirus from the family Picornaviridae, having seven
serotypes: A, O, C, SAT1, SAT2, SAT3, and Asia 1. Serotype variability means that an immune
response is serospecific and vaccines are thus designed to protect against each serotype independently.
A host’s adaptive immune response is key in defence against pathogens; however, this virus uses
successful strategies (along with most microorganisms) enabling it to evade a host’s immune system
to rapidly and efficiently establish itself within such host, and thus remain there. This review has
been aimed at an in-depth analysis of the immune response in cattle and swine regarding FMD virus,
the possible evasion mechanisms used by the virus and describing some immunological differences
regarding these species. Such aspects can provide pertinent knowledge for developing new FMD
control and prevention strategies.

Keywords: foot-and-mouth-disease virus; immune response; immune evasion mechanism

1. Introduction
The immunological system protects organisms against colonization by pathogenic agents and
promotes tissue repair following injury. It uses various mechanisms, such as successive defence layers
or barriers, each being more specific and specialized than the previous one. Such mechanisms can
be grouped into innate immunity and acquired immunity. Concerning innate immunity, the first
barrier consists of physical or anatomical barriers like the skin and the mucous membranes covering the
respiratory, digestive and reproductive tracts; the second barrier contains phagocytic and non-phagocytic
cells which respond rapidly but non-specifically when pathogens have managed to surmount the first

Vaccines 2020, 8, 764; doi:10.3390/vaccines8040764 www.mdpi.com/journal/vaccines

Vaccines 2020, 8, 764 2 of 19

barriers. B- and T-lymphocyte action forms part of the adaptive immune system providing a more
specific though slower response. It is worth stressing that innate and adaptive immune systems depend
on each other [1–4].
Many pathogens’ survival depends on them establishing themselves in a particular host, either in
body cavities (i.e., gastrointestinal parasites, such as helminths) or within cells (i.e., some protozoa,
viruses, and bacteria). Pathogens and their hosts have evolved jointly; whilst pathogens can accumulate
several mutations per generation (that usually involves few hours), the hosts can counteract the attack
through their ability to generate a huge number of T- and B-cell clones with different antigen
specificities [2,5]. In spite of this, viral pathogens particularly mutate rapidly and evolve to counteract
mammals’ non-specific innate responses and their highly specific adaptive immune response [6].
Foot-and-mouth disease (FMD) is a highly infectious viral disease affecting animals from the
order Artiodactyla (even-toed hoofed animals/ungulates) such as cattle, swine, sheep, and goats
and wild hoofed animals, such as deer, antelopes, buffalo, bison, reindeer, and giraffes. Old World
camelids, such as camels and dromedaries, and ones from the New World like llamas, alpacas and
vicuña have been experimentally infected but there are no reports of them becoming infected in their
native environments. Species which do not belong to this order have been shown to be susceptible
to the virus, i.e., hedgehogs, armadillos, chiguiros, kangaroos, rats, and mice; captive Asian and
African elephants have been infected but there have been no reports of them becoming diseased in the
wild [7–9]. FMD is endemic in South America, Africa, Asia, and parts of Europe, causing largescale
economic losses; ulcers form in the animals’ mouths and on their hooves and teats and involves loss of
body condition. This leads to exorbitant spending on veterinary drugs, along with foot-and-mouth
disease-free countries rejecting the import of livestock and livestock-derived products from endemic
countries and/or those affected by FMD [10,11]. FMD is on the World Organization for Animal Health’s
Terrestrial Animal Health Code list and it must be declared. It is the first disease regarding which this
organization has issued an official list of countries or zones certified as being free of the disease with or
without vaccination.
FMD is produced by a virus belonging to the family Picornaviridae, genus Aphthovirus; it is
a positive-sense, single-strand RNA virus. It has ~8300 nucleotides, included in a icosahedral
protein capsid, formed by protomers integrated by four structurally different proteins: VP1, VP2,
VP3, and VP4 [12,13]. Seven immunologically different types can be differentiated by serological
tests; they are known as A, O, C, South African-Type 1 (SAT-1), South African-Type 2 (SAT-2),
South African-Type 3 (SAT-3), and Asia 1 [10,11].
Serospecific and cyclic mass vaccination is used by most nations when trying to control and
eradicate the virus [10,14,15]. However, the virus’ enormous genetic variability and antigenic diversity
has led to its population structure being defined as a quasi-species [16,17], thereby hampering its
control by vaccination [16,18].
A better understanding of virus–host interaction is essential for developing strategies (i.e.,
antiviral approaches and novel vaccines) able to rapidly control the disease and reduce the negative
impact on the economy, bearing in mind the evasion mechanisms used by the virus for escaping the
immune response [19], this being the purpose of this review.

2. Foot-and-Mouth Disease Virus Pathogenesis

The virus’ pathogenic mechanism can be analyzed as a chain of interactions between virus
and host. The specific steps involved may vary depending on the type of virus and host; however,
to explain infection developing with the virus, a general model can be described which is applicable
to most cases:
1. Entry to a susceptible host;
2. Replication for increasing viral load;
3. Dissemination from the entry site to the tissues and/or target organs, developing the infection,
and damage to cells and organs;
Vaccines 2020, 8, 764 3 of 19

4. Elimination, contamination, and dissemination to the environment;

5. Persistence in the environment; and
6. Transmission to new hosts and beginning a new cycle [5].

2.1. Foot-and-Mouth Disease Virus Entry to a Host

Foot-and-mouth disease virus (FMDV) usually enters an animal by aerial route, the respiratory
tract being the main infection route. The virus enters as aerosols produced by animals coughing or
sneezing, infected swine being the most important source for producing them, followed by cattle and
sheep; high infectivity indices have been found in aerosols from milk and feces [20].
FMDV can also enter via the oral cavity by mechanically transmitting infectious nasal secretions
(nose–nose contact) or in saliva (by licking or the shared use of contaminated utensils: watering and
feeding devices, etc.). Fortuitous entry is enabled by injury to the skin in skin folds (such as, interdigital
areas) since intact skin has active mechanisms related to its acid pH, the presence of antibacterial fatty
acids and normal flora, making the skin able to resist many pathogens’ aggression [4,21,22].
Few viral particles are usually needed for infection to begin in most species, except in swine since
this specie is more resistant to infection by respiratory route than cattle or sheep; nevertheless, it is
much more sensitive to infection by oral route. Cattle can thus become infected by 5 to 10 viral particles,
sheep by 15 to 20 particles whereas a much greater amount is required to infect swine by respiratory
route [12]. Experimental studies, like that by Alexandersen et al. [22] required ten 50% tissue culture
infective doses (TCID50 ) for infecting ruminants compared to 103 TCID50 required for infecting swine.
Likewise, viral particle dissemination volume was greater in swine which can disseminate 108 viral
particles, i.e., equivalent to 3000 times more particles than that for cattle or sheep during the same
period of time [12,23–25].

2.2. Foot-and-Mouth Disease Virus Entry to Cells

Once FMDV has entered a host it needs to establish and/or disseminate itself; viruses must bind
to and infect cells on some body surfaces since they are obligate intracellular microorganisms [26].
FMDV has tropism for epithelial cells in which it replicates rapidly from the same entry point, giving
rise to vesicles called primary canker sores which usually pass unnoticed [27]. FMDV particles enter
cells through receptor-mediated endocytosis; this begins with the interaction between the virus’s
proteins binding to cell surface receptors [28]. Viral particles can use different receptors during viral
pathogenesis’ different stages. FMDV is the only virus from the family Picornaviridae which can
use four integrins (αvβ1, αvβ3, αvβ6, and αvβ8) for mediating infection; however, each receptor’s
function during cell infection by FMDV has still not been elucidated [29]. It has been documented that
integrins a5β and avβ5 are involved in cell susceptibility to FMDV infection [28], thereby increasing
the amount of integrins used by the virus to infect epithelial cells (from 4 to 6). Molecular and
immunodiagnostic studies have confirmed avβ6’s role as cell receptor by showing that this integrin’s
expression is restricted to epithelial cells (FMDV’s infection target) located in the upper airways’
epithelium, oral cavity, gastrointestinal tract, and/or the hooves’ coronary bands. Relevantly, it is
expressed in in high levels in the airways tonsillar crypt epithelium in sheep and cattle, the primary
site for viral replication [30,31].
It has also been observed in vitro that FMDV is capable of using heparan sulphate (HS) as
receptor [29]. It was initially considered that HS was a serotype O co-receptor but later studies have
shown that serotypes A, C, Asia 1, and SAT 1 can also bind to HS, thereby facilitating their entry to
cells, even though their peptides and/or residues interacting with HS are located in different parts of
VP1 or VP3 capsid proteins [32–35].
Following integrin binding to cell membrane, FMDV infects target cells by clathrin-mediated
endocytosis and then becomes mobilized via early endosomes. If binding occurs via HS, the virus
enters cells by caveolin-mediated endocytosis, followed by early endosome use (Figure 1). It has
Vaccines 2020, 8, 764 4 of 19

been demonstrated that clathrin-mediated endocytosis develops faster than caveolin-mediated

Vaccines 2020, 8,
endocytosis x
[33,36]. 4 of 19

Figure 1. A scheme showing Foot-and-mouth disease virus (FMDV) entry to target cells. Adapted [36].
Figure 1. A scheme showing Foot-and-mouth disease virus (FMDV) entry to target cells. Adapted
[36]. studies have suggested that cattle and swine dendritic cells (DC) are susceptible to FMDV
Some
infection; however, such experiments were made when little was known about ruminant DC and
Some studies
data should thus behave suggested
interpreted thatcaution
with cattle and
[37].swine
It hasdendritic cells (DC)
been described areFMDV
that susceptible to FMDV
adsorption by
infection; however, such experiments were made when little was known about ruminant
macrophages and DC could be mediated by the crystallisable fraction (Fc) receptor (FcR), an additional DC and
data shouldwhich
mechanism thus could
be interpreted
maintain with caution
pathogen [37].and
activity It has
may been described
be used that FMDV
for infection adsorption
persistence by
in some
macrophages
animals and DCaspect
(an interesting couldstill
be being
mediated by [37,38].
studied) the crystallisable fraction (Fc) receptor (FcR), an
additional mechanism which could maintain pathogen activity and may be used for infection
persistence
2.3. in someDisease
Foot-and-Mouth animals (an interesting
Virus Replication, aspect still being
Dissemination, andstudied)
Effect on[37,38].
Target Organs
Once the virus is inside an endosome, this compartment’s low pH (6.0 to 5.5 pH late endosome, 6.5 to
2.3. Foot-and-Mouth Disease Virus Replication, Dissemination, and Effect on Target Organs
6.0 early endosome) triggers the chemical stripping and release of the viral genome, which translocates
to theOnce the virus
cytosol through is inside an endosome,
the endosome this compartment’s
membrane low pH (6.0 to
[39]; such translocation 5.5 pH late endosome,
is cap-independent [40].
6.5 to 6.0 early endosome) triggers the chemical stripping and
Genomic RNA’s (gRNA) positive polarity functions as messenger RNA (mRNA), is intrinsically release of the viral genome, which
translocates
infectious. Theto50 the
extremecytosol
has through
a protein the
called endosome membraneviral
genome-associated [39]; such (gVP)
protein translocation
followed is by cap-
the
independent
0 [40]. Genomic RNA’s (gRNA) positive polarity functions as
5 UTR region (around 834 nucleotides-long) having a cytidine-rich region (poly(C)) and the internal messenger RNA (mRNA),
is intrinsically
ribosome infectious.
entry site The 5′ extreme
(IRES) binding directly has
to the a protein
ribosomes, called genome-associated
followed viral protein
by the open reading (gVP)
frame (ORF).
followed
The 0
3 UTR byregion
the 5′ UTR region
is located (aroundthe
between 834stop
nucleotides-long) having alength
codon and a variable cytidine-rich
poly(A)region (poly(C))
tail. One RNA
and the internal ribosome entry site (IRES) binding directly to the ribosomes,
molecule is sufficient for initiating infection, implying that it could function as translation template followed by the open
so
reading frame (ORF). The 3′ UTR region is located
that polymerase can be produced along with RNA replication [41–43]. between the stop codon and a variable length
poly(A)
RNAtail.
canOne
begin RNA molecule is
its translation insufficient
2 AUG codons for initiating
located ininfection, implying(Lthat
the L proteinase pro ) it couldterminus,
amino function
as translation template so that polymerase can be produced along with
enabling the encoding of fifteen proteins: four structural ones forming the viral capsid and elevenRNA replication [41–43].
RNA can ones
non-structural beginneeded
its translation
for viralinreplication
2 AUG codons located in some
and inhibiting the L proteinase (Lpro) amino
host cell functions terminus,
[19,40,44].
enabling the encoding of fifteen proteins: four structural ones forming the
Proteolytic cleavage occurs during viral polyprotein processing, during which determined protein viral capsid and eleven
non-structural ones needed for viral replication
pro and inhibiting some
precursors are released: L proteinase (L ) and three polypeptides—P1, P2 and P3 [45]. P1 encodeshost cell functions [19,40,44].
Proteolytic
structural proteins cleavage
VP1, VP2, occurs
VP3,during
and VP4 viral
whichpolyprotein processing,
are assembled to formduring
the viral which
capsiddetermined
whilst P2
protein precursors are released: L proteinase (L pro) and three polypeptides—P1, P2 and P3 [45]. P1
encodes three non-structural proteins 2A, 2B, and 2C [45,46]. P3 encodes four non-structural proteins:
encodes
3A, 3B, 3C,structural
and 3D. proteins VP1, VP2, VP3, and VP4
P3A is membrane-associated in thewhich are assembled
replication complex;toitsform the viral
function capsid
regarding
whilst
viral P2 encodes
genome three non-structural
replication proteins 2A,P3B
has yet to be elucidated. 2B, and
binds2Cto[45,46]. P3 encodes
the viral genomefour and non-structural
is called VPg.
proteins: 3A, 3B, 3C, and 3D. P3A is membrane-associated in the replication complex; its function
regarding viral genome replication has yet to be elucidated. P3B binds to the viral genome and is
called VPg. P3Cpro is a viral protease responsible for post-translational modifications of viral proteins
and P3Dpol functions as RNA-dependent RNA polymerase responsible for viral RNA replication [47]
(Figure 2).
Vaccines 2020, 8, 764 5 of 19

P3Cpro is a viral protease responsible for post-translational modifications of viral proteins and P3Dpol
Vaccines 2020,as
functions 8, xRNA-dependent RNA polymerase responsible for viral RNA replication [47] (Figure 5 of 2).
19

IRES
S
cre
Pks Single ORF(~700 nt) Poly(A)
3B
poly(C)
ssRNA(+) genome 3`UTR
Translation processing

5`UTR

Lpro P1 P2 P3
Proteases/ Clearance site
L pro
P1/2A P2(2BC) 3AB12 3B3CD
2A

3C pro
RNA generates autocleavage 3AB1 3CD

2A 3B123
VP0 VP3 VP1 2B 2C 3A1 3Cpro 3Dpol

Structural proteins Non-structural proteins

Figure 2. Diagram of the FMDV genome, viral polypeptide processing, and structural and non-structural
Figure 2. Diagram of the FMDV genome, viral polypeptide processing, and structural and non-
protein formation. Adapted [14,36,42,43].
structural protein formation. Adapted [14,36,42,43].
FMDV replication is a rapid and efficient process; chromatin condensation can be observed in
hostFMDV
cells onereplication is a rapid (pi),
hour post-infection and efficient
membrane process; chromatin
proliferation withincondensation
infected cells can be observed
three hours pi and in
host
hostcells one hour
cell lysis and newpost-infection
viral output (pi),
six membrane proliferation within infected cells three hours pi
hours pi [30,48].
and host
The first viral replication occurs following natural[30,48].
cell lysis and new viral output six hours pi infection, mainly in oropharyngeal cells, leading to
The first viral
some vesicles calledreplication occurssores
primary canker following
which natural
usually infection, mainly
pass unnoticed in oropharyngeal
[12,23]. Following the cells,
first
leading to some vesicles called primary canker sores which usually pass unnoticed
replication, the virus passes into the bloodstream; the viremia phase becomes developed, characterized [12,23]. Following
the
by first replication,
an animal’s high the virus passes
temperature andinto the bloodstream;
general malaise. FMDV theundergoes
viremia phasea secondbecomes developed,
replication in the
characterized by an animal’s high temperature and general malaise. FMDV
reticuloendothelial cells and the parenchyma of target organs (liver, spleen, bone marrow, and undergoes a second
striated
replication
muscle) duringin thethis
reticuloendothelial
period. The virus cellsreturnsandtothe
theparenchyma
epithelial cellsofintarget organs
the snout, (liver,and
hooves, spleen, bone
mammary
marrow, and striated muscle) during this period. The virus returns to the epithelial
glands, producing the secondary vesicles characteristic of the disease. The mechanism by which viral cells in the snout,
hooves,
particlesand
passmammary
from the bloodglands, producing
to not the secondary
very vascularized areas ofvesicles characteristic
the epithelium has notofyetthe disease.
been The
elucidated;
mechanism by which viral particles pass from the blood to not very vascularized
it may be associated with migration to infected macrophage tissue or the amount of infectious particles areas of the
epithelium has not yet been
entering a host [35,38,49–51]. elucidated; it may be associated with migration to infected macrophage
tissue or the amount of infectious particles entering a host [35,38,49–51].
2.4. Immune Response
2.4. Immune Response
2.4.1. Innate Response
2.4.1. Innate Response
Inhaled particles which might affect the respiratory tract are size-filtered; those greater than 10 µm
Inhaled
remain in theparticles whichlayer
mucocilliary might affect
in the nasalthecavity
respiratory tract airways,
and upper are size-filtered;
whilst thosethose greater
smaller than
than 10
5 µm
μm
canremain in thetomucocilliary
go directly layer in where
the distal airspaces the nasal theycavity
becomeand phagocyted
upper airways, whilst
by the those macrophages.
alveolar smaller than
5Most
μm inhaled
can govirions
directlyareto the distal
retained by theairspaces
mucus and where they become
transported phagocyted
by the action by from
of the cilia the alveolar
the nasal
macrophages.
cavity and upper Most inhaled
airways tovirions are retained
the pharynx by the mucus
to be swallowed and transported
or eliminated by coughing by the
[3].action of the
cilia from the nasal cavity
The respiratory tract’sand uppersurfaces
mucosal airwaysare tocovered
the pharynx to be swallowed
by epithelial cells whichorcan eliminated by
sustain viral
coughing
infection;[3].
defence mechanisms associated with local defence and humoral and cellular immunity are
thusThe respiratory
needed tract’s
to minimize themucosal surfaces[4].
risk of infection areThe
covered by epithelial
mucocilliary cells which
membrane can sustain
is one such viral
local defence
infection; defence mechanisms associated with local defence and humoral and cellular immunity are
thus needed to minimize the risk of infection [4]. The mucocilliary membrane is one such local
defence mechanism; it extends from the nasal cavity to the distal airway in the lungs. It consists of a
layer of mucus which cleans the nostrils and supresses irritants and harmful agents. The respiratory
Vaccines 2020, 8, 764 6 of 19

mechanism; it extends from the nasal cavity to the distal airway in the lungs. It consists of a layer of
mucus which cleans the nostrils and supresses irritants and harmful agents. The respiratory system has
two macrophage types; the former, located in the interstitium, and the alveolar ones located on alveoli
surface to protect them. Both fulfil defence functions against pathogens, such as the phagocytic uptake
of inhaled particles and (less efficiently) bacterial phagocytosis. They are allergen destroyers and act
as T-cell antigen presenters, releasing mediators enabling inflammation to begin and controlling and
remodelling and repairing lung tissue [52–55].
PRRs interacting with their corresponding PAMPs activate phagocytosis or signalling pathways
which stimulate cytokine production, the expression of adhesion molecules and co-stimulators.
Toll-like receptors (TLR) have been the respiratory system PRRs studied in the greatest depth to
date. They have been classified as transmembrane, type 1, membrane receptors; they have an
ectodomain consisting of leucine-rich repeats (LRR), which are responsible for recognising PAMPs,
and cytoplasmatic domain, homologous to the IL-1 receptor cytoplasmic region known as TIR domain
necessary for downstream signalling [56,57]. Greater TLR expression has been identified in the
macrophages, DC, B-lymphocytes, and respiratory tract mucosa epithelial cells.
Evaluating respiratory system response has shown that respiratory tract mucosa epithelial cells are
highly susceptible to direct infection by the virus due to the integrins, specifically αvβ6. Zhu et al. [58]
established the relationship between these integrins and IL-1 secretion by macrophages, monocytes and
DC as a response to tumor necrosis factor (TNF). IL-1 forms part of the initial immune response (fever,
T-lymphocyte, and macrophage activation) intervening in rapid extracellular matrix renewal and
playing a key role in vesicular lesion pathogenesis, leading to the formation of canker sores [55,58–60].
TLR induction activates the signalling control pathways, in turn activating antimicrobial genes,
inflammatory cytokine production inducing leucocyte migration (chemotaxis), specifically neutrophils,
provoking more DC to mature, thereby inducing more co-stimulatory molecules and increasing their
antigen-presenting capability. TLRs thus participate in developing the direct adaptive immunity
response against pathogens [4,55,59].
The members of this family (TLR) have been classified into two subgroups according to their
location; ten TLRs populations have been described in cattle; TLR1, TLR2, TLR4, TLR5, TLR6,
and TLR11 are exclusively expressed on cell surface and TLR3, TLR7, TLR8, and TLR9 are located in
intracellular vesicles, such as endosomes, lysosomes or the endoplasmic reticulum. Such intracellular
TLRs act as viral nucleic acid detectors and activate the innate immune system’s anti-viral response [61].
TLR2 expression has been seen in cells infected by native virus and cells transfected by a DNA vaccine
against FMDV [62,63]. DC and macrophages express TLR3 and express it more forcefully in CD8α+
DCs having high affinity for virus-infected apoptotic bodies. Such expression may also occur in FMDV
infection [61]
The mucocilliary barrier was mentioned as a local defence mechanism at the start of this section;
the mucus secreted by the glands forms part of this. It is made up of 95% water, 4% mucins
(high molecular weight glycoproteins conferring viscosity and elasticity), a series of protection factors
(specific ones forming humoral immunity), and immunoglobulins (mainly mucosal secretory IgA (SIgA)
(80% of immunoglobulins in nasal mucus are SIgA) and systemic circulation IgG, both contributing to
antiviral defence in the lower respiratory airways and in the mucus). Non-specific protection factors
such as lysozymes, lactoferrin, and interferon (IFN) are found to a lesser extent [52].
This defence system also contains accumulations of non-encapsulated lymphatic tissue acting
as secondary or peripheral immune response cell producers, i.e., mucosa-associated lymphoid
tissue (MALT) being lymphocytic aggregates in the gastrointestinal mucosa, genital-urinary tract,
and lower and upper respiratory tracts, the latter being called nasal-associated lymphoid tissue
(NALT) [2,52]. The respiratory apparatus’ immune system also includes bronchus associated-lymphoid
tissue (BALT), the tonsils and adenoids, and alveolar and bronchial macrophage-mediated cellular
immunity. These aggregates function as B-lymphocyte proliferation and differentiation points following
antigen stimulation [55].
Vaccines 2020, 8, 764 7 of 19

DC and plasmacytoid DC (pDC) are the main antigen-presenting cells in mammals; they also
regulate the function of B-, T-lymphocytes, and other immunity cells via cytokine secretion. DC produce
significant amounts of IFN-α following viral infection; immunological studies in humans and mice
have ascertained their function in the association between innate and adaptive immune responses,
concluding that these cells can manipulate the immune system [37,64,65].
Considerable DC heterogeneity has been described in cleft hoof animals; characterising DC
subsets, their role in the immune response and interaction with FMDV could help to clarify their
importance as markers during infection [37,64,66]. In vitro studies by Guzylack-Piriou et al. [67]
demonstrated that swine pDC were not susceptible to FMDV and did not produce IFN-α after being
exposed to it, thereby providing the virus with an opportunity to replicate and spread. However, they
also demonstrated that FMDV infects pDC and initiates an abortive replication cycle resulting in high
IFN-α expression levels by forming immune complexes binding to FcγRII (IgG Fc receptor). They used
these findings to suggest that when the levels of Abs specific against FMDV are low and incapable
of inducing protection during the first stages of adaptive response they could still be sufficient for
mediating pDC viral infection and induce the subsequent release of IFN-α needed for eliminating the
virus [67].
Bautista et al. [68] evaluated the effect of FMDV on swine skin-derived DCs in vitro. Even though
the virus infected the cells, no viral replication, viral protein production or viral progeny were seen in
them, nor was there any phagocytic effect or cell surface co-stimulatory molecule expression; however,
IFN-α and IFN-β were released. A reduction in IFN-α production ex vivo was observed three days
pi in DC and monocytes isolated from swine when stimulated with viral and/or synthetic proteins.
However, the rapid advance of FMDV infection in swine suggested that IFN levels were not high
enough to trigger an antiviral response in neighboring epithelial cells and/or the viral titres produced
by infected cells were not sufficient or efficient for stimulating INFα/β secretion by DCs [68].
Guzylac-Piriou et al. [67] showed that IFN-α secretion in swine did not just depend on FMDV
activity in pigs’ pDC but also triggered a viral abortive replication cycle related to high IFN-α production
when the virus was opsonized by IgG, infecting pDC via the FcγRII receptor (CD32). This could
explain why animals having very low specific Ab levels could be protected in the short-term after
vaccination. These pDCs’ function would then be to retard viral infection until Abs levels could become
more effective and could promote protective immune responses [66,67].
Sei et al. [69] demonstrated that circulating DC and pDC frequency in cattle blood tissue
increased, reaching a maximum peak 3 to 4 days pi following FMDV entry. They also observed that
(unlike that reported by other authors) these cattle had lymphopenia, MHC class II expression in
DC and pDC molecules became drastically reduced and there was increased IL-10 production by
DC and monocytes; these values returned to normal following the elimination of FMDV from the
blood stream. Such observations highlight that FMDV is able to suppress the beginning of an effective
adaptive immune response, stimulating IL-10 production by DC and monocytes, reducing MHC II
expression and inhibiting antigen processing by DCs, even though it stimulates an increase in DC and
monocytes [69].
Interferons, like IFN-α, IFN-β, and IFN-γ, and other cytokines, like interleukin 6 (IL-6), IL-8,
and IL-12, have been seen to be involved in recovering from the infection. Their plasma concentration
becomes increased following vaccination or field challenge, inducing monocyte and/or macrophage
activation, neutrophil chemotaxis, amplified local inflammation, and adhesion molecule regulation [70–72].
The immune response against viral infection begins when interferons are expressed; these induce immune
cell migration to the infection site, so much so that interferons have been used as treatment for controlling
such infection [49,73].
IFN-α inhibits FMDV replication and this cytokine’s release protects swine from subsequent
infection; however, susceptible animals becoming infected by the virus develop viremia one to three
days after infection and rapid clinical presentation of the disease, suggesting that the virus can block or
overcome the initial IFN-α response produced by pDC and maybe other cells [74].
Vaccines 2020, 8, 764 8 of 19

A new interferon family, IFN III or IFN-λ, has been described in many species during the
last decade, including humans, mice, swine, chicken, and cattle (called boINF-λ3 in the latter) [73].
Although its effectiveness is still being studied, INF-λ-induced antiviral activity has been demonstrated
against many viruses, most of them replicating in epithelial cells, such as the influenza virus, respiratory
syncytial virus (RSV), human metapneumovirus (hMPV), and coronavirus. Hepatitis B and C, HIV,
and herpes simplex viruses have also been shown to be sensitive to IFN-λ activity [75,76] and, as FMDV
is a pathogen whose first replication site is the oropharyngeal mucosa, it has been proposed that
boIFN-λ3 is expressed in susceptible animals as a mechanism for preventing and protecting them from
such viral invasion [73,76] (Figure 3).
Some particularities regarding ruminants’ immune system are not clear, such as the large amount
of γδT cells compared to those in humans and mice [48,64] and the situation concerning swine is no less
complex [77]. The greater amount of γδT cells has been attributed to a γδT subpopulation expressing
the workshop cluster 1 (WC1) molecule in ruminants and its orthologue in swine. This population
has only been observed to date in cloven-hooved mammals (Artiodactyla); γδT cells are the first
T-lymphocytes to be developed and are found in epithelial cell-associated tissues, such as the skin and
intestinal and lung mucosa.
It has been stated that γδT response is directed against pathogens (virus, bacteria, and parasites),
reinforcing the immune response at these sites; it has also been observed that they respond to PAMPs,
suggesting that they play an important role in the innate immune response and could bridge immune
and adaptive responses [78,79]. Research regarding swine has shown that emergency vaccination
against FMDV using a high potency vaccine stimulated γδT cells for synthesising cytokines and
chemokines, indicating an innate response-related function for these cells in FMDV infection or
regarding vaccination against it. These cells were isolated from cattle vaccinated against FMDV and
had a cytostatic and cytotoxic effect on the infected cells [80,81].
NK cells are part of the innate immunity mechanism, ensuring the early elimination of
pathogen-infected cells and thereby preventing dissemination amongst hosts and healthy individuals.
NK cells differentiate infected or damaged cells from healthy ones by activating and inhibitory
receptors; these recognize cell surface molecules and promote or inhibit a recognition-type response.
Activating receptors detect molecules expressed by infected cells and stimulate lytic activity in the NK
which initiates infected cell destruction and the inhibitory receptors identify healthy cells for protecting
them [82,83]. The activating receptors located in NK lymphocytes recognize a large variety of ligands;
many of these receptors have been called killer cell immunoglobulin (Ig)-like receptors (KIR) since
they have a structural domain called Ig-fold (a sandwich-like structure). The adaptive immune system
produces IgG1 and IgG3 Abs during infection which bind to the pathogen’s antigens expressed on
infected cells and NK receptors bind to the Abs’ Fc regions, activating NK, and these lymphocytes can
thus destroy infected cells. This mechanism has been called antibody-dependent cellular cytotoxicity
(ADCC) [82,84] (Figure 3).
Much of what is known about NK-mediated immunity against pathogens has come from studies
in humans and mice; these cells’ role in domestic animals is still not clear, mainly due to diversity
amongst species; however, studies have increased regarding cattle and swine. Bovine NK cells are
found in peripheral blood, the liver, lungs, lymph nodes, and bone marrow. As in other species,
bovine NK cells express natural cytotoxic receptors such as CD335, produce IFN-γ, lyse sensitive
targets and seem to have CD335+ /CD2+/− /CD8+/− /CD3− phenotypes; NK are identified in swine as
CD2+ /CD8+ /CD3− [48]. It has been observed that NK-mediated specific responses are greater in
infected cattle than in vaccinated ones when trying to understand how NK cells function during FMDV
infection. In vitro research involving NK cells from FMDV-infected cattle revealed a high level of
cytotoxicity but swine NK cells were minimally cytotoxic, even against FMDV-infected cells [64,84,85].
Vaccines 2020, 8, 764 9 of 19

2.4.2. Acquired Response

Cellular Response
Studies regarding T-helper (Th) cell role have revealed that the immune response to FMDV
is T-dependent and heterotopic, since the T-lymphocytes extracted from infected animals or those
vaccinated with FMDV can proliferate in response to other viral strains [86,87].
An important action has been described for CD4+ and CD8+ T-lymphocytes regarding their role
as eliminators of the virus from infected animals’ tissues; CD4+ T-lymphocytes recruit and activate
macrophages and neutrophils so that they destroy intracellular and some extracellular pathogens
and help B-lymphocytes to produce Abs and CD8+ T-lymphocytes to carry out their cytotoxic action
(killing infected cells and exposing the agent), eliminate pathogens (usually viruses) located in cell
cytosol infecting and reproducing in all cells, including non-phagocytic ones [2,3,5]. Regarding FMDV,
T-lymphocyte action has been seen during infection and vaccination in both cattle and swine; however,
the response has been greater in infected rather than vaccinated animals. CD4+ T-lymphocyte
proliferation has been demonstrated and associated with increased neutralising antibody (nAb)
production and part of the CD8+ T-lymphocyte response. Swine infected by FMDV field strain A24 have
induced a CD8+ T-lymphocyte-specific response; it is thought that this response is directed towards
FMDV A24-specific CD8+ epitopes (such epitopes having recently been identified in cattle) [85,88].
Childestone et al. [85] observed anti-FMDV CD8+ activity five weeks pi, having proposed that
CD8+ lymphocytes participate during the infection’s late phase, bearing in mind that FMDV is a
cytopathic virus, and it is thus unlikely that a CD8+ -type response would occur during the disease’s
acute phase. A protection-inducing response during this stage is mainly Ab-mediated, neutralising
activity playing a predominant role; however, CD8+ lymphocytes could play a more important role
during the late stage [85] (Figure 3).
CD4+ and CD8+ T-lymphocyte antigen recognition is associated with the major histocompatibility
complex (MHC), called bovine leucocyte antigen (BoLA) for cattle and swine leucocyte antigen (SLA) in
pigs. It is an important genetic region influencing resistance to diseases and its level of polymorphism is
associated with host immune capability. Class I and II genes are the most polymorphic ones; regarding
class II genes, the DRB gene is located in region IIa, along with its loci: DRB1, DRB2 and DRB3.
The polymorphism of DRB3 exon 2 (encoding the peptide-binding region) has been related to variability
in the immune response to FMDV inactivated vaccine serotypes O, A and ASIA 1. Gowane et al. [89]
detected eleven alleles having more than 3% frequency in the population being studied and determined
that alleles DRB3/0201/0801 and 1501 always ranked high for the protective immune response [89].
Class I molecules present protein antigen-derived peptides to CD8+ T-lymphocytes (which they
recognize) and class II molecules present peptides to CD4+ T-lymphocytes [90–92].

Humoral Response
Cell and antibody (Ab)-mediated immunity is an essential part of host control of natural infection;
a virus is neutralized within a host by Ab-dependent mechanisms similar to those occurring in
neutralization in vitro; however, it has been suggested that macrophages play an essential role in
clearing the virus from an animal’s tissues via opsonization-enhanced phagocytosis [48,93].
The rapid induction of high IgM nAb titres (2–4 days pi) is characteristic of responses against
cytopathic viruses, like FMDV, usually produced in a thymus-independent form. These Abs are mainly
responsible for controlling infection since they prevent the virus’ systemic dissemination [94] (Figure 3).
A second strong response by nAbs occurs following field infection with FMDV, taking precedence
over recovery; such immunity is against a virus having the same serotype, is prolonged and produced
7 to 14 days pi (IgG). The production of non-protection-inducing Abs against VP2 and VP3 protein
antigenic sites has also been reported, acting against the viral capsid’s pentamer subunit and against
non-structural proteins, especially RNA polymerase [95]. Following vaccination with inactivated virus,
only Ab-mediated humoral immunity is activated and Abs are produced which neutralize infection
 FMD share similarities with that already reported for oral–nasal infection by FMDV in terms of
temporal evolution and isotype profile progression. One study highlighted the finding that the oral–
nasal infection of vaccinated steers did not give rise to the disease but was followed by a slight fall in
nAb titres, correlated with a transitory reduction of FMDV-specific IgM and IgG1 Abs [98].
MALT’s main function is the production of IgA secreted by sensitized lymphocytes. Another
Vaccines 2020, 8, 764
important characteristic concerns IgA being secreted regardless of its serum synthesis; its function is 10 of 19
to impede germs adhering to and entering the mucosa and a harmful inflammatory response is
avoided by the complement pathway not being activated [59]. When IgA binds to a viral particle it
by the viral serotypes
prevents its bindingincluded
to a hostin thebyvaccine
cell blocking[87]. It has
specific been observed
receptors thathappen
and this can protection duration,
within an
nAb titres, and vaccine-induced Ab responses are
epithelial cell when transporting IgA [60] (Figure 3).always lower than those induced by natural infection
and that simply
Figure the presence ofthe
3 summarizes nAbs (in many
expected cases)response
immune is not enough
by cattleforand/or
ensuring protection.
swine This could
immunological
indicate that against
systems cellularFMDV
immunity would play
post-vaccination oran important from
post-recovery role in controlling
field infection. the disease [96].

Figure 3. Ideal
Figure hosthost
3. Ideal immune
immune response
responseto toFMDV.
FMDV. Anatomical, physiological,
Anatomical, physiological, endocytic,
endocytic, phagocytic,
phagocytic,
and inflammatory
and inflammatory barriers initially
barriers initiallyreact
reactagainst
against FMDV:
FMDV: the themucosity
mucosity produced
produced by respiratory
by respiratory tract tract
ciliated
ciliated cellscells retain
retain partpart
of of
thethe viralparticles;
viral particles; neutrophils
neutrophils (Neu),(Neu),eosinophils,
eosinophils, dendritic cells cells
dendritic (DC),(DC),
macrophages
macrophages (M),(M),
and and cytotoxic
cytotoxic (CTL)(CTL)andand natural
natural killer
killer (NK)lymphocytes,
(NK) lymphocytes,complement
complement proteins
proteins and
and other inflammation mediators begin to act. Stress signals trigger gdT in tissue
other inflammation mediators begin to act. Stress signals trigger gdT in tissue to secrete cytokines, to secrete cytokines,
thereby attracting DCs, macrophages and B-lymphocytes which act as antigen-presenting cells (APC).
thereby attracting DCs, macrophages and B-lymphocytes which act as antigen-presenting cells (APC).
A greater amount of DCs and macrophages phagocyte FMDV, process it and express the antigens on
A greater amount of DCs and macrophages phagocyte FMDV, process it and express the antigens on
its membrane with major histocompatibility complex (MHC) intervention, these being recognized by
its membrane with major histocompatibility complex (MHC) intervention, these being recognized
Th-lymphocytes, giving rise to Th1 and Th2 differentiation. The cytokines secreted by these cells
by Th-lymphocytes,
stimulate macrophage,givingneutrophil,
rise to Th1 DC,and Th2 differentiation.
natural killer cell, CTL, and TheB-lymphocyte
cytokines secreted by these
proliferation and cells
stimulate macrophage,
differentiation. neutrophil,
The virus’ solubleDC, natural
antigens (SA)killer
bindcell, CTL, and
to B-cell Abs B-lymphocyte
simultaneously proliferation
with antigen and
differentiation.
presentation The virus’ soluble antigens
by antigen-presenting cells; this(SA) bind tobyB-cell
is followed Abs becoming
these cells simultaneously withinto
transformed antigen
presentation by antigen-presenting
plasma cells secreting IgM, IgG, and cells;
IgA,this is followed
where IgM and by IgGthese cellsviral
opsonize becoming
particles,transformed
stimulating into
plasmaphagocytosis, neutralising
cells secreting IgM, IgG,and and
activating antibody-dependent
IgA, where IgM and IgGcellular cytotoxicity
opsonize (ADCC),stimulating
viral particles, whilst
IgA contributes to retaining viral particles in the mucosa, avoiding infection
phagocytosis, neutralising and activating antibody-dependent cellular cytotoxicity (ADCC), whilst of epithelial cells. IgA
contributes to retaining viral particles in the mucosa, avoiding infection of epithelial cells. Infected
target cells also express viral proteins from their cytosol along with MHC class I intervention and
CTL are recognized, enabling Th1 stimulus to induce infected cell apoptosis. Antigen binding to B-
and T-lymphocyte receptors triggers their activation; this is mediated by a complex cytokine network
(interleukins (IL), tumor necrosis factors (TNF), interferons (IFN), colony-stimulating factors (CSF) and
chemokines) connecting cells to each other, regulating their function by inducing or supressing their
synthesis or that of other cytokines and their receptors. Activated lymphocytes multiply by clonal
expansion, producing two types of cell: effector and memory cells. Effector cells have a short lifespan
and effect and regulate response to a pathogen. Memory cells live for a long time, increasing immune
response speed and intensity if the same pathogen is found again.

Mulcay et al. [97] have demonstrated IgG subclass dynamics. High IgG1 levels were seen when
evaluating anti-FMDV vaccine protection which, in vitro, were more efficient for complement fixation than
IgG2 subclass, and equally for high affinity interaction with phagocyte Fc receptors, thereby promoting
opsonization and phagocytosis by antigen-Ab complex, providing protection far beyond their neutralising
ability (Figure 3).
Vaccines 2020, 8, 764 11 of 19

Results to date have indicated that humoral responses following systemic vaccination against FMD
share similarities with that already reported for oral–nasal infection by FMDV in terms of temporal
evolution and isotype profile progression. One study highlighted the finding that the oral–nasal
infection of vaccinated steers did not give rise to the disease but was followed by a slight fall in nAb
titres, correlated with a transitory reduction of FMDV-specific IgM and IgG1 Abs [98].
MALT’s main function is the production of IgA secreted by sensitized lymphocytes. Another important
characteristic concerns IgA being secreted regardless of its serum synthesis; its function is to impede
germs adhering to and entering the mucosa and a harmful inflammatory response is avoided by the
complement pathway not being activated [59]. When IgA binds to a viral particle it prevents its binding to
a host cell by blocking specific receptors and this can happen within an epithelial cell when transporting
IgA [60] (Figure 3).
Figure 3 summarizes the expected immune response by cattle and/or swine immunological
systems against FMDV post-vaccination or post-recovery from field infection.

2.5. Evasion Mechanisms

FMDV is very small, having a 25 to 30 nm diameter, thereby enabling particles to evade the upper
respiratory tract’s physical barriers and pass from the upper airways to the lower ones [99]. Even though
macrophage, DC and polymorphonuclear infection mechanisms (via Fc receptors) have been proven in
RNA viruses (such as flavivirus), it has been suggested that they have been adopted by FMDV and
other picornaviruses, facilitating the virus becoming established in the lower airways [37,38].
Lymphopenia has been observed in swine, followed by FMDV infection, depending on serotype
and virulence; lymphopenia has not been detected in cattle; however, a constant amount of leucocytes
within normal physiological ranges has been observed during the disease’s period of viraemia and
clinical phase. Leucocytes have been evaluated in studies like that by Bautista et al. [89] but no changes
have been observed in them; by contrast, evaluation in swine has revealed a significant reduction in
CD4+ and CD8+ cells [68,100].
2B and 2C protein expression has been associated with blocking protein secretion in host cells and
increased plasmatic membrane permeability [45,46]. The Lpro protein is essential for rapid FMDV genome
replication since it is involved in autocleavage of nascent peptide and inactivates eukaryotic translation
initiation factor 4F (eIF-4G) which is fundamental for host cell mRNA translation. Lpro ’s function
is to inhibit host cell protein synthesis and promote viral protein synthesis, thereby compromising
host cells’ ability to develop an antiviral response due to their inability to synthesize new protein
molecules [42,66,101]. For example, blocking host cell IFN-α and IFN-β expression promotes the virus’
rapid multiplication and propagation [102].
P3Cpro or 3Cpro viral proteins have been associated with cell protein cleavage, even though it is
still not clear whether they act directly or indirectly in activating cell proteases. Immunofluorescence
studies have demonstrated that 3Cpro is only found in infected cell cytoplasm [19]. Capozzo [103]
demonstrated that this protein is concentrated in the perinuclear region and its expression induces
H3 histone cleavage thereby reducing host mRNA levels. Briones et al. [104] described 3CD0 (a 3Cpro
precursor) in FMDV-infected cells’ nuclei and that this could also produce H3 cleavage, contributing
even more towards inhibiting host cell RNA synthesis [19]. Wang et al. [62] have shown that this protein
can impede IFN type I receptor signalling, specifically, STAT1 and STAT2 nuclear translocation. These
molecules have hundreds of possible transcriptional objectives, including many antiviral effectors and
immunostimulatory genes [29,51].
Tumor cell line studies have detected alterations in cell signalling induced by FMDV virion protein
1 (VP1) binding to target cells, thereby causing apoptosis in tumor cells by integrins modulating Akt
signalling pathways and reducing COX-2 over-expression and thus tumor progression. Studies have also
been evaluating whether FMDV infection reduces the inflammatory response [51,105]. Taken together,
these studies have highlighted the varied and powerful way in which FMDV manipulates a host’s immune
system to enable its replication and propagation (Figure 4 graphically summarizes such mechanisms).
Vaccines 2020, 8, 764 12 of 19
Vaccines 2020, 8, x 12 of 19

Figure 4. Foot-and-mouth disease virus mechanisms for evading an immune response. (A) FMDV
Figure 4. Foot-and-mouth disease virus mechanisms for evading an immune response. (A) FMDV
entersenters
the airways,
the airways,taking
taking advantage
advantageofofits itssmall
small size whichseems
size which seems to to enable
enable it toitevade
to evade the airways’
the airways’
lysozymes
lysozymes and lactoferrins. It enters the epithelial cells by receptor-mediated endocytosis: i.e., i.e.,
and lactoferrins. It enters the epithelial cells by receptor-mediated endocytosis:
integrins andand
integrins heparan
heparan sulphate
sulphate(HS).(HS).Capsid
Capsid protein viralpolypeptide
protein viral polypeptide 1 (VP1)
1 (VP1) is the
is the most most antigenic
antigenic
viral protein; a region
viral protein; in this
a region polypeptide’s
in this polypeptide’s G-H G-Hloop
loopconsists
consists of
of 140
140 to 160
160 aa;
aa;itithas
hasbeen
beenidentified
identified as
as the predominant
the predominant epitopeepitope stimulating
stimulating nAb production
nAb production by B-cells.
by B-cells. Comparing
Comparing the structures
the structures of
of various
various FMDV serotypes has shown that the main differences
FMDV serotypes has shown that the main differences regarding formation are produced in VP1, regarding formation are produced in VP2,
VP1, VP2, and VP3 loop and C-terminal region, thereby contributing
and VP3 loop and C-terminal region, thereby contributing to the virus’ antigenic variation. Dynamicto the virus’ antigenic variation.
Dynamic simulations of FMDV’s molecular structure have revealed that the G-H loop protrudes from
simulations of FMDV’s molecular structure have revealed that the G-H loop protrudes from the capsid’s
the capsid’s surface and actively fluctuates as a tentacle in its natural state, suggesting that such
surface and actively fluctuates as a tentacle in its natural state, suggesting that such “flexibility” could
“flexibility” could ensure the virus’ correct binding to Abs and cell receptors. The VP2 polypeptide’s
ensure the virus’ correct binding to Abs and cell receptors. The VP2 polypeptide’s Cys 130 region of the
Cys 130 region of the virus’ O serotype could covalently bind to VP1 Cys 134 region using a
virus’disulphide
O serotype could
bond for covalently
increasing the bind to VP1
extent Cys 134in
of variation region
the VP1using
loop.a disulphide
(B) The endosome’sbond forlow increasing
pH
the extent of variation in the VP1 loop. (B) The endosome’s low pH
promotes the viral genome’s stripping and release which becomes translocated to host cell cytosol. promotes the viral genome’s
stripping and release
Positive-sense which
genomic RNAbecomes
(gRNA) translocated
functions as to host cellRNA
messenger cytosol.
(mRNA);Positive-sense
it has the VPg genomic
proteinRNA
(gRNA) functions as followed
messenger RNA (mRNA); it has 0
at the 5′ extreme, by the 5UTR region with thethe VPg
PolyC protein
region andat the 5 ribosome
internal extreme,entryfollowed
site by
(IRES)region
the 5UTR which withbinds the
to the ribosomes,
PolyC regionfollowed by theribosome
and internal open reading entryframe
site(ORF).
(IRES)The 3UTRbinds
which regionto the
and thefollowed
ribosomes, PolyA tailbyare theat open
the 3′reading
extreme.frame Proteinase
(ORF).L (L pro) and P1, P2, and P3 polypeptides are
The 3UTR region and the PolyA tail are at
released
0 by viral polyprotein processing
pro by
the 3 extreme. Proteinase L (L ) and P1, P2, and P3 polypeptides proteolytic cleavage; P1 encodes structural
are released proteins
by viral VP1,
polyprotein
VP2, VP3, and VP4 which assemble to form the viral capsid. P2 encodes three non-structural proteins:
processing by proteolytic cleavage; P1 encodes structural proteins VP1, VP2, VP3, and VP4 which
2A, 2B, and 2C. P3 encodes four non-structural proteins: 3A, 3B, 3C, and 3D. 2B and 2C expression is
assemble to form the viral capsid. P2 encodes three non-structural proteins: 2A, 2B, and 2C. P3 encodes
related to blocking host cell protein secretion and greater permeability of their plasmatic membrane.
four non-structural proteins: 3A, 3B, 3C, and 3D. 2B and 2C expression is related to blocking host
Lpro promotes rapid replication of the viral genome, inactivates (eIF-4G) host cell mRNA translation,
cell protein secretion and greater permeability of their plasmatic membrane. Lpro promotes rapid
inhibits host cell protein synthesis, blocks IFNα and IFNβ expression and promotes viral protein
replication
synthesis of thereby
the viral genome,
reducing hostinactivates
cell ability to (eIF-4G)
develophost cell mRNA
an antiviral translation,
response. Viral proteinsinhibits
P3Cpro host
or cell
protein
3C synthesis,
pro are relatedblocks IFNα and
to cell protein IFNβinexpression
cleavage infected cell and promotes
cytoplasm, induceviral protein synthesis
H3 cleavage, lowering host thereby
reducing host cell
cell mRNA ability
levels. When to 3CD
develop an antiviral
or 3Cpro are foundresponse.
in host cellViral proteins
nucleus P3CproH3,
this inhibits 3Cpro are
orreducing related
host
to cellcell
protein
mRNAcleavage
synthesis in even infected
more so.cell Thiscytoplasm, induce IFN
could also impede H3 Icleavage,
signalling lowering
by alteringhost STATcell mRNA
1 and
STAT
levels. When 2 nuclear
3CD ortranslocation,
3Cpro are found affectingin antiviral
host celleffectors
nucleusand thisimmunostimulatory
inhibits H3, reducing genes. (C).cell
host It has
mRNA
synthesis even more so. This could also impede IFN I signalling by altering STAT 1 and STAT 2 nuclear
translocation, affecting antiviral effectors and immunostimulatory genes. (C). It has been estimated that
FMDV also affects the mechanism for antigen presentation by DCs and monocytes; these cells increase
IL-10 production, reduce MHC II expression which decreases antigen processing by these cells.
Vaccines 2020, 8, 764 13 of 19

3. Conclusions and Perspectives

Despite reports in the pertinent literature about FMDV’s immunological aspects [6,64,106],
this document has contributed towards constructing knowledge in the immunological area, specifically
regarding pathogen evasion. It is clear that reducing outbreaks and viral permanency in the environment
requires the use of strategies such as mass vaccination of the population in combination with
other control measures, thereby enabling Europe and North America to be currently considered
FMDV-free [61,107]. In fact, revising prioritization models about the disease in Europe, control measure
scores are low, specifically for vaccines (–1), being considered good (www.discontools.eu), and have
managed to reduce FMDV incidence. Nevertheless, there are reports of cases in regions having
regular vaccination [108] since limited virus replication in the oropharynx is one of the limitations of
vaccination enabling vaccinated animals to become infected, making them carriers for a long period of
time. The virus’ biological characteristics, mainly regarding its biology (i.e., genetic derivation/drift
and recombination), make controlling the disease a greater challenge [107].
Many studies have stressed the significance of FMDV’s infection and evasion mechanisms as one
of the greatest challenges for protecting susceptible production systems, starting from vaccination.
Studying the innate immune response may cast the greatest light on this mechanism and answer
plentiful questions about FMDV. Toka and Golde’s studies in 2013 have clearly shown that early
protection can be achieved with very little Ab production, indicating that the humoral immune response
plays an important role during the infection’s early phase and should be evaluated in greater depth.
DC and pDC mechanisms of action (MoA), along with IFN I production and variation regarding
the response to FMDV amongst bovine and swine species, leads to it being thought that such expression
is also key to a better understanding of the innate immune response. FMDV’s ability to infect DC and
antigen presentation by them and stimulating the immune response is still poorly understood.
Although advances have been made regarding the virus’ structure and its structural and
non-structural proteins’ relationship with target cells, questions still remain unanswered about
the pathways and/or mechanisms by which genetic material is released from the endosomes to
the cytoplasm.
FMDV has developed mechanisms for breaking or overcoming antiviral response induction and
activation, using its non-structural proteins for survival and replicating itself in host cells. Lpro and
3Cpro are the main proteins modifying host immune responses; Lpro has been one of the most studied
proteins. It can cleave many host proteins, inhibit cell protein expression and deubiquitinate some
crucial molecules for activating antiviral pathways and signal transduction. However, some questions
still need to be answered about the different forms of Lpro and the pathways involved in Lpro -mediated
antagonist effects. More studies are needed for clarifying such questions and the multifunctional role
of Lpro in FMDV infection.
FMDV control and prevention programmes can be improved via better understanding and
in-depth knowledge concerning pertinent host immune response mechanisms, as well as the virus’
evasion mechanisms; this would include producing new vaccines contributing towards such virus
control having wider and better coverage regarding susceptible species.

Author Contributions: Conceptualization, I.R.-H., C.C.-G., C.A. and M.A.P.; writing—original draft preparation,
I.R.-H., C.C.-G. and C.A.; writing—review and editing, C.A., M.E.P. and M.A.P. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: We would like to thank the Universidad del Rosario for assuming open access charges.
We would like to thank Jason Garry for translating this manuscript.
Conflicts of Interest: The authors declare that there are no conflict of interest regarding the publication of
this paper.
Vaccines 2020, 8, 764 14 of 19

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