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What are arboviral diseases?

Arboviral disease is a general term used to describe infections caused by a group of

viruses spread to people by the bite of infected arthropods (insects) such as
mosquitoes and ticks. These infections usually occur during warm weather months,
when mosquitoes and ticks are active. Examples include California encephalitis,
Chikungunya, dengue, Eastern equine encephalitis, Powassan, St. Louis
encephalitis, West Nile, Yellow Fever, and Zika.

What are the signs and symptoms of arboviral infections?

Symptoms of arboviral infections can range from very mild to very severe. Most
people infected experience no symptoms or mild symptoms of a slight fever,
headache, muscle or joint pain, and/or a skin rash, which resolve with no serious
health problems. Severe infections are marked by a rapid onset, headache, high
fever, confusion, tremors, seizures, paralysis, coma, or death. Symptoms usually
appear 3 to 14 days after a bite from an infected mosquito or tick but can vary
depending on specific infection.

 Flaviviridae Family
 Genus Flavivirus
 The genomic such as dengue virus (DEN)
m7GpppN like those of cellular mRNAs but .
 It is transmitted by primarily Aedes aegypti followed by Aedes albopictus
and other species of Aedes.
 Aedes aegypti mosquito, a daytime feeder mosquito, is the primary vector.
 Aedes albopictus is a secondary dengue vector. These species are active for
approximately two hours after sunrise and several hours before sunset but
can bite at night in well-lit areas. This mosquito can bite people without
being noticed because it approaches from behind and bites on the ankles and
elbows.
 The dengue virus has 5 antigenically distinct serotypes; DENV-1, DENV-2,
DENV-3, and DENV-4, DENV-5.
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 40-60 nm in diameter.

 The dengue virus genome consists of a single-stranded positive-sense RNA.

 The genome consists of 10 genes that are translated into a single
polyprotein that encodes for three structural proteins, namely, capsid
(C), pre membrane (prM/M), envelope(E), and 7 nonstructural proteins
(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). (2 & 4 has A, B
division.)

 The C protein is essential for specific encapsidation of its RNA genome, M

protein plays an important role in the arrangement and maturation of the
DENV particle and the E protein present on the viral surface is essential for
the initial attachment of the virus to the host cell.
 The nonstructural proteins help in viral replication and assembly.
 The dengue virus is roughly spherical in its symmetry with approximately
50nm diameter.
 The nucleocapsid consists of the viral RNA and C proteins that form the
core of the virus.
 The nucleocapsid is surrounded by a lipid bilayer of the viral envelope
that is embedded with 180 E and M proteins across the layer.
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 The dengue virus exhibits different conformations and surface structures

during its maturation and infection, made possible by the flexibility of the
envelop protein.
 The E protein is the major exposed antigen of the dengue virus against
which the antibodies develop during the infection.
 The E protein of the four different serotypes of DENV shares 60-70%
similarity in their amino acid sequence.

Morphology of Dengue virus:

 Dengue virus is spherical enveloped viruses with single-protein capsids,

two membrane proteins, envelope and membrane.
 It has a single-stranded Positive-sense RNA genome of ~10,700
nucleotides, surrounded by a nucleocapsid and covered by a lipid envelope
that contains the viral glycoproteins.
 The RNA genome lacks poly A tail at 3‟end and contains a single open
reading frame (ORF) flanked by two untranslated regions (5’ and
3’UTRs).
 The 5’ and 3’ terminal RNA sequences of the genome form large stem
loop structures known as stem loop A (SLA) and 3’ stem loop (3’ SL)
respectively, both essential for viral replication.
 The single ORF encodes a precursor polyprotein, which is co- and post-
translational cleavage resulting in the formation of three structural proteins,
Capsid (C), membrane (M), and envelope (E), and seven non-structural
proteins, NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5.
 NS2B and NS3 form the proteases, and NS3 also has helicase and RNA
triphosphate activity.
 NS5 is the RNA dependent RNA polymerase (RdRp) and also has
methyltransferase activity.
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Capsid:
 Icosahedral capsid, 20-30 nm in diameter
 Spherical shape
Envelope:
 Envelope contains glycoprotein spike which has haemagglutinin
(HA) activity.

Mode of transmission:

 Dengue is transmitted by female Aedes aegypti and Aedes albopictus

 In order to transmit dengue between person, Mosquitoes must feed blood of
patient during 5 days period when large amount of virus are present in blood.
 Inside mosquito, virus requires additional 8-12 days incubation period (EIP)
before it can be transmitted to healthy person.
 The dengue virus mixed with saliva of mosquito and transmitted
intradermally during biting.
 In rare cases dengue is transmitted through blood transfusion, organ
transplant and from infected mother to fetus.
OR
Transmission of Dengue Virus
 The transmission of the dengue virus occurs through the bite of female
mosquitoes of the genus Aedes, primarily Aedes aegypti followed by Aedes
albopictus and Aedes polynesiensis depending on the geographical region.
 After feeding on a DENV-infected individual, the virus replicates in the
midgut of the mosquito, reaches the hemocoel and hemolymph, and further
disseminates to other secondary tissues like the salivary glands.
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 Studies have shown the presence of the viral particles within the nervous
system, salivary glands, foregut, midgut, fat body, epidermal cells, ovary,
and internal body wall lining cells of the mosquito whereas their absence
from muscle, hindgut, and Malpighian tubules.
 The extrinsic incubation period (EIP), which is the time taken from entry
of the virus to actual transmission to the new host, takes about 8-12 days
with temperatures between 25-28°C.
 Once a mosquito is infected, it is capable of transmitting the virus
throughout its lifetime.
 While the primary mode of transmission of DENV involves mosquito
vectors, there is evidence of the possibility of maternal transmission (from
pregnant mother to her child) that seems to be linked to the timing of the
infection during the pregnancy. The babies conceived from mothers with
DENV infection during pregnancy may suffer from pre-term birth, low birth
weight, and fetal distress.
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Figure: Life Cycle of Aedes albopictus.

Dengue Virus Replication

1. Virus entry: The virus enters into the host cell through receptor-mediated
endocytosis although the exact cell-surface receptors have not yet been
identified. Some possible cellular receptors include various glycoproteins (i.e.
heparin sulfates), dendritic cell-specific intercellular adhesion molecule-3-
grabbing non-integrin, or a mannose receptor. The viral E glycoprotein (acts as
ligand) interacts with the receptors and mediates attachment and entry through
the formation of endosomes.
2. Membrane fusion and release of nucleocapsid: Once internalized, the low pH
of the endosome induces a conformational change in the E protein and
mediates fusion of the viral and endosomal membrane of the host cells, and
releases the nucleocapsid into the cytoplasm.
3. Translation & replication of genome (vRNA): The nucleocapsid opens to
uncoat the viral genome which then uses the host cell‟s machinery to replicate
itself. It uses the ribosomes on the host’s ER and translates the positive-
sense viral RNA into a single polyprotein. The polyprotein is further cleaved
into individual structural and nonstructural proteins by NS2B or NS3 viral
protease and host proteases. Non-structural proteins replicate the viral RNA.
Viral replication occurs in two steps, first the positive-mRNA is copied to
negative sense RNA, which, in turn, serves as a template for the synthesis of
multiple strands of positive sense RNAs. Then the positive-sense RNA can be
used for translation.
4. Virus assembly: The newly synthesized viral RNA gets enclosed in the C
protein forming the nucleocapsid in the ER membrane and entering the rough
ER where it gets enveloped and surrounded by the M and E proteins.
5. Transport of immature viral particles: Immature virus particles travel in
vesicles to the Golgi apparatus where they undergo glycosylation and in the
acidic environment of the trans- Golgi network (TGN), furin-protease mediated
cleavage of prM in M generates maturation of the virus.
6. Release of mature virus: The mature dengue viruses are then finally released
from the cell and become capable of infecting other host cells. Mature virus is
released from the cell by exocytosis.
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Figure: Dengue Virus Replication.

Pathogenesis of Dengue

 Most primary infections of dengue with a certain serotype are asymptomatic

and only lead to mild febrile illness, although some individuals can develop
hemorrhagic fever.
 Secondary infection with a different serotype may however lead to severe
clinical manifestations like dengue hemorrhagic fever (DHF) or dengue
shock syndrome (DSS).
 An individual develops immunity after infection from a serotype against it,
however, they are still prone to future infections from the other serotypes of
the virus as the heterologous immunity declines gradually in 1-2 years.
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 Many viral, as well as host factors, contribute to the pathogenesis of DENV

such as:
 The non-structural protein 1 (NS1) viral antigen
 Dengue virus genome variations
 Subgenomic RNA
 Antibody-dependent enhancement (ADE)
 T-cells
 Anti-DENV NSI antibodies
 Autoimmunity.
 After the virus enters the bloodstream of an individual through the mosquito
bite, it infects the nearby skin cells and specialized immune cells known as
Langerhans cells.
 The viral antigen is then presented on the surface of Langerhans cells that
travel to the lymph nodes and help activate the innate immune response.
 The dengue virus further infects the macrophages and monocytes and
further disseminates throughout the body which results in viremia in the
individual.

Clinical Features:

 Infection with any one of the four DENV serotypes has the potential to
involve all human organ systems and cause a wide variety of clinical
manifestations ranging from mild febrile illness to severe and fatal disease.
 Symptomatic dengue disease is separated into 2 different clinical
syndromes, dengue fever (DF), and the more severe dengue hemorrhagic
fever (DHF); DF was described as a nonspecific febrile illness with
prominent constitutional symptoms, while DHF was defined as a distinct
syndrome characterized by increased vascular permeability, altered
hemostasis and hemorrhage. DF is also referred to as the „break bone‟
disease due to its nature of severe joint and muscular pain.
 Following an infectious mosquito bite there is an incubation period of
up to 2 weeks (commonly 5-7 days), after which the individual develops
symptoms suddenly and the illness typically follows 3 phases-
i. An initial febrile phase
ii. A critical phase starts 4-5 days from fever onset
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iii. A spontaneous recovery phase.

 In febrile phase, patient experiences sudden onset of high fever (39-40° C)
accompanied by nonspecific constitutional symptoms including headache,
general malaise, nausea, vomiting, myalgia (muscle pain), and joint pain.
 In recovery phase, the increased vascular permeability and abnormal
hemostasis are transient and usually resolve within 48-72 hours. Spontaneous
reabsorption of fluid starts around 6-8 day of illness and progress rapidly,
usually concurrent with improvement in the patient‟s symptoms. Loss of hair
has been reported during convalescence.

Primary infection:

 Primary infection with dengue virus results in a self-limiting disease

characterized by mild to high fever lasting 3 to 7 days, severe headache
with pain behind the eyes, muscle and joint pain, and a rash.
 Complications often occur within two days after the fall in temperature.
Epistaxis (bleeding from nose), bleeding of gums, passage of black stools,
rashes (petechiae, maculopapular, bruises, etc.) and sub-conjunctival
hemorrhage are indicators of increasing disease severity.
 A maculopapular rash usually appears 3 – 4 days after the onset of fever.

Primary infection:

 Secondary infection with a different dengue virus serotype is the more

common form of the disease in many parts of Southeast Asia and South
America.
 The major clinical symptoms can include high fever, hemorrhagic events,
and circulatory failure, and the fatality rate can be as high as 30%. Early
diagnosis of dengue shock syndrome is particularly important, as patients
may die within 12 to 24 hours if appropriate treatment is not administered.

The major host factors that influence clinical manifestations are:

• Age: constitutional symptoms become more prominent with increasing age, that
adults complain of headache, retro-orbital pain, and severe myalgia and arthralgia
more frequently than children.

[Arthralgia is joint pain w/o inflammation, whereas Arthritis is joint pain with inflammation.]
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• Gender: Females have a lower threshold for vascular leakage than males.
Although dengue is diagnosed more frequently in male than female patients,
female patients have a higher risk of developing DSS and of dying from this
complication than male patients.

• Pregnancy and transplacental infection: With the increasing burden of dengue

seen among young adults, exposure to infection during pregnancy is becoming
more frequent.

Host Immune Response

Primary response

IgM antibodies appear about 5 days after symptoms onset and continue to
rise to 21 days and decrease gradually.
IgG antibodies appear about 14 days after symptoms onset and persist at a
low level for life.

Secondary response

Weaker and shorter IgM response

Rapid IgG response (usually 2 days after reinfection)
High IgG level persists for 30-40 days

Laboratory Diagnosis:

 Clinical findings alone are not sufficient to make an accurate diagnosis of

DENV as several other infectious diseases may present with similar
findings, which requires the need for laboratory testing for the dengue
confirmation.
 Leucopenia, thrombocytopenia, increased hematocrit and liver enzyme
levels are common laboratory findings suggestive of DENV infection.

Sample: Blood or Tissue

1. Microscopy and staining: Direct visualization of the virus in the sample (using
electron microscopy or via fluorescent staining technique) can be done. This is
not the preferred method in diagnostic laboratories.
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2. Culture: Virus isolation in cell culture is difficult and is not the commonly used
method in diagnostic laboratories because it is a demanding procedure.

Virus may be recovered from serum, plasma, and peripheral blood mononuclear
cells. Inoculation of a mosquito cell line with patient serum, coupled with
nucleic acid assays to identify the recovered virus is a commonly used approach in
the research lab.

3. Serology
 Detection of antigen: Dengue NS1 antigen detection is useful for the
diagnosis of acute dengue infections up to 0-7 days of symptoms but
not recommended after 7 days.
 NS1 antigen has been detected in the serum of DENV infected patients
as early as 1-day post-onset of symptoms (DPO), and up to 18 DPO.
 NS1 ELISA based antigen assay is commercially available.
 NS1 assay may also be useful for differential diagnostics between
flaviviruses because of the specificity of the assay.

Result interpretation

 A positive NS1 test result confirms dengue virus infection but does not
provide serotype information.
 A negative NS1 test result does not rule out infection. People with
negative NS1 results should be tested for the presence of dengue IgM
antibodies to determine possible recent dengue exposure
 Detection of antibody

Detection of anti-dengue antibodies in serum or other body fluids by ELISA

or other rapid tests: Various methods (IgM/IgG ELISA, Hemagglutination
Inhibition Test, or rapid diagnostic kits) are available to detect anti-dengue
antibodies; IgM detection:

 Useful for the diagnosis of primary dengue infection and in distinguishing

dengue from other flavivirus infections.
 IgM antibodies are detectable in 99% of patients by day 10 after the onset of
illness.
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 IgM levels peak about two weeks after the onset of symptoms and then
decline to undetectable levels over 2–3 months.
 Sensitivity: 65-75% sensitive in a single acute serum sample.
4. Molecular diagnosis: RT PCR.

The viral disease chikungunya (CHIK) is caused by the chikungunya virus

(CHIKV), which is transmitted through the bite of infected day-biting Aedes
mosquitoes mainly Aedes aegypti & Aedes albopictus.

CHIKV virions are spherical, enveloped particles of ∼70 nm in diameter.

CHIKV is a member of the family Togaviridae, genus Alphavirus, which

comprises enveloped, positive single-stranded-RNA viruses. In humans, CHIKV
infection is of rapid onset and is typically cleared in 5–7 days.

 Genus: Alphavirus
 Family: Togaviridae
 +ve sense SS (single stranded) RNA virus
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 Linear, positive-sense, single stranded RNA molecule

 Genome size: 11.6 kb
 +SS RNA virus
 CHIKV encodes 4 nonstructural proteins (nsP1-nsP4) at the 5‟ end and 5
structural proteins encoded at the 3′ end of the genome, including 3
glycosylated proteins, namely E1, E2, E3, a small 64 amino-acids
glycoprotein 6K, and 1 non-glycosylated nucleocapsid protein C.
 E1 and E2 form heterodimeric spikes on the viron surface.
 The proper folding of p62, which is the precursor of E2, and formation of
the E1-p62 heterodimers are controlled by E3, which is therefore essential
for producing mature spikes on the Alphavirus surface.
 6K, a small 64 amino-acids glycoprotein, assists in the translocation of
structural polyproteins to the endoplasmic reticulum (ER) and in the
cleavage of p62 into mature structural proteins E2.
 The most common symptoms of CHIKV infection are fever and joint pain.
Other symptoms may include headache, muscle pain, joint swelling, or rash.

 E2 binds to cellular receptors in order to enter the host cell through receptor-
mediated endocytosis.
 E1 contains a fusion peptide which, when exposed to the acidity of the
endosome in eukaryotic cells, dissociates from E2 and initiates membrane
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fusion that allows the release of nucleocapsids into the host cytoplasm,
promoting infection.
 Subsequently, the nucleocapsid disassembles in the cytoplasm, releasing the
viral genomic RNA. The mature virion contains 240 heterodimeric spikes of
E2/E1, which after release, bud on the surface of the infected cell, where
they are released by exocytosis to infect other cells.
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Replication Strategy:

FIGURE: Chikungunya virus (CHIKV) cell entry and replication.

(1) E2 glycoprotein binds to the membrane receptor Mxra8, inducing the

translocation of clathrin molecules to the plasma membrane.
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(2) GAG, DC-SIGN, CD147, and TIM are described as CHIKV co-receptors.
CHIKV entry occurs via clathrin-mediated endocytosis pathway.

(3) Once the early endosome is formed, clathrin molecules dissociate from the
endocytic vesicle.

(4) The endosome proceed in the endocytic pathway. The pH acidification of

endocytic vesicles triggers the detachment of E1-E2 heterodimers, exposing the
fusion loop, which will culminate in the fusion of the endosome with the viral
membranes.

(5) Then, the nucleocapsid is released in the cytoplasm, genomic RNA is exposed,
and translation of the non-structural polyprotein nsP1234 will take place.

(6) The nsP1234 protein is thus cleaved by the viral protease nsP2, releasing the
individual non-structural proteins, which will form the viral replicase complex.

(7) The replicase complex is responsible for the synthesis of the negative-strand
RNA.

(8) The –ve strand RNA will be the template for new positive-strand RNA.

(9) The –ve strand RNA will also be the template for the synthesis of 26S
subgenomic RNA.

(10) The subgenomic RNA, in its turn, is translated into the structural polyprotein
C-pE2-6K-E1 in the rough endoplasmic reticulum (RER).

(10b) The C protein, which contains a protease domain responsible for its self-
cleavage, dissociates from the polyprotein just after its translation.

(11) The C protein will attach to the positive polarity genomic RNA to form
the nucleocapsid in the cytoplasm.

(10a) In this meantime, the pE2-6K-E1 precursor will be addressed to the lumen of
the ER.

(13) In the lumen of ER, pE2-6K-E1 maturation process will take place.

(14) The structural proteins will proceed in the exocytic pathway.

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(15) The pathway continues until the end of E1-E2 heterodimers is mature.

(16) E1-E2 dimers will be deposited in the cell membrane forming the „virus
budding microdomain‟, a membrane domain where the budding process will occur.

(17) The recently assembled nucleocapsid migrates to this region, and new virions
will be released to the extracellular milieu by budding.
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Transmission

Figure: Transmission patterns of CHIKV in primates and human. The sylvatic

cycle resides in primates but the urban cycle consists of man mosquito man cycle.
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Figure: Dissemination of chikungunya virus in vertebrates. Transmission of

chikungunya virus (CHIKV) occurs following a mosquito (Aedes aegypti or Aedes
albopictus) bite. CHIKV then replicates in the skin, in fibroblasts, and
disseminates to the liver, muscle, joints, lymphoid tissue (lymph nodes and spleen)
and brain. The target cells are indicated for each tissue.

Clinical Features
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Diagnosis

 Anti CHIKV IgM and IgG on blood samples

 Reverse transcription–polymerase chain reactions (RT-PCR)
 Enzyme immunoassay
 Immunofluorescence assays

Figure: Time frame for various laboratory diagnosis of Chikungunya

 Flaviviridae family
 Genus: Flavivirus

Structure of Zika Virus

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 Spherical shape
 The virus is 50 nm/40 nm in size/diameter with 180 copies of E and
M protein present in its viral membrane.
 It consists of a positive-stranded RNA genome with 3 structural
proteins and a lipid envelope.
 Icosahedral
 Contains envelope proteins E and M

Genome Structure of Zika Virus

Figure: Genome organization and polyprotein processing: ZIKV genome

(approximately 10.8 kb) is translated as a single polyprotein that is cleaved co- and
post-translationally by viral (arrows) and host (diamonds) proteases to yield the
three structural proteins C, M and E; and seven NS proteins NS1, NS2A, NS2B,
NS3, NS4A, NS4B and NS5. The 5′ and 3′ untranslated regions (UTR) are
indicated as black lines at the end of the viral genome.
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 The ZIKV consists of a single positive-stranded RNA genome of

approximately 10.7kbp genome size which encodes a single open reading
frame.
 It consists of 2 untranslated regions (UTRs) in each of its terminals.
 The single polyprotein codes for 3 structural proteins which include the
capsid(C), pre-membrane (prM), and envelope (E), and seven non-
structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
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 The structural proteins form the structure of the virus while the non-
structural proteins are involved in genome replication, viral polyprotein
processing, and regulation of host response.

Transmission of Zika Virus

The ZIKV transmission in humans mainly takes place via the bite of the infected
Aedes aegypti mosquito.

Other species of Aedes like Aedes albopictus, A. africanus, A. luteocephalus, A.

furcifer, and A. taylori are also seen to aid in the transmission of the virus.

Other routes of transmission of the virus include:

 Through sexual intercourse with the infected individual

 Through blood transfusions and organ transplantation
 Congenital infection from the mother to fetus as the virus is capable of
crossing the placental barrier
 Through accidental exposure to laboratory workers

Sign and Symptoms of Zika Virus

These symptoms are mild and usually last for 2-7 days.

 Headache
 Fever
 Skin rashes (exanthema)
 Pink eye
 Conjunctivitis
 Muscle and joint pain
 Malaise

Pathogenesis of Zika Virus

 Incubation period in mosquitoes is about 10 days.

 The vertebrate hosts of the virus are primarily monkeys and humans.
 The pathogenesis of the virus is hypothesized to start with an infection of
dendritic cells near the site of inoculation, followed by a spread to lymph
nodes and the bloodstream.
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 Flaviviruses generally replicate in the cytoplasm, but Zika virus

antigens have been found in infected cell nuclei.
 Infection with the virus appears to be linked to the development of
unusually small heads and brain damage in newborns (microcephaly). The
most dangerous time is thought to be during the first trimester of
Pregnancy– when some women do not realize they are pregnant. Experts
do not know how the virus enters the placenta and damages the growing
brain of the fetus.

Replication of Zika Virus

1. Attachment/Adsorption: The viral attachment occurs through the viral E

proteins present in its envelope with the host receptors like the C-type lectin
receptors (CLRs) expressed in myeloid cells, including monocytes,
macrophages, and dendritic cells.
2. Penetration: The virus is endocytosed through clathrin-mediated
endocytosis aided by the mechanism of molecular mimicry.
3. Uncoating: The clathrin-coated vesicle gets transported away from the
plasma membrane, and the clathrin gets removed from it. The endocytic
vesicle is then delivered to the early endosomes that mature into late
endosomes. Due to the variation of the pH environment, the viral membrane
then fuses with the endosomal membrane releasing the viral genome into the
cytoplasm.
4. Biosynthesis: The ssRNA genome is then translated to a single polyprotein
that is cleaved into the structural and non-structural proteins. The viral
replication occurs at the surface of the endoplasmic reticulum (ER) and a
dsRNA genome is synthesized from the positive-stranded ssRNA. The
dsRNA yields the viral mRNA and new ssRNA products through
transcription and replication.
5. Assembly: The viral assembly occurs at the endoplasmic reticulum from
where it buds off and then enters the Golgi apparatus.
6. Maturation: Viral maturation occurs in the Golgi apparatus where the prM
protein of the immature virion gets cleaved and the mature viral particle is
released into the cytoplasm.
7. Release: The viral particle is released from the cell through the process of
exocytosis.
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Diagnosis of Zika Virus

Culture: The virus can be cultured and isolated by inoculation in chicken embryo
yolk sacs, allantoic sacs, and chorioallantoic membrane, as well as cell cultures in
Vero, rhesus monkey kidney, and pig kidney cells. It can also be inoculated in
suckling mice but shows less sensitivity than the cell cultures. The ZIKV has been
successfully cultured from human blood, semen, and urine.

Molecular Detection of ZIKV RNA: The ZIKV RNA viruses can be detected by
gene amplification in a two-step procedure through the reverse transcription of
genomic RNA into a single-stranded DNA (cDNA), followed by the conversion to
double-stranded DNA and the amplification of the DNA. Real-time PCR can be
performed for a faster detection than conventional PCR.

Serological Diagnosis: The serological test for ZIKV is performed by ELISA and
the test is confirmed by PRNT (Plaque Reduction Neutralization Test) according to
the standard protocols. PRNT is considered the “gold standard” for anti-Flavivirus
antibody differentiation.

The IgM antibody response in primary Flavivirus was found to be specific for
ZIKV with limited cross-reactivity with other flaviviruses. However, a high degree
of serological cross-reactivity was observed during the secondary Flavivirus
infection with other flaviviruses with both IgM ELISA and PRNT. PRNT is,
however, expensive and performed only in highly specialized laboratories.

Diagnosis of ZIKV in Endemic Countries: In countries with limited access to

molecular testing methods, diagnosis is often performed by serological testing by
IgM ELISA or rapid tests. A combined NS1 antigen and IgM antibody test
increase the sensitivity and specificity of dengue fever diagnosis. If several
patients test negative by DENV NS1 test, Zika and other Flavivirus infections
are suspected.

PCR: It is useful in the first 3-5 days after the onset of symptoms. It helps in the
direct detection of Zika virus RNA or specific viral antigens in clinical specimens.
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Serology Test: It detect the presence of antibodies but are useful only after five
days.

 Family Flaviviridae
 The disease occurs in tropical Africa and South America, never been
reported in Asia despite the presence of the vector
 It is an acute viral hemorrhagic disease transmitted by infected mosquitoes.
The “yellow” in the name refers to jaundice that affects some patients.
 Flavivirus Genus
 Transmitted by Aedes & Haemagogus spp.

Morphology and genome:

 positive-sense single-stranded RNA virus

 Viral particles are 43 nm in size; they are made up of a ribonucleoprotein
core and a lipoprotein envelope
 Heterogeneity among yellow fever strains.
 Nearly spherical
 Genome is 11,000 nucleotides long (11 kb) and has a single open reading
frame encoding a polyprotein.
 Host proteases cut this polyprotein into three structural (C, prM, E) and
seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B,
NS5.

NCR= UTR
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Replication:

 Yellow fever virus‟s surface E protein has role in binding, fusion and cell
entry is most important.
 The genome of the YFV has two distinct non-coding regions; the 3′ NCR
of these regions is a control mechanism for the virus‟s replication and
level of virulence.
 After entering the host cell, the viral genome is replicated in the rough
endoplasmic reticulum (ER) and in the so-called vesicle packets.
 At first, an immature form of the virus particle is produced inside the
ER, whose M-protein is not yet cleaved to its mature form, so is
denoted as precursor M (prM) and forms a complex with protein E.
 The immature particles are processed in the Golgi apparatus by the host
protein furin, which cleaves prM to M.
 This releases E from the complex which can now take its place in the
mature, infectious virion.
 Virus is released by exocytosis.
 SHABUJ

Figure: YFV Replication.

Pathogenesis:

 The incubation period is generally 3 to 6 days but maybe longer.

 A short incubation period followed by an acute biphasic illness.
 Severity ranges in severity from non-specific, self-limited symptoms of
fever, malaise, photophobia and headache to an illness of sudden onset
with fever, vomiting, and prostration which may progress to jaundice
and hemorrhage.
 After transmission from a mosquito, the viruses replicate in the lymph
nodes and infect dendritic cells in particular.
 SHABUJ

 From dendritic cells, they reach the liver and infect hepatocytes (probably
indirectly via Kupffer cells), which leads to eosinophilic degradation of
these cells and to the release of cytokines.
 Apoptotic masses known as Councilman bodies appear in the cytoplasm
of hepatocytes.
 Fatality may occur when cytokine storm, shock, and multiple organ
failure follow.

Clinical manifestation:

The initial phase of illness:

 Viremia renders the patient infectious to biting mosquitoes and is also

responsible for the acute constitutional symptoms (nonspecific febrile
illness, headache, malaise, nausea, muscle pain).
 Signs and symptoms of yellow fever may be a flushing (rednees) of the
head and neck, conjunctival injection, strawberry tongue and a relative
bradycardia.

The second phase:

 Severity in the second phase of the acute illness

 Death usually occurs 7 to 10 days after the onset of illness

Symptoms:

 Most people will not have symptoms.

 Some people will develop yellow fever illness with initial symptoms
including:
 Sudden onset of fever
 Chills
 A severe headache
 Back pain
 General body aches
 Nausea
 Vomiting
 Fatigue (feeling tired)
 Weakness
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Severe symptoms include:

 High fever
 Yellow skin (jaundice)
 Bleeding
 Shock
 Organ failure

Among those who develop severe disease, 30-60% die.

Diagnosis:

Yellow fever is difficult to diagnose, especially during the early stages. A more
severe case can be confused with severe malaria, leptospirosis, viral hepatitis
(especially fulminant forms), other hemorrhagic fevers, infection with other
flaviviruses (such as dengue hemorrhagic fever), and poisoning.

1. The first diagnosis comes from neutropenia, the decrease in circulating

neutrophils.
2. The IgM antibody capture ELISA is used.
3. Immunofluorescence tests can also determine its presence.
4. HAI (HemAgglutination Inhibition Test), CF and NT (Neutralization Test)
tests are further used to detect the virus.
5. Isolation of virus by intracerebral or intraperitoneal inoculation of
suckling mice or by the intrathoracic inoculation of male Aedes aegypti or
Toxorhynchites mosquitoes.
6. Real-time reverse transcription polymerase chain reaction (RT-PCR).

Vaccine (LAV) available & provide protection for 10 years.

o Family Flaviviridae
o The Japanese encephalitis virus causes Japanese encephalitis (JE), an
infection of the brain.
o Symptoms include headache, vomiting, fever, confusion, and seizures. This
occurs between 5 and 15 days following infection.
o JEV is typically transmitted by mosquitoes, specifically the Culex species.
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o Pigs and wild birds act as the virus‟ reservoir.

o Most important one for human infection is Culex tritaeniorrhynchus, which
breeds in pools of stagnant water.
o Can be Zoonotic as pig and birds can transmit the disease to human.

Structure of JEV:

o Spherical particles that are approximately 50 nm in diameter.

o Surrounded by a lipid bilayer envelope.

Genome of Japanese Encephalitis (JE) Virus

 +SS RNA Genome, approximately 11kb long.

 This RNA contains a 5′ cap (m7G5‟ppp5‟A) at the 5′ end and lacks a
polyadenylate tail.
 Single ORF translates into a polyprotein.
 Surrounding the ORF are 5′ and 3′ noncoding regions (NCRs) of around 100
nucleotides (nt) and 400 to 700 nt, respectively.
 The genome is translated as a large polyprotein that is processed co- and
post–translationally by cellular proteases and a virally encoded serine
protease into at least 10 discrete products.
 SHABUJ

 The N-terminal one quarter of the polyprotein encodes the structural

proteins, and the remainder contains the nonstructural (NS) proteins, in the
following order: C-prM-E-NS1-NS2A-NS3-NS4A-NS4B-NS5.
 3 viral proteins are structural: the E (envelope), M (membrane), and C
(capsid) proteins.
 The E protein (50kd) is the major surface protein of the viral particle,
probably interacts with viral receptors, and mediates virus–cell membrane
fusion.
 M protein is a small proteolytic fragment of prM protein (26kd), which is
important for maturation of the virus into an infectious form.
 C protein (about 11 kd) is highly basic, consistent with its proposed role in
forming a ribonucleoprotein complex with packaged genomic RNA.

Transmission of disease

 Human is an accidental host of the JEV.

 After an infected mosquito bites a human, the earliest viral replication
occurs in local and regional lymph nodes. Viruses likely invade the central
nervous system through the blood, producing infection and disease.
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Replication:

o Attachment of the viral envelope protein E to host receptors mediates

internalization into the host cell by clathrin-mediated endocytosis, or by
apoptotic mimicry.
o Fusion of virus membrane with host endosomal membrane and results
in release of RNA genome into the cytoplasm.
o The positive-sense genomic ssRNA is translated into a polyprotein, which is
cleaved into all structural and non structural proteins to yield the replication
proteins.
o Following translation and processing of the viral proteins, a viral replicase is
assembled from NS proteins, the viral RNA, and presumably some host
factors.
o Replication begins with the synthesis of a genome-length minus-strand
RNA, which then serves as a template for the synthesis of additional plus-
strand RNAs.
o Virus assembly occurs at the endoplasmic reticulum.
o The virion buds at the endoplasmic reticulum and is transported to the
Golgi apparatus.
o The prM protein is cleaved in the Golgi, thereby maturing the virion which
is fusion competent.
o Release of new virions by exocytosis.
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Signs and symptoms of Japanese Encephalitis (JE) Virus

Japanese encephalitis (JE) is a viral infection that can cause inflammation of the
brain. Symptoms of JE virus infection can range from mild to severe, and some
people may not have any symptoms at all. Common signs and symptoms of JE
virus infection include:

 Fever
 Headache
 Nausea and vomiting
 Fatigue and weakness
 Muscle pain and joint pain
 Encephalitis (inflammation of the brain)
 Seizures
 Stiff neck
 Confusion or disorientation
 Paralysis or muscle weakness
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 Tremors or jerky movements

 Coma

Symptoms usually develop 5 to 15 days after being bitten by an infected mosquito.

In some cases, JE virus infection can lead to severe complications such as brain
damage, coma, and even death.

The clinical picture of the Japanese encephalitis includes 3 phases:

1. Prodromal phase: The incubation period is 1-6 days and the

characteristic features of disease condition include malaise, anorexia,
headache, fever and vomiting. In children, diarrhea and abdominal
pain may be prominent.
2. Acute encephalitic phase: The incubation period is 7-13 days. The signs
and symptoms includes photophobia, hyper excitability, focal and
neurologic signs, muscular rigidity, dull, mask like face, tremulous eye
movements, cranial nerve palsies, speech impairment ad stiff neck.
3. Late convalescent phase: The incubation period is 14-15 days. The
clinical picture demonstrated as mental impairment, neurological signs
and symptoms, epilepsy, abnormal movements, and CNS injury.

Pathogenesis and pathology of Japanese Encephalitis (JE) Virus

The portal of entry for the JE virus is through the bite of mosquito which
contain virus.
After the bite on skin the virus enter the Reticuloendothelial system (RES)
and follows transient phase of viremia.
After the transient viremia the virus invades the central nervous system.
The virus enters the neuroparenchyma by crossing the capillary walls of
brain and distributes itself in hypothalamus, hippocampus, substantia nigra
and medulla oblongata regions of brain via vascular endothelial cells by the
mechanism of endocytosis.
The mechanism of endocytosis is either cholesterol or clathrin mediated
pathway.
 SHABUJ

Virus then replicates in neurons and matures in the neuronal secretory

system.
JE typically develops in patients after an incubation period of 5–15 days.
It is possible that during this time, the virus resides and multiplies within
host leukocytes, which act as carriers to the CNS.
T lymphocytes and IgM play a major role in the recovery and clearance
of the virus after infection.
Besides neuronal cells, researchers have shown that astrocytes are also
infected by JEV.
Crossing the blood brain barrier is an important factor in the increased
pathogenesis and clinical outcome of the neurotropic viral infection.
Astrocytes, being a component of the blood–brain barrier, may help in the
transmission of JEV from peripheral tissues to the cerebrospinal fluid.
It is known that JEV causes neuronal cell death in two ways: direct
neuronal killing , wherein viral multiplication within neuronal cells leads to
cell death, and the indirect mode of killing, wherein massive inflammatory
response causes an up-regulation of reactive oxygen species and cytokines
such as tumor necrosis factor α (TNFα), which, in turn, causes neuronal
death.

Laboratory Diagnosis of Japanese Encephalitis (JE) Virus

 Specimens: CSF, Blood, Plasma, Serum, Tissue(rare)

 Detection of JE virus–specific IgM by using a JE virus–specific IgM-
capture ELISA on CSF or serum. JE virus–specific IgM can be measured
in the CSF of most patients by 4 days after onset of symptoms and in
serum by 7 days after onset.
 Plaque reduction neutralization tests can be performed to confirm the
presence of JE virus–specific neutralizing antibodies and discriminate
between cross-reacting antibodies from closely related flaviviruses.
 Detection of JE antigen in tissue by immunofluorescence or
immunohistochemistry.
 Detection of JE virus genome in serum, plasma, blood, CSF or tissue by
RT-PCR.
 SHABUJ

Vaccine: One JE vaccine is licensed and available in the United States which is an
inactivated Vero cell culture derived vaccine.

Table: Arboviruses.

Dengue Chikungunya Zika Yellow Fever Japanese

Encephalitis
Family Flavivirid Togaviridae
Flaviviri Flaviviridae Flaviviridae
ae dae
Genus FlavivirusAlphavirus Flaviviru Flavivirus Flavivirus
s
Transmi Aedes Aedes aegypti, Aedes Aedes & Culex
tted by aegypti, Aedes aegypti, Haemagogus tritaeniorrhyn
Aedes albopictus Aedes spp. chus
albopictus albopictu
s,
Envelop Yes Yes Yes Yes Yes
ed?
Size 50 nm 70 nm 50 nm 43 nm 50 nm
(Diamet
er)
Shape Spherical Spherical Spherical Nearly Spherical
spherical
Capsid Icosahedr Icosahedral Icosahedr Icosahedral Icosahedral
Shape al al
Genome +SS RNA +SS RNA +SS +SS RNA +SS RNA
/NA (Linear) RNA
Genome 10.7 Kb 11.6 Kb 10.7/10.8 11 Kb 11 Kb
Size Kb
Presenc On both On both ends On both On both ends On both ends
e of ends ends
UTR
Stem Yes 3'UTR forms Yes Yes Yes
loop a highly stable
structur and conserved
e on 5’ Y-shaped
& 3’ stem-loop.
Termina
l
Number 1 2 1 1 1
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of ORF
Occurre Yes Yes Yes Yes Yes
nce of
Cap at
the 5’
End
Occurre No Yes No No No
nce of
Poly (A)
tail at 3’
end
Structur 3 (C, 5 (C, E3, E2, 3 (C, 3 (C, PrM, E) 3 (C, PrM, E)
al PrM, E) 6K, E1) PrM, E)
Proteins
Non– 7 (NS1, 4 (NS1, NS2, 7 (NS1, 7 (NS1, NS2a, 7 (NS1, NS2a,
Structur NS2a, NS3, NS4) NS2a, NS2b, NS3, NS2b, NS3,
al NS2b, NS2b, NS4a, NS4b, NS4a, NS4b,
Proteins NS3, NS3, NS5) NS5)
NS4a, NS4a,
NS4b, NS4b,
NS5) NS5)
Major E protein C, E2 E E E
Antigen
Entry is E E2 E E E
mediate
d by
Protease NS2b, NSP2, NS2b, NS2b, NS3 NS2b, NS3
by NS NS3 C protein has NS3
proteins self–cleaving
protease
RdRp NS5 NSP4 NS5 NS5 NS5
by NS
protein
Detectio 1. Micros 1. Anti 1. 1. 1. ELISA
n copy CHIKV IgM Culture Neutropenia (IgM)
(Not and IgG on in Vero 2. ELISA 2. PRNT
preferr blood samples cell, (IgM) 3.
ed) 2. Reverse Rhesus 3. Immunofluore
2. Cultur transcription– monkey Immunofluore scence test
 SHABUJ

e polymerase & Pig scence tests. 4. RT PCR

(Unco chain Kidney 4. HAI
mmon) reactions (RT- cell (HemAgglutin
3. Serolo PCR) 2. RT ation
gy: 3. Enzyme PCR Inhibition
NS1, immunoassay 3. Test), CF and
IgM, (EIA) Serodiag NT
IgG 4. nosis by (Neutralizatio
4. RT Immunofluore ELISA & n Test)
PCR scence assays confirme 5. RT-PCR
d by
PRNT.