MOLECULAR TECHNIC OF VIRUS IDENTIFICATION

By:
Name : Pratiwi Kusuma Kurniawati
SID : B1B017007
Entourage : III
Group :8
Assistant : Agung Wiriat Putra Pratama Hadi

VIROLOGY LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGI
PURWOKERTO

2019
 I. INTRODUCTION

A. Background

Dengue is an acute febrile disease caused by the mosquito-borne dengue

viruses (DENVs). Dengue virus is transmitted by the Aedes aegypti and Aedes
albopictus mosquitoes, consisting of four serotypes (DENV 1 to 4), that are members
of the flaviviridae family, genus flavivirus and included into group IV (+) single strain
RNA. All four DENV serotypes have emerged from sylvatic strains in the forests of
South-East Asia (Abdullah, 2013). The DENV particle is approximately 500 Å in
diameter and includes a positive-sense RNA genome with ~10,700 nucleotides and 3
structural proteins: capsid (C, 100 amino acids), precursor membrane (prM, 75 amino
acids), and envelope (E, 495 amino acids). The capsid protein and the viral RNA
genome form a nucleocapsid that buds at the endoplasmic reticulum (ER) in
association with 180 copies of prM and E and carries host-derived lipids to form the
immature virion. The structural proteins are the capsid (C) protein, the envelope (E)
glycoprotein and the membrane (M) protein, itself derived by furine-mediated
cleavage from a prM precursor. The E glycoprotein is responsible for virion
attachment to receptor and fusion of the virus envelope with the target cell membrane
and bears the virus neutralization epitopes. In addition to the E glycoprotein, only one
other viral protein, NS1, has been associated with a role in protective immunity. NS3
is a protease and a helicase, whereas NS5 is the RNA polymerase in charge of viral
RNA replication. In the structural proteins, the RNA genome of dengue virus encodes
7 nonstructural proteins that are essential for viral replication (NS1, NS2A, NS2B,
NS3, NS4A, NS4B and NS5) (Chen et al., 2018).
The structural proteins include a capsid protein rich in arginine and lysine
residues and a non-glycosylated prYi protein produced from a glycosylated precursor
in a late step of virus maturation. The major structural envelope protein is involved in
the main biologic functions of the virus particle such as cell tropism, acid catalyzed
membrane fusion, and the induction of hemagglutination-inhibiting, neutralizing, and
protective antibodies (Kautner et al., 1997). The first nonstructural protein is NS1, a
glycoprotein with a function in the virus life cycle that is unknown. NS1 proteins are
detected in high titers in patients with secondary dengue infections, but are rarely
found in primary infections. The NS2 region codes for two proteins (NSYA and
NSYB) that are thought to be implicated in polyprotein processing whereas NS5 is
probably the viral proteinase that functions in the cytosol. The NS4 region codes for
two small hydrophobic proteins that seem to be involved in the establishment of the
membrane bound RNA replication complex. The protein encoded by basis of the
amino acid sequence, this protein is believed to be the virus-encoded RNA-dependent
RNA polymerase (Zeidler et al, 2017).
DF or Dengue Fever follows both primary and secondary infections, and is
most frequently encountered in adults and older children. Typical dengue fever is
characterized by abrupt onset of fever, headache, severe myalgia (‘breakbone’ fever),
and arthralgia. Gastrointestinal symptoms may also be prominent. A rash is noted in
about half the cases. Early generalized erythema may be noted; a maculopapular rash
may appear later. Minor bleeding phenomena, such as petechiae and nosebleeds, may
also be present. The incubation period is typically 4–7 days (range 3–14). Although
clinical manifestations may vary with the specific serotype or genotype, most
infections are asymptomatic or mild, and most infections are self-limited, with fever
lasting 2–7 days. The ratio of asymptomatic to symptomatic infections ranges from 2
: 1 to 10 : 1. Complicated forms, dengue hemorrhagic fever (DHF) and dengue shock
syndrome (DSS), are increasing in number as more populations in larger regions of the
world have already been infected with one or more dengue serotypes and are at
increased risk for complicated dengue. Onset of symptoms is characterized by a
biphasic, high-grade fever lasting for 3 days to 1 week. Severe headache (mainly
retrobulbar), lassitude, myalgia and painful joint, metallic taste, apetite loss, diarrhea,
vomiting, and stomachache are the other reported manifestations (Hasan et al., 2016).
DHF is frequently seen during a secondary dengue infection. However, in
infants it may also occur durring a primary infection due to maternally attained dengue
antibodies. Clinical parameters: Acute-onset febrile phase – high-grade fever lasting
from 2 days to 1 week. Hemorrhagic episodes (at least one of the following forms):
Petechiae, purpura, ecchymosis, epistaxis, gingival and mucosal bleeding, GIT or
injection site, hematemesis and/or malena. Laboratory parameters: Thrombocytopenia
(platelet count <100,000/cu mm). The clinical course of DHF is characterized by three
phases: Febrile, leakage, and convalescent phase. High-grade fever of acute onset
along with constitutional signs and facial erythema characterizes the commencement
of the febrile illness. The initial febrile illness is marked by a morbilliform rash and
hemorrhagic tendencies. The fever persists for 2 days to 1 week and then drops to
normal or subnormal levels when the patient either convalesces or advances to the
plasma leakage phase. High plasma escape cases are marked by frank shock with low
pulse pressure, cyanosis, hepatomegaly, pleural and pericardial effusions, and ascites.
Severe ecchymosis and gastrointestinal bleeding followed by epistaxis may also be
noted in a few cases. Bradycardia, confluent petechial rashes, erythema, and pallor are
seen during this phase (Chen et al., 2018).
DSS (Dengue Shock Syndrom) is defined as DHF accompanied by a unstable
pulse, narrow pulse pressure (<20 mmHg), restlessness, cold, clammy skin, and
circumoral cyanosis. Progressively worsening shock, multiorgan damage, and
disseminated intravascular coagulation account for a high mortality rate associated
with DSS. The patient may die within 12–24h of going into shock or recover rapidly
with volume replacement therapy (Khaterpal & Khanna, 2016).
The severity of DENV infection is modulated by multiple risk factors such as
age, the genetic background of the host, viral serotype and genotype, and secondary
DENV infection by a heterologous serotype. Finally, the virus serotype and genotype
also influence the symptomatic picture of disease and outcome. These observations
were initially based on epidemiological findings, but accumulating laboratory and
experimental data have contributed to the recognition of DENV virulence as an
important risk factor (Back & Lundkvist, 2013).
The infection of dengue virus prevention can have been done in several ways
there are by GIS mapping of Dengue Foci. GIS Mapping of Dengue Foci Among the
advanced techniques used for location of DENV, GIS mapping has been efficient in
locating dengue concentrations. By locating dengue seri-positive cases within the
study area, dengue transmission can be prevented by locating dengue foci, and then
treating them with diverse preventive strategies. The next way is focused and effective
surveillance, GIS Mapping of Dengue Foci Among the advanced techniques used for
location of DENV, GIS mapping has been efficient in locating dengue concentrations.
By locating dengue seri-positive cases within the study area, dengue transmission can
be prevented by locating dengue foci, and then treating them with diverse preventive
strategies. Determination of oviposition sites, the oviposition sites have been
determined, introduction of strategies that eliminate mosquito population at a later
developmental stage will increase the efficacy of control strategies. Next way is made
community-based control program to educate the community about the measures for
extermination of mosquito breeding sites. Last is made education of prevention
strategies. Education serve as a basis for an ability of an individual to identify and deal
with vector habitats, and use preventive measures (Widoyono, 2011).
Another ways to prevent the infection of dengue virus is through vaccine.
Development of dengue vaccine candidates has progressed over the last decade and
some of these have entered clinical trials in both endemic and non-endemic areas.
There are two times of dengue vaccine strategies, replicating viral vaccines and non-
replicating viral vaccines. Replicating viral vaccines include live-attenuated viruses
(LAV) that are created by reducing the virulence of a pathogen without compromising
its viability. Current methods of producing live-attenuated viruses for dengue vaccines
include attenuation by serial passage in cell lines and targeted mutagenesis and by
constructing chimeric vaccine viruses (Khaterpal & Khanna, 2016).

The objective of this laboratory activity is to find out the steps of molecular
detection of the Dengue Virus gene from a mosquito sample.
 II. MATERIAL AND METHOD
A. Materials

The materials that used in this lab activity are mosquito (Aedes aegypti),
RNAse free water 100 µL, proteinase k 20 µL, lysis buffer & carrier RNA 420 µL,
ethanol 96-100% 25 µL, wash buffer 500 µL, RNAse free water 50 µL, 2x reaction
mix 7,5 µL, RNA template (100 µg/µL) 1 µL, primer forward (10 µg/µL) 1 µL,
primer reverse (10 µg/µL) 1 µL, superscriftTM III RT/platinumTM taq mix 2 µL,
ddH2O 2,5 µL, agarosa 0,5 g + TAE buffer 50 mL, +5 µL sybr die, TAE 1x, and
DNA ladder 100 pb 5 µL.
The tools that used in this lab activity are sterile tube, grinder, vortex,
incubator, sentrifuge, viral spin column, collection tube, recovery tube, PCR
amplification, erlenmeyer, micropipette along with the tip, oven, beaker glass, a
set of electrophoresis tools, and UV transluminator.

B. Methods

The method used in this laboratory activity are:

1. Isolation of RNA Virus Dengue
The mosquito of Aedes aegypti was putted into tube steril and pounded
with grinder until smooth. (+)RNAse free water 100 µL, (+)proteinase k 20
µL, and (+)lysis buffer & carrier RNA 420 µL was added into tube steril and
was homogenized by vortex until 15 second. After being homogenized, the
tube steril was incubated in 56oC until 15 minutes and was sentrifuged in 5000
rpm until one minutes. Ethanol 96-100% 25 µL was added, so was
homogenized by vortex any more until 29 second, and waited at room
temperature about 5 minutes. The sample was taken about 675 µL, putted into
viral spin column together with the collection tube, added wash buffer 500 µL,
sentrifuged in 6800 rpm about 1 minutes, and repeated about 2 times. The
sample was dried by sentrifuge in 13.000 rpm about 1 minute and was
transferred into recovery tube. (+)RNAse free water 50 µL was added into
recvoery tube, sentrifuged in 13.000 rpm about 1 minutes and stored in -20o C
until -80o C.
2. Making PCR Mix
Mixture of PCR mix is made in a 0.2 mL PCR tube which is nuclease
free and worked in ice with a composition 2x reaction mix (a buffer containing
 0.4 mM of each dNTP, 3.2 mM MgSO4) about 7,5 µL, RNA template (100
µg/µL) about 1 µL, primer forward (10 µg/µL) about 1 µL, primer reverse (10
µg/µL) about 1 µL, superscriftTM III RT/platinumTM taq mix 2 µL, ddH2O
(autoclaved distilled water) about 2,5 µL. So, the total volume of solution is 15
µL.
3. PCR Amplification
The amplification stage was carried out on PCR machines in 35 cycles
with various kinds of PCR conditions. The stage set on the PCR machine are
cDNA synthesis at 45oC for 30 minutes and pre denaturation at 94oC for 2
minutes, both stages are carried out once. Denaturation stage at 94oC for 15
seconds, anneal at 63.4oC for 30 seconds, extension at 68oC for 1 minute and
final extension at 68oC for 5 minutes. The stage of dentauration until the final
extension is done in 35 cycles.
4. Visualize PCR Results with Agarosa Gel Electrophoresis (1%)
The results of gene amplification were visualized using the gel
electrophoresis method. 1% agarose gel is made by mixing 0.5 gr agarose and
50 mL TBE buffer. The suspension is then oven-heated for 1 minute until the
agarose powder dissolves completely. The electrophoresis results are then
cooled while being shaken slowly. After the temperature is around 50-60ºC,
0.5 µL EtBr are added. The suspension was then poured into an electrophoresis
tray which had previously been combed and insulated, then waited until it
hardened for 25-45 minutes. The hardened gel is then placed into an
electrophoresis tank and soaked by TAE 1x until all parts of the gel are
completely submerged. DNA marker (DNA ladder 100 pb) of 5 µL. Running
is carried out for 60 minutes with a voltage of 80 V and a strong current of 200
A. The results of visualization are carried out with UV translator and
documented.
 III. DISCUSSION

Reverse transcription coupled to the polymerase chain reaction (RT–PCR) is

commonly used to detect the presence of mRNAs, pre-mRNAs, or other types of RNA
such as noncoding RNAs. The method involves using a primer annealed to the RNA
of interest. For mRNA, the primer is usually a synthetic oligo (dT), a random hexamer
mixture (dN), or a synthetic DNA oligonucleotide that is complementary to a specific
transcript (a gene-specific primer) (Rio, 2014). Due to the low stability of RNA outside
the cell, the use of reverse transcriptase polymerase chain reaction (RT-PCR) for
detecting RNA is considered as a suitable marker for tracking the living cells. At the
same time, the stability of different RNAs varies outside the cell (Moalee et al., 2015).
Reverse transcription PCR (RT-PCR) is a modification of conventional PCR,
whereby RNA molecules are first converted into complementary DNA (cDNA)
molecules that can then be amplified by PCR. Norovirus and HAV are both enteric
RNA viruses, so RT-PCR has been the best (and nearly only) means for the detection
of these pathogens. RT-PCR requires more time than conventional PCR due to the
reverse transcription step; however, detection can generally be performed in 1 day.
RT-PCR can be used clinically to screen for the expression of low copy transcripts in
several body fluids and tissues. RT-PCR as a relatively simple, inexpensive, extremely
sensitive and specific tool to determine the expression level of target genes (Mo et al.,
2012). The disadvantages of RT-PCR include its complexity and problems associated
with its sensitivity, reproducibility, and specificity. Moreover, it suffers from the
problems inherent in traditional PCR when it is used as a quantitative method. The
several advantages of RT-PCR over other PCRbased quantification approaches,
including elimination of post amplification handling, easier automation, and
processing of large numbers of samples. In addition, it has a very large dynamic range
of template determination (around 6 orders of magnitude). Thus, real-time RT-PCR
offers an enormous potential for the quantification of a range of microorganisms of
medical, alimentary, and environmental importance (Bleve et al., 2003).
The first stage was isolation from the Aedes aegypti mosquito, then detected
the DENV itself in the Aedes spp. begins with the viral RNA extraction process using
QIAmp® Viral Mini Spin Kit (Qiagen) according to the QIAmp® kit protocol.
Detection of the DEN virus uses the primary generic virus D1 as the primary D2
forward primary primer as a reverse primer and uses type-specific primers TS1, TS2,
and TS3 respectively to amplify the region at 482, 119 and 290 pb. From DEN-1,
DEN-2, DEN-3, and DEN-4. There are 2 RT-PCR methods used in detecting DENV,
namely single-tube multiplex RT-PCR and nested RT-PC. The most widely used PCR
based method for detecting DENV nucleic acid in the serum is the nested RTPCR
developed and modified. The nested RT-PCR is considerably economical and has the
advantage of sero-typing, which may be relevant for public health and for patient
management as well. Many of these shortcomings can be taken care of in single-tube
multiplex reverse transcriptase-PCR (M-RT-PCR); which has been developed for use
in the detection, quantitation, and serotyping of dengue viruses from patient serum or
plasma. There is evidence in recent literature that this test is having high sensitivity
and specificity, and that reaction set-up to result time that is much shorter (Fadilla et
al., 2015).
Single-tube multiplex RT-PCR. Extraction results were used as RT-PCR
templates. Amplification was carried out using Super Script III One-Step RT-PCR kit
(Invitrogen). In the amplification process, the DNA target to be amplified was prepared
at a volume of 25 µl with a mixture composition of 2.76 µl dH2O Master Mix, 2x 12.5
µl buffer, Primary D1 (25 pmole), Primary TS1 (25 pmole), Primary TS2 (12, 5
pmole), and Primary TS3 (12.5 pmole), Enzyme 1 µl. The tube is inserted into the PCR
machine. The PCR stage begins with the reverse transcription stage (50oC, 60minutes)
followed by 40 cycles of denaturation (94°C, 30s) annealing (55°C, 1minute), and
extension (68°C, 2minutes). Nested RT-PCR method. The first amplification was
performed using the Super Script III One-Step RT-PCR kit (Invitrogen). In the
amplification process, the RNA target was prepared to be amplified at a volume of 25
µl with a mixture of Master Mix 4 µl dH2O, 2x buffer 12.5 µl, Primary D1 (25 pmole),
Primary D2 (25 pmole), Enzyme 1 µl and 5 µl template. The tube is inserted into the
PCR machine. The second amplification process uses the DyNAzeme EXT DNA
Polymerase kit with a total volume of 25µl. In the second amplification process the
Master Mix was prepared with a mixture of 19.3 µl dH2O, 10x 2.5 µl buffer, dNTP
0.5 µl Primary D1 (12 pmole), TS3 Primer (12 pmole), 0.5 µl DyNAzeme and
amplicon the first amplification. The PCR stage begins with the reverse transcription
stage (50°C, 60m) followed by 35 cycles of denaturation (94°C, 30s), annealing (55°C,
1minutes), extension (68°C, 2 minutes). Then there is the electrophoresis stage,
amplification results were electrophoresed on 1% agarose gel with power supply at
120 V for 45 minutes, loading buffers and leader markers were used using
Fermentation products, after electrophoresis, agarose gel was inserted into an ultra
violet transluminator to see DEN virus DNA tape, which is the stage of data analysis
(Fadilla et al., 2015).
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the discussion it can be concluded that there are steps to find out the
steps of molecular detection of the Dengue Virus gene from a mosquito sample, which
are isolation of RNA virus dengue, making PCR mix, PCR amplification, visualization
PCR results with agarosa gel electrophoresis (1%).

B. Suggestion

Students must be careful in using all the tools and materials used during this
practice, because there are several hazardous materials and students must be careful in
operating laboratory equipment because laboratory equipment has a highly price.

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