UTS

Introduction
 Arbovirus is a term used to refer to a group of viruses that
are transmitted by arthropod vectors
 The word arbovirus is an acronym (ARthropod-BOrne
virus)
 Many arboviruses are transmitted by bloodsucking
arthropods from one vertebrate host to another.
 The vector acquires a lifelong infection through the
ingestion of blood from a viremic vertebrate.
 The viruses multiply in the tissues of the arthropod without
evidence of disease or damage.
 Some arboviruses are maintained in nature by
transovarian transmission in arthropods.
 The major arbovirus diseases worldwide are yellow
fever, dengue, Japanese encephalitis, St. Louis
encephalitis, western equine encephalitis, eastern
equine encephalitis, Chickungunya, Russian
spring-summer encephalitis, West Nile fever, and
sandfly fever
Classification
 Arboviruses are classified within six families
 Most are members of families Togaviridae,
Flaviviridae and Bunyaviridae
 Some are assigned to families Reoviridae,
Orthomyxoviridae and Rhabdoviridae
 Within Togaviridae, only one (Alphavirus) of the
two genera contains arthropod borne viruses
while the other genus (Rubivirus) does not
contain arthropod borne virus
 Likewise, the Flaviviridae contains three genera
(Flavivirus, Pestivirus and Hapacivirus), but
only Flavivirus contains arthropod borne
viruses
 Bunyaviridae consists of 5 genera (Bunyavirus,
Hantavirus, Nairovirus, Phlebovirus and
Tospovirus) and none of them contains
arthropod borne viruses; genus Hantavirus
contains viruses that are transmitted by rodents
 Most alphaviruses and flaviviruses survive in nature by
replicating alternately in a vertebrate host and a hematophagous
arthropod (mosquitoes or, for some flaviviruses, ticks).
 Arthropod vectors acquire the viral infection by biting a
viremic host, and after an incubation period during which the
virus replicates in the vector's tissues, they transmit virus
through salivary secretions to another vertebrate host.
 Virus replicates in the vertebrate host, causing viremia and
sometimes illness.
 The ability to infect and replicate in both vertebrate and
arthropod cells is an essential quality of alphaviruses and
flaviviruses.
 The principal vertebrate hosts for most are various
species of wild mammals or birds.
 The natural zoonotic cycles that maintain the virus do
not usually involve humans
 However, a few viruses (yellow fever virus, dengue
virus types 1, 2, 3 and 4 and chikungunya virus) can be
transmitted in a human-mosquito-human cycle.
Togaviridae Alphavirus
Alphavirus
 In the Togaviridae family, the Alphavirus genus consists of more
than 30 viruses, 60 to 70 nm in diameter, enveloped and possess a
single-stranded, positive-sense RNA genome
 The envelope surrounding the particle contains two glycoproteins.
 Alphaviruses often establish persistent infections in mosquitoes
and are transmitted between vertebrates by mosquitoes or other
blood-feeding arthropods.
 They have a worldwide distribution.
 All alphaviruses are antigenically related.
 The viruses are inactivated by acid pH, heat, lipid solvents,
detergents, bleach, phenol, 70% alcohol, and formaldehyde.
 Most possess hemagglutinating ability
Structure and genome of alphavirus
 Structure
 Virions are spherical, 60 to 70 nm in diameter, with an icosahedral
nucleocapsid enclosed in a lipid-protein envelope
 Alphavirus RNA is a single stranded strand, that is capped and
polyadenylated.
 Alphavirus genomes that have been sequenced in their entirety are
approximately 11.7 kilobases long.
 Virion RNA is positive sense: it can function intracellularly as
mRNA, and the RNA alone has been shown experimentally to be
infectious.
 The alphavirus envelope consists of a lipid bilayer derived from
the host cell plasma membrane and contains two viral
glycoproteins (E1 and E2)
 A small third protein (E3) remains virion-associated in
Semliki Forest virus but is dispatched as a soluble protein
in most other alphaviruses.
 The only proteins in the envelopes of alphaviruses are the
viral glycoproteins, each anchored in the lipid
 On the virion surface, E1 and E2 are closely paired and
appear as "spikes"
 Classification and Antigenic Types
 Classification is based upon antigenic relationships.
 Viruses have been grouped into seven antigenic complexes;
typical species in four medically important antigenic
complexes are Venezuelan equine encephalitis, eastern
equine encephalitis, western equine encephalitis, and
Semliki Forest viruses.
 Genome sequence information typically obtained after viral
RNA has been amplified by polymerase chain reaction
(PCR) is used with increasing frequency in the
identification and classification of new viruses.
 The capsid protein induces antibodies, some of which
are widely cross-reactive within the genus by
complement fixation and fluorescent-antibody tests.
 Similarly, hemagglutination-inhibiting antibodies
may react with either E2 or E1.
 Neutralization assays are virus-specific, and species
or subtypes are defined principally on the basis of
neutralization tests
Replication of
Alpha virus
 Multiplication
 Alphaviruses attach to cells, probably via interactions
between E2 and a poorly defined family of cellular
receptors found on many vertebrate and invertebrate cells.
 A few receptors (for example, dendritic cell-specific
CD209), liver and lymph node-SIGN (L-SIGN; also known
as CLEC4M), heparan sulphate, laminin and integrins)
 Entry takes place in mildly acidic endosomal vacuoles
where glycoprotein spikes undergo conformational
rearrangements and an acid-dependent fusion event
(principally a function of E1) delivers genomic RNA to the
cell cytoplasm.
 Viral replication occurs in the cytoplasm.
 Initial translation of virion RNA produces a polyprotein that
is proteolytically cleaved into an RNA polymerase.
 Transcription of the virion RNA through a negative-strand
RNA intermediate produces a 26S positive-strand mRNA
which encodes only the structural proteins, as well as
additional 42S RNA, which is incorporated into progeny
virions.
 Translation from the 26S mRNA (which represents the 3'
one-third of genomic RNA) produces a polyprotein that is
cleaved proteolytically into three proteins: C, PE2, and E1;
PE2 is subsequently cleaved into E2 and E3.
 Envelope proteins formed by posttranslational cleavage
are glycosylated and translocated to the plasma
membrane.
 Virion formation occurs by budding of preformed
icosahedral nucleocapsids through regions of the plasma
membrane containing E1 and E2 glycoproteins
 Host Defenses
 Differences in susceptibility between individuals and species are
not easily ascribed to specific immune responses, and a variety of
non-specific defense mechanisms may be important.
 Alphaviruses are efficient inducers of interferon, the production of
which probably plays a role in modulating or resolving infections.
 Antibodies are important in disease recovery and resistance.
 The appearance of neutralizing antibodies in serum coincides with
viral clearance, and immune serum can diminish or prevent
alphavirus infection.
 Although their precise roles are not clearly established, T-cell
responses are also demonstrable and may contribute substantially to
immunity.
 Lasting protection is generally restricted to the same
alphavirus, and is associated with (but not solely
attributable to) the presence of neutralizing antibodies.
 Cross-reactive immunity among different alphaviruses is
sometimes observed in the absence of cross-neutralizing
antibodies.
 In experimental animals, such immunity can be mediated
by cytolytic nonneutralizing antibodies.
 The role of T cells is less clear but has been inferred from
cytotoxic and other effector activities in vitro that may be
alphavirus specific or cross-reactive
 Epidemiology
 Eastern and western equine encephalitis viruses are maintained
in natural ecologic cycles involving birds and, principally, bird-
feeding mosquitoes such as Culiseta melanura.
 Eastern equine encephalitis (EEE) virus is enzootic in fresh
water swamps and causes sporadic equine and rare human cases.
 Cs. melanura mosquitoes usually do not feed on humans,
transmission to horses and humans is potentiated when less
fastidious Aedes species feed upon an adequate natural reservoir
of infected birds.
 EEE virus outbreaks are typically recognized upon the
occurrence of severe equine or human encephalitis in a discrete
geographic area.
 Western equine encephalitis (WEE) virus whose principal vector,
Culex tarsalis, is a common mosquito, especially in irrigated regions.
 Eight or more antigenic subtypes of Venezuelan equine encephalitis
(VEE) virus exist; they have differing virulence and epidemic
potentials.
 At least 10 different species of mosquitoes, including Culex and
Aedes species, may transmit VEE virus, and vector competence varies
for enzootic versus epizootic subtypes.
 Birds do not seem to play an important reservoir role in nature.
 The enzootic, less pathogenic strains are maintained in mosquito-
rodent-mosquito cycles.
 Enzootic strains are ecologically restricted to cycles between small
mammals and mosquitoes.
 Sporadic and sometimes severe human cases have been described.
 In contrast to other alphavirus encephalitides, epizootic strains of
VEE are mainly amplified in horses, so that equine cases occur
prior to reports of human disease
 Chikungunya virus exists in Africa in a forest cycle involving
baboons and other primates and forest species of mosquitoes.
 It can also be transmitted in a human-mosquito-human cycle by
Aedes aegypti.
 This mode of transmission has caused massive epidemics in
Africa, India, and Southeast Asia.
 The virus is endemic throughout much of south and Southeast
Asia.
 The antigenically similar Mayaro virus exists in the Amazon
Basin.
 Its cycle involves new world primates and
hematophagous mosquitoes and causes outbreaks of
human disease through exposure to the forest cycle.
 Ross River virus is endemic in Australia and has
spread in epidemic form to several islands of the
Western Pacific.
 Pathogenesis and Clinical Manifestations
 Human illness caused by alphaviruses is exemplified by agents
that produce three markedly different disease patterns.
 Chikungunya virus is the prototype for those causing an acute (3-
to 7-day) febrile illness with malaise, rash, severe arthralgias, and
sometimes arthritis.
 O'nyong'nyong, Mayaro, and Ross River viruses, which are
closely related (antigenically) to chikungunya virus, cause similar
or identical clinical manifestations; Sindbis viruses cause similar
but milder diseases known as Ockelbo (in Sweden), Pogosta
(Finland), or Karelian fever (Russia).
 Virus introduced by the bite of an infected mosquito replicates and
causes a viremia coincident with abrupt onset of fever, chills,
malaise, and joint aches.
 The viremia subsides in 3 to 5 days, and antiviral antibodies
appear in the blood within 1 to 4 days of the onset of
symptoms.
 A macular-papular rash typically develops around the third
to fifth day of illness, when the patient is defervescing.
 The migratory arthralgia, which is so characteristic of these
viral diseases, involves mainly the small joints and occurs
more prominently in adults than children.
 In more severe cases the involved joints are swollen and
tender, and rheumatic signs and symptoms may persist for
weeks or months following the acute illness.
 The pathogenesis of eastern equine encephalitis and western
equine encephalitis virus infection of humans (as well as of
equines) similarly involves percutaneous introduction of virus
by a vector and development of viremia; however, the majority
of human infections with these viruses are either asymptomatic
or present as a nonspecific febrile illness or aseptic meningitis.
 The ratio of neurologic disease per human infection is
estimated for eastern equine encephalitis as 1:23.
 For western equine encephalitis this ranges from about 1:1000
in adults to nearly 1:1 in infants, respectively.
 Symptoms usually begin with malaise, headache, and fever,
followed by nausea and vomiting.
 Over the next few days the symptoms intensify, and
somnolence or delirium may progress into coma.
 Seizures, impaired sensorium, and paralysis are common.
 The severity of neurologic involvement and sequelae is
greater with decreasing age.
 Histopathologic findings resulting from neuronal invasion
and replication are similar to those of most other acute viral
encephalitides, and include inflammatory cell infiltration,
perivascular cuffing, and neuronal degeneration.
 All regions of the brain may be affected.
 Venezuelan equine encephalitis virus infection in humans
routinely produces an acute febrile illness with pronounced
systemic symptoms, whereas the central nervous system
disease occurs only infrequently and usually is much less
severe than in eastern and western equine encephalitis.
 Following an incubation period of 2 to 6 days, patients
typically develop chills, high fever, malaise, and a severe
headache.
 A small percentage of human infections (less than 0.5% in
adults and up to 4% in children, but probably varying with
virus subtype) will progress to neurologic involvement with
lethargy, somnolence or mild confusion, and possibly nuchal
rigidity.
 Seizures, ataxia, paralysis, or coma herald more severe
central nervous system invasion.
 Overt encephalitis is more commonly seen in infected
children, where case fatalities range as high as 35% in
comparison to 10% for adults.
 However, for those who survive encephalitic
involvement, neurologic recovery is usually complete.
Diagnosis
 Diagnosis of alphavirus infection is suggested by clinical evidence
and known risk of exposure to virus.
 It can be confirmed only by laboratory tests
 In conjunction with laboratory serologies, the geographic locale and
patient's travel history are of major importance in diagnosing an
arboviral encephalitis
 Laboratory diagnosis can be established by isolating virus from the
blood during the viremic phase or by antibody determination.
 A variety of serologic tests, especially neutralization, but also
enzyme-linked immunosorbent assay (ELISA), hemagglutination
inhibition, complement fixation are used by public health
laboratories to diagnose alphavirus infections.
 Testing by ELISA for specific IgM is particularly useful in
discriminating recent infection with one alphavirus from
previous exposure to another alphavirus.
 An increasing number of laboratories have the capacity to
diagnose alphavirus infections by detection of viral RNA
(e.g. using polymerase chain reaction, PCR)
 Control
 Control of alphavirus diseases is based on surveillance of
disease and virologic activity in natural hosts and, when
necessary, on control measures directed at reducing
populations of vector mosquitoes.
 These measures include control of larvae and adult
mosquitoes, sometimes by using ultra-low-volume aerial
spray techniques.
 In some areas, insecticide resistance (for example, resistant C
tarsalis) is a major limitation to effective control.
 Inactivated vaccines are used to protect laboratory workers
from eastern, western, and Venezuelan equine encephalitis
viruses.
 An effective live attenuated Venezuelan equine encephalitis
vaccine has been employed extensively in equines as an
epidemic control measure, and a similar vaccine is used to
protect laboratory workers.
 A live attenuated chikungunya vaccine has proven safe and
immunogenic in investigational human trials.
 Inactivated vaccines are used to protect laboratory workers from
eastern, western, and Venezuelan equine encephalitis viruses.
 An effective live attenuated Venezuelan equine encephalitis
vaccine has been employed extensively in equines as an
epidemic control measure, and a similar vaccine is used to
protect laboratory workers.
 A live attenuated chikungunya vaccine has proven safe
Flaviviridae Flavivirus
 Family Flaviviridae
 Genus Flavivirus
 Tick-borne viruses
 Mammalian tick-borne virus group
 Kyasanur forest disease virus (KFDV)
 Tick-borne encephalitis virus (TBEV)
 Mosquito-borne viruses
 Dengue virus group
 Dengue virus (DENV)
 Japanese encephalitis virus group
 Japanese encephalitis virus (JEV)
 Murray Valley encephalitis virus (MVEV)
 St. Louis encephalitis virus (SLEV)
 West Nile virus (WNV)
 Spondweni group
 Spondweni virus
 Zika virus (ZIKV)
 Yellow fever virus group
 Yellow fever virus (YFV)
 Flaviviruses were initially included in the togavirus family as "group B
arboviruses" but were moved to a separate family because of differences
in viral genome organization, replication strategies, structure and
biochemistry
 Flaviviridae contains three genera (Flavivirus, Pestivirus and
Hapacivirus), of which only Flavivirus contains arthropod borne viruses
 They are primarily spread through arthropod vectors (mainly ticks and
mosquitoes).
 The family Flaviviridae gets its name from the Yellow Fever virus, the
type virus of Flaviviridae; flavus means yellow in Latin. (Yellow fever
in turn was named because of its propensity to cause jaundice in victims.
 Viruses such as JEV, Dengue viruses and Yellow fever viruses belongs to
genus Flavivirus
Flavivirus
Structure
 The virus particles are spherical, with a diameter of 40–60
nm in diameter and consist of a nucleoprotein capsid
enclosed in a lipid envelope.
 The RNA is a single, positive-sense strand and is capped
at the 5' end, but, unlike alphaviruses, has no poly A
segment at the 3' end
 The virion has a single capsid protein (C)
 The envelope consists of a lipid bilayer, a single envelope
protein (E) of 51,000-59,000 daltons, and a small non-
glycosylated protein (M) of approximately 8,500 daltons.
 All flaviviruses are antigenically related by sharing common or
similar antigenic determinants on C and E proteins.
 The single envelope glycoprotein, E, is the viral hemagglutinin and
antibodies against E are involved in virus neutralization and
hemagglutination inhibition.
 The antigenic determinants that induce neutralizing antibody are
specific, and species or subtypes of flaviviruses are distinguished
principally by neutralization tests.
 Hemagglutination inhibition tests reveal a broad range of cross-
reactions among the flaviviruses.
 The nonstructural proteins also are antigenic, and at least one
nonstructural protein, NS-1, contains both virus-specific and cross-
reactive epitopes.
Viral genome
 Viral genome
 The nucleic acid of flaviviruses consists of a single molecule of
positive sense ssRNA.
 A single open reading frame(ORF) on the genomic RNA is
translated directly into a polyprotein, which is further processed
into the three structural proteins., these are the internal RNA
associated C protein and then the two envelope proteins, pre-M
and E.
 The pre-M protein is a glycosylated precursor protein which is
cleaved during or shortly after release from the cell into the
non-glycosylated M membrane
 The E membrane protein is usually glycosylated and is
considerably larger, with a molecular weight of 51–59k Da
 The core protein C is rich in arginine and lysine, with a
molecular weight of 14–16kDa.
 Following the translation of the three structural proteins,
seven non structural proteins are produced—the
glycoproteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and
NS5. Two of these proteins, NS3 and NS5, are components
of the RNA replicase.
 The gene order is thus 5’ – C –pre –M–E–NS1–NS2A –
NS2B–NS3–NS4A–NS4B–NS5-3’
Replication of
Flavivirus
Replication
 Flaviviruses enter cells by receptor-mediated endocytosis and
fuse their membrane with that of the endosome.
 The mechanism by which flaviviruses enter cells involves an
interaction between the E protein and cellular receptors,
followed by a post-attachment fusion event that occurs in
acidic intra-cytoplasmic vacuoles
 The acidic pH of this compartment triggers an irreversible
conformational change in the viral fusion protein E that drives
the fusion of the viral membrane with the endosomal
membrane, resulting in the release of the viral genome into the
cytoplasm
 Naked genomic RNA is then introduced into the cytoplasm and
it serves as mRNA for all proteins.
 Structural proteins are encoded at the 5' end of the genome, and
nonstructural proteins (e.g., NS-1 and RNA-dependent RNA
polymerase) are encoded in the 3' two-thirds.
 Flavivirus proteins arise by co- or post-translational cleavage of
the polyprotein encoded by the genome.
 Complementary (negative-sense) RNA is made from genomic
RNA that serves as a template to generate genomic RNA.
 Assembly of flaviviruses takes place in the endoplasmic
reticulum (ER) leading to the formation of immature virions
that are transported through the exocytotic pathway of the cell.
 Virus maturation occurs in the Trans-Golgi network (TGN)
 The precursor of M (prM) is cleaved late in viral
morphogenesis and is thought to stabilize the E protein
during early events of viral assembly and transport.
 Unlike alphaviruses, no evidence of budding has been seen
in flavivirus-infected cells
Flavivirus and Diseases
 Complications

Case fatality
Incubation Does
Arbovirus

distribution
Geographic
Duration of
infection

Vector(s)

Primary
symptoms
Symptoms provide
Disease

host(s)
period
lifelong
immunity

rate
Asymptomatic Encephalitis,
Culex
in most cases; seizures,
mosquitoes Domestic
Japanese fever, paralysis, 20-30% in Southeast
especially pigs and
encephalitis JE 5–15 days headache, coma, and encephalitis and East Yes
Culex wading
virus (JEV) fatigue, long-term cases Asia
tritaeniorhy birds
nausea, and brain
nchus
vomiting damage
 Does

Duration of

Primary host
Incubation
Arbovirus

Case infection

symptoms
Complicati Geographic
Disease Symptoms fatality Vector(s) provide
ons distribution
period
rate lifelong
immunity

<1%
with
Asymptomatic
Shock, treatmen Aedes

Humans
in most cases;
internal t, 1-5% mosquitoes Near the
Dengu Dengue fever, 7–10
3–14 days bleeding, without; , especially equator Varies
e virus fever headache, days
and organ about Aedes globally
rash, muscle,
damage 25% in aegypti
and joint pains
severe
cases
 Primary host
Does
Duration Case infection
Incubation Complicati Geographic
Arbovirus Disease Symptoms of fatality Vector(s) provide
period ons distribution
symptoms rate lifelong
immunity

Fever,
Jaundice, 3% in
headache Tropical
liver general;
, back Aedes and
damage, 20% in

Primates
Yellow pain, loss mosquitoes subtropical
Yellow gastrointe cases
fever 3–6 days of 3–4 days , especially regions of Yes
fever stinal with
virus appetite, Aedes South
bleeding, severe
nausea, aegypti America
recurring complica
and and Africa
fever tions
vomiting
Japanese encephalitis
 Japanese encephalitis (JE), formerly known as Japanese B
encephalitis is a disease caused by the mosquito-borne Japanese
encephalitis virus (JEV)
 Japanese Encephalitis Virus (JEV) is a flavivirus maintained in a
zoonotic cycle which involves pigs , birds and Culex species of
mosquitoes causing fatal encephalitis
 JE originated reportedly in Indonesia and Malaysia long back
(Weaver et al.,1999; Sinniah,1989).
 JE has spread extensively to several countries in Asia including both
temperate- Japan, Korea, Taiwan, China and tropical countries like
India, Sri Lanka, Bangladesh and Nepal (Bista and Shrestha, 2005).
 24 countries in the WHO South-East Asia and Western Pacific
regions have endemic JEV transmission, exposing more than 3
billion people to risks of infection.
 The principle vector is Culex mosquito, most important being
Culex tritaenorhynchus, present in greatest density in rainy
season (June to November)
 Humans are accidental dead-end-hosts as they do not develop
a level of viraemia sufficient to infect mosquitoes.
 The natural cycle of JEV consists of pig-mosquito-pig or
bird-mosquito-bird and pigs serve as a biological amplifiers
and reservoirs.
 The risk for Japanese encephalitis varies by appropriate
ecological conditions and season to cause epidemics and
epizootics.
 Disease control by vaccination is considered to be most
effective.
transmission
 Viral structure and genome
 Japanese encephalitis virus is an RNA virus of Flaviviridae family.
 It measures around 40-50 nm in diameter and structurally it is
spheroidal and of cubic symmetry.
 It is an enveloped virus having single stranded RNA as a genome
which is infectious.
 The genome is single stranded positive sense
 The genome can be divided into two parts: structural and
Nonstructural (NS) genes.
 Structural genes are three in number and are involved in antigenicity
since they are expressed on the virus coded by capsid protein and
involved in capsid formation: Core (C), pre Membrane (prM) and
Envelope (E).
 Among all three the E gene is the most important and is the
most studied one.
 There are seven NS genes: NS1, NS2a, NS2b, NS3, NS4a,
NS4b, NS5 and these are involved in virus replication
 JEV replicates exclusively in the cytoplasm of infected cells,
in a perinuclear location and matures on intracellular
membranes
Genome of JEV
Geographic Distribution of JE Virus
Disease epidemiology
 JEV is transmitted to humans through bites from infected
mosquitoes of the Culex species (mainly Culex
tritaeniorhynchus).
 Humans, once infected, do not develop sufficient viraemia to
infect feeding mosquitoes.
 The virus exists in a transmission cycle between mosquitoes,
pigs and/or water birds (enzootic cycle).
 The disease is predominantly found in rural and periurban
settings, where humans live in closer proximity to these
vertebrate hosts.
 In most temperate areas of Asia, JEV is transmitted mainly
during the warm season, when large epidemics can occur.
 In the tropics and subtropics, transmission can occur year-
round but often intensifies during the rainy season and pre-
harvest period in rice-cultivating regions, during which vector
populations increase
 The annual incidence of clinical disease varies both across and
within endemic countries, ranging from <1 to >10 per 100 000
population or higher during outbreaks.
 A literature review estimates nearly 68 000 clinical cases of JE
globally each year, with approximately 13 600 to 20 400
deaths.
 JE primarily affects children. Most adults in endemic
countries have natural immunity after childhood infection, but
individuals of any age may be affected.
 24 countries in the South-East Asia and Western Pacific
regions have JEV transmission risk, which includes more than
3 billion people.
 JEV is the main cause of viral encephalitis in many countries
of Asia with an estimated 68 000 clinical cases every year.
 Safe and effective vaccines are available to prevent JE.
 WHO recommends that JE vaccination be integrated into
national immunization schedules in all areas where JE disease
is recognized as a public health issue.
 Epidemiology of JE in Nepal
 Nepal has the second highest prevalence of Japanese encephalitis
(JE) in South East Asia
 One in five cases of JE results in death and those who survive
frequently suffer from residual neuropsychiatric disorders
 JE is endemic in the Terai (southern Nepal that borders with India),
with maximum number of cases occurring in the western districts of
Banke, Kanchanpur and Kailali.
 The disease was first recorded in Nepal in 1978 as an epidemic in
Rupandehi district of the Western Development Region (WDR) and
Morang of the Eastern Region (EDR).
 JE has also been reported and is now endemic in the Kathmandu
valley in the hill region
 The species Cx tritaeniorhyncus is suspected to be the principal
vector of JE in Nepal as the species is abundantly found in the
rice-field ecosystem of the endemic areas during the transmission
season
 About half of all JE cases are reported in children under the age
of 15 and the incidence rate is the highest amongst children age
5-15 years
 Of those infected, about 60 percent are male
 The most fatalities and residual neurological and psychiatric
disorders are seen in children under the age of 10
 Immunization programs targeting children in the JE affected
districts of Nepal have decreased the number of cases in these
areas.
 Since 2009, JE immunization is included in the national
immunization program in JE endemic districts.
 The highest risk months for JE are August, September and
early October of each year
 Those not using insecticide treated bed nets (ITNs), living
amongst animal reservoirs of the disease and practicing poor
agricultural practices in JE-endemic areas are most at risk
Disease
pathogenesis
 Pathogenesis
 JE typically develops in patients after an incubation period of 5–15
days.
 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
 The failure of the host to produce antibodies against the virus is
associated with an increased likelihood of the disease to turn lethal
 Crossing the blood–brain barrier is an important factor in the
increased pathogenesis and clinical outcome of the neurotropic
viral infection
 After entering the body through a mosquito bite, the virus reaches the
central nervous system (CNS) via leukocytes (probably T lymphocytes),
where JEV virions then bind to the endothelial surface of the CNS and
are internalized by endocytosis
 In other flaviviral infections, such as WNV, macrophages could serve as
a reservoir, spreading the virus from the peripheral areas to the CNS
 Studies have shown that WNV is capable of entering the CNS through
axonal transport
 Because both WNV and JEV belong to the same family of viruses,
macrophage and axonal transport may play a critical role in JEV
pathogenesis; however, convincing evidence is still lacking.
 JEV causes extensive neuronal damage in the brain, though in many
cases, the virus is probably not directly involved in the destruction of
brain tissue but may cause damage indirectly by triggering cell-
mediated immune response by activating microglia
 Microglias are the resident immune cells of the CNS and have a
critical role in host defense against invading microorganisms.
 Activated microglia release neuroprotective factors to facilitate the
recovery of injured neurons and they also phagocytose dying or
damaged neurons
 JEV infection has been shown to activate microglia both
morphologically and functionally, in vivo, that leads to an elevation
of pro-inflammatory mediators, such as IL-6, TNF-α, RANTES
(Regulated on Activation, Normal T Cell Expressed and Secreted)
and MCP-1(monocyte chemotactic protein-1)
RANTES is a chemokine expressed by many hematopoietic and non-
hematopoietic cell types that plays an important role in homing and
migration of effector and memory T cells during acute infections
 These pro-inflammatory mediators and cytotoxins released
from activated microglia are responsible in inducing neuronal
death that accompanies JE.
 Nitric oxide (NO) also plays an important role in inflammation
during JE infection, although NO itself is a strong
antimicrobial agent researchers have shown that it profoundly
inhibits viral RNA synthesis, viral protein accumulation and
virus release from infected cells
 Thus, NO may play a crucial role in the innate immunity of the
host and its ability to restrict the initial stages of JEV infection
in the CNS.
 Besides neuronal and microgial cells, researchers have shown
that astrocytes are also infected by JEV
 Astrocytes are known to maintain homeostasis in the CNS and
to support the survival and information processing functions of
neurons.
 They respond promptly to CNS infection and help regulate
neuro inflammation.
 Though astrocytes are activation, the infection overwhelms the
capacity of even activated astrocytes to maintain metabolic
homeostasis, resulting in an over accumulation of toxic by
products of metabolism that are detrimental to neuronal
viability.
 Signs and symptoms
 Most JEV infections are mild (fever and headache) or without
apparent symptoms, but approximately 1 in 250 infections results
in severe clinical illness.
 Severe rigors may mark the onset of this disease.
 Severe disease is characterized by rapid onset of high fever
(100.4–105.8 °F), headache, neck stiffness, disorientation, coma,
seizures, spastic paralysis and ultimately death.
 The case-fatality rate can be as high as 30% among those with
disease symptoms.
 Of those who survive, 20%–30% suffer permanent intellectual,
behavioural or neurological problems such as paralysis, recurrent
seizures or the inability to speak.
 Laboratory diagnosis
 Laboratory diagnosis of JE is generally accomplished by
testing of serum or cerebrospinal fluid (CSF) to detect virus-
specific IgM antibodies.
 JE virus IgM antibodies are usually detectable 3 to 8 days after
onset of illness and persist for 30 to 90 days, but longer
persistence has been documented.
 Serum collected within 10 days of illness onset may not have
detectable IgM, and the test should be repeated on a
convalescent sample.
 Routine diagnosis is usually carried out by using HI, IF, CF or
ELISA techniques
 Virus isolation from blood is rarely successful during the
acute illness because the viraemic phase is probably over by
the time central nervous system symptoms appear
 Virus isolation from the CSF is done by variety of isolation
techniques including intra-cerebral inoculation of suckling
mice, intra-thoracic inoculation of live mosquitoes, the use
of common mammalian cell lines such as Vero and LLC-
MK2 and mosquito cell lines, especially those of A.
albopictus and A. pseudoscutellaris.
 Other Clinical laboratory findings might include a moderate
leukocytosis, mild anemia, and hyponatremia.
 Cerebrospinal fluid (CSF) typically has a mild to moderate
pleocytosis with a lymphocytic predominance, slightly elevated
protein, and normal ratio of CSF to plasma glucose.
 Magnetic resonance imaging (MRI) of the brain is better than
computed tomography (CT) for detecting JE virus-associated
abnormalities such as changes in the thalamus, basal ganglia,
midbrain, pons, and medulla.
 Prevention and control
 Safe and effective JE vaccines are available to prevent
disease.
 WHO recommends having strong JE prevention and control
activities, including JE immunization in all regions where the
disease is a recognized public health priority, along with
strengthening surveillance and reporting mechanisms.
 Even if the number of JE-confirmed cases is low, vaccination
should be considered where there is a suitable environment
for JE virus transmission.
 There is little evidence to support a reduction in JE disease
burden from interventions other than the vaccination of
humans.
 There are 4 main types of JE vaccines currently in use: inactivated
mouse brain-derived vaccines, inactivated Vero cell-derived
vaccines, live attenuated vaccines, and live recombinant vaccines.
 Over the past years, the live attenuated SA14-14-2 vaccine
manufactured in China has become the most widely used vaccine in
endemic countries
 All travellers to Japanese encephalitis-endemic areas should take
precautions to avoid mosquito bites to reduce the risk for JE.
 Personal preventive measures include the use of repellents, long-
sleeved clothes, coils and vaporizers.
 Travellers spending extensive time in JE endemic areas are
recommended to get vaccinated.
 Dengue virus &
Dengue Fever (DF)

Dengue Hemorrhagic Fever (DHF)

Dengue Shock Syndrome (DSS)
 Dengue virus (DEN) is a small single-stranded RNA virus
comprising four distinct serotypes (DEN-1 to -4).
 Dengue serotypes 1, 3 and 4 show a closer antigenic and genetic
relationship to each other than dengue 2.
 However, within each of the serotypes, considerable
heterogeneity and strain variation is demonstrable on nucleic acid
sequence analysis and DNA/RNA hybridization studies
 These closely related serotypes of the dengue virus belong to the
genus Flavivirus, family Flaviviridae.
 Dengue virus is transmitted by female mosquitoes mainly of
the species Aedes aegypti and, to a lesser extent, A.
albopictus, Aedes polynesiensis and Aedes scutellaris
 The only vertebrate hosts of dengue virus in nature are
man and several species of Asian and African sub human
primates
 The mature particle of the dengue virus is spherical with a
diameter of 50nm containing multiple copies of the three
structural proteins, a host-derived membrane bilayer and a
single copy of a positive-sense, single-stranded RNA
genome.
 The genome is cleaved by host and viral proteases in three
structural proteins (capsid, C, prM, the precursor of
membrane, M, protein and envelope, E) and seven
nonstructural proteins (NS).
 Infection with any of the DENV serotypes may be asymptomatic in
the majority of cases or may result in a wide spectrum of clinical
symptoms, ranging from a mild flu-like syndrome, known as
Dengue Fever [DF], to the most severe forms of the disease, which
are characterized by coagulopathy, increased vascular fragility, and
permeability, known as Dengue Hemorrhagic Fever [DHF]
 The latter may progress to hypovolemic shock known as Dengue
Shock Syndrome [DSS]
 In Asia the risk of developing severe disease is greater in DENV-
infected children (≤15 years) than in adults
 In contrast, in the Americas mainly the adult population is affected,
resulting in mild disease, although an increasing trend of cases
progressing toward DHF/DSS has also been observed in adults
there
Transmission of
Dengue virus
Disease Pathogenesis
 During the feeding of mosquitoes on humans, DENV is injected into
the bloodstream, with spillover in the epidermis and dermis, resulting
in infection of Langerhans cells (epidermal dendritic cells [DC]), and
keratinocytes
 The virus enters the cells through binding between viral proteins and
membrane proteins on the Langerhans cell, specifically the C-type
lectins called DC-SIGN, mannose receptor and CLEC5A
 DC-SIGN, a non-specific receptor for foreign material on dendritic
cells, seems to be the main point of entry
 The dendritic cell moves to the nearest lymph node, where
monocytes and macrophages are recruited, which become targets
of infection.
 Consequently, infection is amplified and virus is disseminated
through the lymphatic system.
 As a result of this primary viremia, several cells of the mononuclear
lineage, including blood-derived monocytes, myeloid DC and
splenic and liver macrophages are infected.
 DENV has also been shown to have tropism for circulating
mononuclear cells in blood and for cells residing in the spleen,
lymph nodes, and bone marrow
 The immunopathology of dengue hemorrhagic fever/dengue shock
syndrome remains incompletely understood.
 However, most patients who develop dengue hemorrhagic fever or
dengue shock syndrome have had prior infection with one or more
dengue serotypes.
 When an individual is infected with another serotype (ie,
secondary infection) and produces low levels of non-neutralizing
antibodies, these antibodies, directed against 1 of 2 surface
proteins (precursor membrane protein and envelope protein), when
bound by macrophage and monocyte Fc receptors, have been
proposed to fail to neutralize virus and instead form an antigen-
antibody complex.
 This results in increased viral entry into macrophages bearing IgG
receptors, allowing unchecked viral replication with higher viral
titers and increased cytokine production and complement
activation, a phenomenon called antibody-dependent
enhancement.
 The affected macrophages release vasoactive mediators that
increase vascular permeability, leading to vascular leakage,
hypovolemia, and shock.
 This mechanism, along with individual host and viral
genome variations, plays an active role in pathogenesis.
 Infants born to mothers who have had dengue, as maternally
derived dengue neutralizing IgGs wane, are also thought to
be at risk for enhanced disease.
 Host immunity
 After an incubation period of 4-10 days, infection by any of the
four virus serotypes can produce a wide spectrum of illness,
although most infections are asymptomatic or subclinical
 Primary infection is thought to induce lifelong protective
immunity to the infecting serotype
 Individuals suffering an infection are protected from clinical
illness with a different serotype within 2--3 months of the
primary infection but with no long-term cross-protective
immunity.
 The acquired immune response following a dengue infection
consists of the production of IgM and IgG antibodies primarily
directed against the virus envelope proteins.
 The immune response varies depending on whether the
individual has a primary (first dengue or other flavivirus
infection) versus a secondary (had dengue or other flavivirus
infection in past) dengue infection.
 A primary dengue infection is characterized by a slow and low
titer antibody response. IgM antibody is the first
immunoglobulin to appear.
 Anti-dengue IgG is detectable at low titer at the end of the first
week of illness, and slowly increases.
 In contrast, during a secondary infection, antibody titers
rise extremely rapidly and antibody reacts broadly with
many flaviviruses.
 High levels of IgG are detectable even in the acute
phase and they rise dramatically over the proceeding
two weeks.
 IgM levels are significantly lower in secondary
dengue infections
 Epidemiology
 Dengue fever (DF) is caused by any of four closely related viruses, or
serotypes: dengue 1-4.
 Dengue is transmitted between people by the mosquitoes Aedes aegypti
and Aedes albopictus, which are found throughout the world
 In rare cases dengue can be transmitted in organ transplants or blood
transfusions from infected donors, and there is evidence of transmission
from an infected pregnant mother to her fetus, but in the vast majority of
infections, a mosquito bite is responsible.
 In many parts of the tropics and subtropics, dengue is endemic, that is, it
occurs every year, usually during a season when Aedes mosquito
populations are high, often when rainfall is optimal for breeding.
 Dengue epidemics require a coincidence of large numbers of vector
mosquitoes, large numbers of people with no immunity to one of the
four virus types (DENV 1, DENV 2, DENV 3, DENV 4), and the
opportunity for contact between the two.
 The incidence of dengue has grown dramatically around the world in
recent decades.
 The actual numbers of dengue cases are under reported and many
cases are misclassified.
 One recent estimate indicates 390 million dengue infections per year
(95% credible interval 284–528 million), of which 96 million (67–
136 million) manifest clinically (with any severity of disease).
 Before 1970, only 9 countries had experienced severe dengue
epidemics
 The disease is now endemic in more than 100 countries in the
WHO regions of Africa, the Americas, the Eastern
Mediterranean, South-East Asia and the Western Pacific.
 The America, South-East Asia and Western Pacific regions are
the most seriously affected.
 In 2013, cases have occurred in Florida (United States of
America) and Yunnan province of China.
 Dengue also continues to affect several South American
countries, notably Costa Rica, Honduras and Mexico.
 In Asia, Singapore has reported an increase in cases after a
lapse of several years and outbreaks have also been reported in
Laos.
 In 2014, trends indicate increases in the number of cases in the
People's Republic of China, the Cook Islands, Fiji, Malaysia and
Vanuatu, with Dengue Type 3 (DEN 3) affecting the Pacific
Island countries after a lapse of over 10 years.
 Dengue was also reported in Japan after a lapse of over 70
years.
 In 2015 an increase in the number of cases was reported in
Brazil and several neighbouring countries.
 The Pacific island countries of Fiji, Tonga and French Polynesia
have continued to record cases.
 An estimated 500 000 people with severe dengue require
hospitalization each year, a large proportion of whom are
children.
 About 2.5% of those affected die.
Dengue in Nepal
 Dengue, a mosquito borne disease emerged in Nepal in the form of
Dengue Fever [DF], Dengue Hemorrhagic Fever [DHF] and
Dengue Shock Syndrome [DSS]
 The earliest cases were detected as early as 2005.
 The sporadic cases continued and outbreak occurred in 2006 and
2010
 Initially most of the reported cases had travel history to neighboring
country (India), however lately indigenous cases were also reported
 The affected districts were Kanchanpur, Kailali, Banke, Bardiya,
Dang, Kapilvastu, Parsa, Rupandehi, Rautahat, Sarlahi, Saptari and
Jhapa, indicating spread throughout the country from west to east
lying in the terai plain
 In 2011, 79 confirmed cases were reported from 15 districts
with the highest case incidence in Chitwan
 Aedes aegipti has been identified in 5 peri-urban areas of
Terai region ( Kailali, Dang, Chitwan, Parsa and Jhapa)
Disease symptoms
 Dengue infection is a systemic and dynamic disease.
 It has a wide clinical spectrum that includes both severe
and non-severe clinical manifestations
 After the incubation period of 4-10 days, the illness begins
abruptly and is followed by the three phases -- febrile,
critical and recovery
 Febrile phase
 Patients abruptly develop high-grade fever (40°C/104°F)
 This acute febrile phase usually lasts 2–7 days and is often
accompanied by facial flushing, skin erythema, generalized
body ache, myalgia, arthralgia and headache
 Some patients may have sore throat, injected pharynx and
conjunctival injection.
 Anorexia, nausea and vomiting are common.
 It can be difficult to distinguish dengue clinically from non-
dengue febrile diseases in the early febrile phase.
 A positive tourniquet test in this phase increases the probability
of dengue
Tourniquet test procedure
 The tourniquet test is part of the new WHO case definition for dengue. The test is
a marker of capillary fragility and it can be used as a triage tool to differentiate
patients with acute gastroenteritis, for example, from those with dengue. Even if a
tourniquet test was previously done, it should be repeated if
 It was previously negative
 There is no bleeding manifestation
 How to do a Tourniquet Test
 1. Take the patient's blood pressure and record it, for example, 100/70.
 2. Inflate the cuff to a point midway between SBP and DBP and maintain for 5
minutes. (100 + 70) ÷ 2 = 85 mm Hg
 3. Reduce and wait 2 minutes.
 4. Count petechiae below antecubital fossa
 A positive test is 10 or more petechiae per 1 square inch.
 In addition, these clinical features are indistinguishable
between severe and non-severe dengue cases.
 Mild hemorrhagic manifestations like petechiae and
mucosal membrane bleeding (e.g. nose and gums) may be
seen
 Massive vaginal bleeding (in women of childbearing age)
and gastrointestinal bleeding may occur during this phase
but is not common
 The liver is often enlarged and tender after a few days of
fever
 The earliest abnormality in the full blood count is a
progressive decrease in total white cell count
 Critical phase
 Around the time of defervescence, when the temperature drops to
37.5–380C or less and remains below this level, usually on days
3–7 of illness, an increase in capillary permeability in parallel
with increasing haematocrit levels may occur
 This marks the beginning of the critical phase.
 The period of clinically significant plasma leakage usually lasts
24–48 hours
 Progressive leukopenia followed by a rapid decrease in platelet
count usually precedes plasma leakage.
 At this point patients without an increase in capillary permeability
will improve, while those with increased capillary permeability
may become worse as a result of lost plasma volume.
 The degree of plasma leakage varies.
 Pleural effusion and ascites may be clinically detectable
depending on the degree of plasma leakage and the volume
of fluid therapy.
 Hence chest x-ray and abdominal ultrasound can be useful
tools for diagnosis.
 The degree of increase above the baseline haematocrit often
reflects the severity of plasma leakage.
 Shock occurs when a critical volume of plasma is lost
through leakage.
 The body temperature may be subnormal when shock occurs.
 With prolonged shock, the consequent organ hypoperfusion
results in progressive organ impairment, metabolic acidosis
and disseminated intravascular coagulation.
 This in turn leads to severe haemorrhage causing the
haematocrit to decrease in severe shock.
 Instead of the leukopenia usually seen during this phase of
dengue, the total white cell count may increase in patients
with severe bleeding.
 In addition, severe organ impairment such as severe
hepatitis, encephalitis or myocarditis and/or severe bleeding
may also develop without obvious plasma leakage or shock
 Those who improve after defervescence are said to have non-
severe dengue.
 Some patients progress to the critical phase of plasma leakage
without defervescence and, in these patients, changes in the
full blood count should be used to guide the onset of the
critical phase and plasma leakage.
 Recovery phase
 If the patient survives the 24–48 hour critical phase, a gradual
reabsorption of extravascular compartment fluid takes place in
the following 48–72 hours.
 General well-being improves, appetite returns, gastrointestinal
symptoms abate, haemodynamic status stabilizes and diuresis
ensues.
 Some may experience generalized pruritus.
 The haematocrit stabilizes or may be lower due to the dilutional
effect of reabsorbed fluid.
 White blood cell count usually starts to rise soon after
defervescence but the recovery of platelet count is typically
later than that of white blood cell count.
 Respiratory distress from massive pleural effusion and
ascites will occur at any time if excessive intravenous
fluids have been administered.
 During the critical and/or recovery phases, excessive fluid
therapy is associated with pulmonary oedema or
congestive heart failure
Laboratory diagnosis
 Laboratory diagnosis methods for confirming dengue
virus infection may involve detection of the virus,
viral nucleic acid, antigens or antibodies, or a
combination of these techniques.
Laboratory
diagnosis
Comparison of diagnostic tests according to their accessibility and confidence
 Virus isolation
 Specimens for virus isolation should be collected early in the course
of the infection, during the period of viraemia (usually before day 5).
 Virus may be recovered from serum, plasma and peripheral blood
mononuclear cells and attempts may be made from tissues collected
at autopsy (e.g. liver, lung, lymph nodes, thymus, bone marrow).
 The mosquito cell line C6/36 (cloned from A. albopictus) or AP61
(cell line from A. pseudoscutellaris) are the host cells of choice for
routine isolation of dengue virus.
 Several mammalian cell cultures, such as Vero, LLCMK2, and
BHK21, may also be used but are less efficient
 Virus isolation followed by an immunofuorescence assay for
confirmation generally requires 1–2 weeks
 Clinical specimens may also be inoculated by intracranial
route in suckling mice or intrathoracic inoculation of
mosquitoes.
 Newborn animals can develop encephalitis symptoms but
with some strains mice may exhibit no signs of illness.
 Virus antigen is detected in mouse brain or mosquito head
squashes by staining with anti-dengue antibodies.
 Nucleic acid detection
 RT-PCR
 The specific dengue virus genome is identified by
reverse transcription-polymerase chain reaction (RT–
PCR) from serum or plasma, cerebrospinal fluid, or
autopsy tissue specimens
 Compared to virus isolation, the sensitivity of the RT-
PCR methods varies from 80% to 100% and depends
on the region of the genome targeted by the primers
 Serological tests
 MAC-ELISA
 IgM antibody-capture enzyme-linked immunosorbent assay (MAC-
ELISA)
 Total IgM in patients’ sera is captured by anti-µ chain specific
antibodies (specifc to human IgM) coated onto a microplate.
 Dengue-specific antigens, from one to four serotypes (DEN-1, -2, -
3, and -4), are bound to the captured anti-dengue IgM antibodies
and are detected by monoclonal or polyclonal dengue antibodies
directly or indirectly conjugated with an enzyme that will transform
a non-coloured substrate into coloured products
Principle of a MAC-ELISA test
 IgM/IgG ratio
 A dengue virus E/M protein-specifc IgM/IgG ratio can be
used to distinguish primary from secondary dengue virus
infections.
 IgM capture and IgG capture ELISAs are the most
common assays for this purpose.
 In some laboratories, dengue infection is defined as
primary if the IgM/IgG OD ratio is greater than 1.2 (using
patient’s sera at 1/100 dilution) or 1.4 (using patient’s sera
at 1/20 dilutions).
 The infection is secondary if the ratio is less than 1.2 or 1.4
 IgG ELISA
 The IgG ELISA is used for the detection of recent or past
dengue infections (if paired sera are collected within the
correct time frame).
 This assay uses the same antigens as the MAC-ELISA.
 The use of E/M-specific capture IgG ELISA (GAC) allows
detection of IgG antibodies over a period of 10 months after
the infection.
 IgG antibodies are lifelong as measured by E/M antigen-
coated indirect IgG ELISA, but a fourfold or greater increase
in IgG antibodies in acute and convalescent paired sera can
be used to document recent infections
 Haemagglutination-inhibition test
 The haemagglutination-inhibition (HI) test is based on
the ability of dengue antigens to agglutinate red blood
cells
 Anti-dengue antibodies in sera can inhibit this
agglutination and the potency of this inhibition is
measured in an HI test
 Detection of antigens
 Envelope/membrane (E/M) antigen and the non-structural
protein 1 (NS1) can be detected
 High concentrations of these antigens in the form of
immune complexes could be detected in patients with both
primary and secondary dengue infections up to nine days
after the onset of illness.
 Commercial kits for the detection of NS1 antigen are now
available, though they do not differentiate between dengue
serotypes.
 Haematological tests
 A drop of the platelet count below 100 000 per µL may be
observed in dengue fever but it is a constant feature of
dengue haemorrhagic fever.
 Thrombocytopaenia is usually observed in the period
between day 3 and day 8 following the onset of illness.
 Haemoconcentration, as estimated by an increase in
haematocrit of 20% or more compared with convalescent
values, is suggestive of hypovolaemia due to vascular
permeability and plasma leakage.
 Treatment
 There is no specific treatment for dengue fever.
 For severe dengue, medical care by physicians and nurses
experienced with the effects and progression of the disease
can save lives – decreasing mortality rates from more than
20% to less than 1%.
 Maintenance of the patient's body fluid volume is critical
to severe dengue care.
 Immunization
 There is no vaccine to protect against dengue.
 However, major progress has been made in
developing a vaccine against dengue/severe dengue.
 Three tetravalent live-attenuated vaccines are under
development in phase II and phase III clinical trials,
and 3 other vaccine candidates (based on subunit,
DNA and purified inactivated virus platforms) are at
earlier stages of clinical development.
 Blood feeding Aedes aegypti

Aedes aegypti
Yellow Fever
 Yellow fever virus
Virus classification

Group IV
Group:
((+)ssRNA)

Order: Unassigned

Family: Flaviviridae

Genus: Flavivirus

Species: Yellow fever virus

Introduction
 Yellow fever virus (YFV) is an arthropod-borne virus
belonging to the family Flaviviridae, genus Flavivirus
 Yellow fever is an acute viral disease that causes
hemorrhagic fever and jaundice.
 The virus is transmitted between humans by the Aedes
aegypti mosquito
 YFV has a tropism for the liver and causes a
viscerotropic disease whereas many other mosquito-
borne flaviviruses have a tropism for the brain, or in the
case of the DEN viruses they target cells of
reticuloendothelial origin
 The YFV genome is an 11kb single-stranded positive-
sense RNA genome coding for a polyprotein, which is
post- and co-translationally processed into three structural
proteins and seven non-structural proteins.
 The largest of the structural proteins, the envelope (E)
protein, is the major component of the virion surface.
 It is the primary immunogen and plays a central role in
receptor binding and membrane fusion
 REPLICATION
 Attachement of the viral envelope protein E to host receptors
mediates internalization into the host cell by clathrin-mediated
endocytosis, or by apoptotic mimicry
 Fusion of virus membrane with host endosomal membrane.
 RNA genome is released into the cytoplasm.
 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).
 Replication takes place at the surface of endoplasmic reticulum in
cytoplasmic viral factories.
 A dsRNA genome is synthesized from the genomic
ssRNA(+).
 The dsRNA genome is transcribed/replicated thereby
providing viral mRNAs/new ssRNA(+) genomes.
 Virus assembly occurs at the endoplasmic reticulum.
 The virion buds via the host ESCRT (Endosomal sorting
complexes required for transport) complexes at the
endoplasmic reticulum, is transported to the Golgi apparatus.
 The prM protein is cleaved in the Golgi, thereby maturing
the virion which is fusion competent.
 Release of new virions by exocytosis.
Transmission
cycles of YF
 Transmission
 Mosquito is the primary vector of yellow fever
 It carries the virus from one host to another, primarily between
monkeys, from monkeys to humans, and from person to person.
 Several different species of the Aedes and Haemogogus
mosquitoes transmit the virus.
 The mosquitoes either breed around houses (domestic), in the
jungle (wild) or in both habitats (semi-domestic).
 There are three types of transmission cycles.
 Sylvatic (or jungle) yellow fever cycle
 Intermediate yellow fever cycle
 Urban yellow fever cycle
 Sylvatic (or jungle) yellow fever cycle:
 In tropical rainforests, yellow fever occurs in monkeys that are
infected by wild mosquitoes.
 The infected monkeys then pass the virus to other mosquitoes
that feed on them.
 The infected mosquitoes bite humans entering the forest,
resulting in occasional cases of yellow fever.
 The majority of infections occur in young men working in the
forest (e.g. for logging).
 Aedes africanus (in Africa) or mosquitoes of the genus
Haemagogus and Sabethes (in South America) serve as vectors
 Intermediate yellow fever cycle :
 In humid or semi-humid parts of Africa, small-scale
epidemics occur.
 Semi-domestic mosquitoes (that breed in the wild and
around households) infect both monkeys and humans.
 In Africa, this infectious cycle known as "savannah
cycle" or intermediate cycle, occurs between the jungle
and urban cycles.
 Different mosquitoes of the genus Aedes are involved.
 In recent years, this has been the most common form
of transmission of yellow fever in Africa
 Urban yellow fever cycle:
 Large epidemics occur when infected people
introduce the virus into densely populated areas with
a high number of non-immune people and Aedes
mosquitoes.
 Infected mosquitoes transmit the virus from person to
person.
 In this cycle, which is confined to urban areas, A
aegypti, a domestic mosquito is the primary vector.
Distribution of Yellow fever virus

Areas of risk: South America

Areas of risk: Africa
 Populations at risk
 Forty-four endemic countries in Africa and Latin America, with a
combined population of over 900 million, are at risk.
 In Africa, an estimated 508 million people live in 31 countries at risk.
 The remaining population at risk are in 13 countries in Latin America,
with Bolivia, Brazil, Colombia, Ecuador and Peru at greatest risk.
 According to WHO estimates from the early 1990s, 200 000 cases of
yellow fever, with 30 000 deaths, are expected globally each year, with
90% occurring in Africa.
 A recent analysis of African data sources due to be published later this
year, estimates similar figures, but a slightly lower burden of 84 000–
170 000 severe cases and 29 000–60 000 deaths due to yellow fever in
Africa for the year 2013.
 Without vaccination, the burden figures would be much higher.
 Small numbers of imported cases occur in countries free of yellow
fever.
 Although the disease has never been reported in Asia, the region is
at risk because the conditions required for transmission are present
there.
 In the past centuries (XVII to XIX), outbreaks of yellow fever were
reported in North America (Charleston, New Orleans, New York,
Philadelphia, etc) and Europe (England, France, Ireland, Italy,
Portugal and Spain).
 There is no risk of yellow fever in Nepal.
 The government of Nepal requires proof of yellow fever vaccination
from people arriving from a country with risk of yellow fever.
 Pathogenesis
 An infected female mosquito inoculates approximately 1000 to
100,000 virus particles intradermally during blood feeding.
 Virus replication begins at the site of inoculation, probably in
dendritic cells in the epidermis, and spreads through lymphatic
channels to regional lymph nodes.
 Lymphoid cells, particularly monocyte-macrophages and large
histiocytes, appear to be the preferred cell types for primary
replication.
 The virus reaches other organs via the lymph and then the
bloodstream, seeding other tissues.
 Large amounts of virus are produced in the liver and
spleen and released into the blood.
 Disease symptoms are associated with hepatic
damages and presence of virus in blood
 During the viremic phase (days three to six), infection
may be transmitted to blood-feeding mosquitoes.
Clinical features
 Clinical features
 Incubation period: 3-6 days
 YF presents with a variety of clinical signs and outcomes, ranging
from mild to severe and fatal cases.
 The WHO-recommended case definition for suspect YF is as
follows:
 Any case presenting with acute onset of fever, with jaundice
appearing within14 days after the onset of the first symptoms.
 YF in human beings has the following characteristics:
 An acute phase lasting four to five days and presenting with:
 sudden onset of fever;
 headache or backache;
 muscle pain;
 nausea;
 vomiting;
 red eyes (infected conjunctiva).
 This phase can be confused with other diseases that also
present with fever, headache, nausea and vomiting,
because jaundice may not be present in less severe (or
mild) cases of YF.
 Less severe cases are often non-fatal.
 Temporary remission, lasting up to 24 hours, follows the acute phase in
5% to 20% of cases.
 A toxic phase may follow the period of remission, presenting with:
 jaundice;
 dark urine;
 reduced amounts of urine production;
 bleeding from the gums or nose, or blood in the stool;
 vomiting of blood;
 hiccups;
 diarrhoea;
 despite a rising or persistent temperature the pulse may decrease (Faget’s
signs)
 The severe form eventually leads to shock and failure of multiple organs.
 Laboratory diagnosis
 Preliminary diagnosis is based on the patient's clinical features,
vaccination status, and travel history, including destination, time of year,
and activities.
 Case confirmation requires that one of the following laboratory criteria be
fulfilled:
 presence of YF virus-specific IgM antibody;
 a fourfold or greater rise in serum IgG antibody levels in paired acute
and convalescent sera
 propagation of YF virus in cell culture or laboratory animals;
 detection of YF genetic sequences in blood or organs by molecular
diagnostic techniques, e.g. the reverse transcriptase polymerase chain
reaction (RT-PCR)
 positive postmortem liver histopathology;
 detection of YF virus antigen in tissues by immunohistochemistry
 Virus isolation
 YF virus may be isolated from blood collected during the initial three-
day febrile illness or from liver tissues of fatal cases.
 After isolation the virus can be confirmed in the cell culture supernatant
by RT-PCR, neutralization or ELISA tests using appropriate polyclonal
or monoclonal antibodies, or in the virus-infected cells by indirect
immunofluorescence or RT-PCR
 YF virus can be grown in a wide variety of primary and continuous cell
cultures.
 YF virus grow to high titre and produce CPE and plaques in monkey
kidney (MA-104, Vero, LLC-MK2), rabbit kidney (MA-111), baby
hamster kidney (BHK) and porcine kidney cell lines as well as in
primary chick and duck embryo fibroblasts.
 Likewise, Mosquito cell cultures are useful for primary
isolation and are more sensitive than Vero cells or infant
mice.
 Aedes pseudoscutellaris (AP-61), cloned Aedes albopictus
(C6/36) and Aedes aegypti cells are all susceptible to
infection. AP-61 cells have proved particularly useful for YF
isolation
Treatment
 No specific treatment is available for YF.
 In the toxic phase, supportive treatment includes therapies
for treating dehydration and fever.
 In severe cases, death can occur between the seventh and
tenth days after the onset of the first symptoms.
 Vaccination to prevent yellow
fever
 Vaccine
 Yellow fever vaccine is recommended for persons aged ≥ 9
months who are traveling to or living in areas at risk for yellow
fever virus transmission in South America and Africa.
 The 17D vaccine, which is based on a live, attenuated viral
strain, is the only commercially available yellow fever vaccine.
 It is given as a single subcutaneous (or intramuscular)
injection.
 Yellow fever vaccine is highly effective (approaching 100%).
 All individuals aged 9 months or older and living in countries
or areas at risk should receive yellow fever vaccine
Prevention by avoiding mosquito
bites
 Use insect repellent.
 To prevent outdoors mosquito bite, use of insect repellent such as those
containing DEET, picaridin, IR3535, or oil of lemon eucalyptus on
exposed skin is recommended
 Even a short time outdoors can be long enough to get a mosquito bite.
 Wear proper clothing to reduce mosquito bites. When weather
permits, wear long-sleeves, long pants and socks when outdoors.
 Mosquitoes may bite through thin clothing, so spraying clothes with
repellent containing permethrin or another repellent will give extra
protection.
 Clothing pre-treated with permethrin is commercially available.
 Mosquito repellents containing permethrin are not approved for
application directly to skin.
 Be aware of peak mosquito hours.
 The peak biting times for many mosquito species is dusk to
dawn.
 However, Aedes aegypti, one of the mosquitoes that transmits
yellow fever virus, feeds during the daytime.
 Extra care is required to use repellent and protective clothing
during daytime as well as during the evening and early morning.
 Staying in accommodations with screened or air-conditioned
rooms, particularly during peak biting times, will also reduce
risk of mosquito bites.