Dengue virus

Submitted to:- Dr Pawas Goswami

Submitted by:- Aman jaiswal and

Abhishek behera
 Classification of Dengue
unranked: Virus
Kingdom: Riboviria
Phylum: incertae sedis
Family: Flaviviridae
Genus: Flavivirus
Species:Dengue virus
Epidemiology
Epidemiology
• Dengue viruses are distributed worldwide in tropical regions. Most subtropical and tropical regions
around the world where Aedes vectors exist are endemic areas.
• In the past 20 years, epidemic dengue has emerged as a problem in the Americas. In 1995, more than
200,000 cases of dengue and more than 5500 cases of dengue hemorrhagic fever occurred in Central and
South America. The changing disease patterns are probably related to rapid urban population growth,
overcrowding, and lax mosquito control efforts.
• In 2008 dengue was the most important mosquito borne viral disease affecting humans.
• There are an estimated 50 million or more cases of dengue annually worldwide, with 400,000 cases of
dengue hemorrhagic fever. The latter is a leading cause of childhood death in several Asian countries. The
risk of the hemorrhagic fever syndrome is about 0.2% during the first dengue infection but is at least 10-
fold higher during infection with a second dengue virus serotype. The fatality rate with dengue
hemorrhagic fever can reach 15% but can be reduced to less than 1% with proper treatment.
• Dengue epidemics start during the rainy season, when the vector mosquito, A aegypti, is
abundant . The mosquito breeds in tropical or semitropical climates in water-holding receptacles
or in plants close to human dwellings. A aegypti is the primary vector mosquito for dengue in the
Western Hemisphere. The female acquires the virus by feeding upon a viremic human. After a
period of 8–14 days, mosquitoes are infective and probably remain so for life (1–3 months). In the
tropics, mosquito breeding throughout the year maintains the disease.
• World War II was responsible for the spread of dengue from Southeast Asia throughout the
Pacific region. Only dengue type 2 was present in the Americas for years. Then, in 1977, a dengue
type 1 virus was detected for the first time in the Western Hemisphere.
• In 1981, dengue type 4 was first recognized in the Western Hemisphere followed in 1994 by
dengue type 3. The viruses are now spread throughout Central and South America, and dengue
hemorrhagic fever is endemic in many countries.
Aedes aegypti mosquitoes originated in Africa, but they have spread through tropical and
subtropical regions around the world.
Aedes aegypti mosquitoes first spread outside Africa during the slave trade between the 15th and 19th centuries. They
also spread through trade with Asia during the 18th and 19th centuries, and then again following troop movements in
World War II.
Global Dengue Risk Determination of the risk status was based on combined reports from the World Health
Organization, the Canters for Disease Control and Prevention, Gideon online, ProMED, Dengue Map, Euro surveillance, and
published literature. Risk exclusions were made on the basis of a biologic model of temperature suitability and areas of
excessive aridity defined according to the Glob Cover “bare areas” land cover classification. Within areas at risk,
environmental suitability for dengue transmission was modelled with the use of a boosted regression-tree algorithm
Transmission cycles of dengue viruses

These viruses have enzootic

maintenance cycles involving Aedes
vectors and nonhuman primates.
Dengue viruses are transmitted
principally between humans and
Aedes aegypti that breed in domestic
water containers. In Africa, sylvatic
vectors are responsible for monkey–
monkey and inter-human virus
transmission, and there is frequent
involvement of A aegypti in urban
and dry savanna regions.
Genome Organization Of Dengue Virus
Dengue is a positive-sense ssRNA virus belonging to the Flaviviridae family. There are four distinct
serotypes — dengue virus serotype 1 (DENV1) to DENV4 — and several genotypes within each serotype.

Dengue virus is a relatively simple positive-sense single stranded RNA virus that is 50 nm in diameter
and has 3 structural proteins -- capsid (C) protein, precursor membrane (prM) protein and envelope (E)
protein — as well as 7 non-structural (NS) proteins termed NS1, NS2A, NS2B, NS3, NS4A, NS4B and
NS5. NS5 is the largest non-structural among them all.

E and prM protein form the glycoprotein shell of the virus, and E protein is responsible for host cell
binding and entry. Dengue E protein has 2 N-linked glycosylation sites and is divided into 3 domains
termed EDI, EDII and EDIII.

During viral assembly in the ER, 180 copies of E protein associate with 180 copies of prM protein to
form 60 trimeric (heterohexameric) spikes, which gives immature virions their characteristic spiky
appearance.
 In the trans-Golgi network, prM protein is cleaved at a membrane proximal site by host-encoded furin protease,
resulting in a membrane-anchored M  stump and a pr molecule that remains associated with the virion until it is
secreted. Upon secretion from the host cell, pr protein disassociates to leave the ‘mature virion’, which has a
smooth structure.

 The cleavage of prM protein is not complete in all dengue virions, leaving a proportion of partially mature dengue
virions that contain a varying amount of cleaved and uncleaved prM protein.

 The cleavage of prM protein is more efficient in certain cell types, particularly primary human cells such as dendritic
cells (over expression of furin), compared with the cleavage event in insect cells or tumor cell lines

 Fully immature dengue viruses contain regular trimeric E–prM protein spikes and are non-infectious. By contrast, the
partially mature forms, some of which are infectious, have a less regular structure, with areas that are spiky and
contain prM protein–E protein trimers and areas that are smooth and are proposed to contain E protein dimers.

 Finally, virions attach to cells, possibly through EDIII. Several host proteins — such as heparan sulfate, dendritic cell-
specific ICAM3-grabbing non-integrin (DC-SIGN; also known as CD209), heat shock protein 70 kDa (HSP70), HSP90,
mannose receptor, CD14, T  cell immunoglobulin and mucin domains (TIMs; also known as HAVCRs), TAM receptor
protein tyrosine kinases and laminin — have role in dengue virus binding, but there is no single receptor yet defined
that is necessary for entry.
• Following endocytosis, acidification in the early endosome triggers a dramatic reorganization
of the viral envelope, from the dimer form to a new trimeric conformation. This exposes the
fusion loop at the tips of the trimeric spikes, which then fuses with the endocytic membrane and
allows the viral nucleic acid to be released into the host cell cytoplasm.
life cycle of dengue virus
 The life cycle of dengue virus
Steps in life cycle of dengue virus is as follows:-
 Dengue virus exists as a number of different viral forms depending on the degree of precursor
membrane (prM) protein cleavage. Fully immature dengue virus particles contain a full complement
of prM proteins and are non-infectious, whereas all prM proteins are cleaved in fully mature virus
particles. A number of intermediate, partially mature forms exist in which some prM proteins have
been cleaved and some remain intact. Fully mature and some of the partially mature virus particles
are infectious.
 The dengue viral replication process begins when a virion attaches directly to a diverse group of host
cell receptors or when the Fc portion of a dengue virus-containing immune complex attaches to a Fc
receptor on the target cells. DC-SIGN (also known as CD209 is a protein and act as C type lectin
receptor present on dc and macrophages), dendritic cell-specific ICAM3-grabbing non-integrin; NS
proteins, non-structural proteins.
 Subsequently it enters the cell by receptor-mediated endocytosis.
 Acidification of the endosomal vesicles triggers conformational changes in the virion, resulting in an
irreversible trimerization of the viral envelope (E) protein. This exposes the fusion peptide and
mediates fusion between the viral and the endosomal membranes, allowing the release of the
nucleocapsid into the cytoplasm.
The viral RNA is released into the cytoplasm and presented to the RER. At the ER, viral RNA is
translated into a single polyprotein that is processed by viral and host proteases.
 After the viral replication complex is synthesized, viral RNA translation switches off, and RNA
synthesis begins by the transcription of an antisense viral RNA followed by the amplification of viral
RNA.
The newly synthesized RNA is subsequently packaged by capsid (C) protein, forming a nucleocapsid.
Virus assembly occurs on the surface of the ER when the nucleocapsid buds into the ER lumen,
resulting in non-infectious, immature viral particles.
Immature viral particles are transported through the Golgi into the trans-Golgi network, where
acidification induces conformational changes of the virion and exposes the furin cleavage sites. The
host protease furin cleaves between pr protein and M protein, with the pr protein remaining
associated until the virion is released in the neutral pH of the extracellular fluid environment.
 T cell responses to dengue virus
Dengue virus proteins are translated from the viral genomic RNA as a single polyprotein, which is subsequently
cleaved to yield the 3 structural proteins and 7 non-structural proteins. This generate equimolar amounts of all 10
proteins.

T cell epitopes are found throughout the dengue virus polyprotein. These epitopes follows general principles of
T cell epitope immunogenicity, and they show MHC molecule binding kinetics.

Most of the identified T cell epitopes, for both CD4+ and CD8+ T cells, reside in the NS3 protein.

Dengue virus-specific T cells recognize virus-infected cells and respond with a diverse set of effector functions,
including proliferation, target cell lysis and the production of a range of cytokines.

In vitro, both CD4+ and CD8+ human T cells with specificity for dengue virus have demonstrated the capacity to lyse
MHC-matched virus-infected cells.

Mechanism of viral peptide presentation by MHC class II molecules in this in vitro system has not been defined. A
broad array of cytokines is produced by dengue virus-specific T cells in response to the recognition of peptide–MHC
complexes on target cells.
• In case for dengue virus-specific antibodies, amino acid differences between serotypes can affect the
avidity of the interaction between peptide–MHC complexes on dengue virus-infected cells and the T cell
receptors of individual dengue virus-specific T cell clones, and this has functional consequences.
Fig:-Dengue virus polyprotein is shown at the top and the locations of
well-defined epitopes that are recognized by human T cells are marked
by arrows.
Fig:- T cell epitopes are shown to demonstrate the incomplete Fig:- Variant epitopes alter the T cell functional
sequence conservation of typical T cell epitopes. The location of response
the epitope, its recognition by CD4+ or CD8+ T cells and its HLA
restriction are indicated at the top. The predominant sequences
for each of the four serotypes are shown. Residues that are
completely conserved are shown in black and residues that are
not completely conserved are shown in red.
 Immune involvement of dengue
une involvement of dengue pathoge
pathogenesis
• Primary infection occurs with Serotype 1 of DENV, resulting in adaptive immune responses where
Serotype 1–specific T cells are selected, activated, and clonally expanded to combat infection.
During the resolution of primary infection, memory Serotype 1–specific T cells are formed and are
retained in higher frequency in the T cell repertoire than other T naive cells.
• A secondary challenge Serotype 1 would evoke a memory recall response and effective containment
of infection by highly specific T cells.
• A secondary challenge with a heterologous strain, Serotype 2, has the potential to reactivate
memory T cells that are of greater specificity for Serotype 1 than for Serotype 2. These memory
Serotype 1–specific T cells outcompete naive T cells that would be more specific for Serotype 2,
resulting in an expanded memory T cell pool that is low specificity for Serotype 2 and poor viral
clearance in vivo. (Original antigenic sin, also known as the Hoskins effect)
• Antibody-dependent enhanced replication has the potential to occur during a secondary
heterologous infection.
• During primary infection, B cell selection occurs, promoting Serotype 1–specific antibody
production. These pre-existing antibodies are present during the secondary challenge. If the
secondary challenge is with Serotype 1 again, antibody-mediated neutralization of DENV occurs,
limiting infection.
• If the secondary challenge is heterologous, as with Serotype 2, antibody specificity may be low and
weakly neutralizing antibodies can promote Fc receptor–mediated uptake of virus-antibody
complexes.
• Increased uptake of virus into the cell without efficient antibody-mediated neutralization results in
production of higher viral titers and increases activation of pro-inflammatory intracellular signalling
pathways.
• Cytokine storm can occur during either primary or secondary infection when infected cells produce
high levels of cytokines or may also be derived from non infected immune cells, such as activated T
cells.
• Cytokines act directly on the host vasculature and promote vascular leakage when they reach
pathological levels. Mast cell also release cytokines and additional de novo synthesized and pre-
stored vasoactive mediators when they are activated by DENV.
• Prior to secondary infection, mast cell may also be sensitized by binding DENV-specific antibodies,
which can also mediate MC activation in response to DENV.
• Mast cell derived mediators act directly on the host vasculature to promote vascular leakage.
• Dengue virus may cause increased vascular permeability through binding to the endothelial glycocalyx
layer, which lines the luminal surface of microvessels, providing vital barrier functions to capillaries.
• Both the virus and NS1 protein have been shown to bind to heparan sulfate, a major component of the
glycocalyx layer, which may in turn alter the permeability of capillaries.
• NS1-mediated complement activation may also play a part in the altered capillary permeability
through the generation of the anaphylatoxins C3a and C5a and the terminal complement complex C5b–
C9.
• High C5b–C9 levels in plasma and pleural fluid have been demonstrated in patients with dengue
shock, and patients who were high producers of C5b–C9 had more signs of plasma leakage. Furthermore,
NS1 may have an immunomodulating role by binding to and reducing the functional capacity of C4,
thereby altering the classical and lectin complement pathways.
• Innate immune cells also have a role in dengue pathogenesis; for example. mast cells have a role in both
protection against and pathogenesis of dengue. Upon interaction with dengue virus, mast cells
release chemokines that recruit T  cells, natural killer (NK) cells and NKT cells to control the infection. By
contrast, the interaction also leads to the secretion of mediators such as chymases — proteases
that may contribute to vascular leakage. The level of chymases is correlated with disease severity.
vector
Vector life cycle
Transmission cycle of vector Borne disease
Vector Control
Transmission to humans
 Vector life cycle

Culex and Culiseta species, the eggs are stuck together in rafts of up to 200. Anopheles, Ochlerotatus and Aedes , as well as many other
genera, do not make egg rafts, but lay their eggs singly.
Culex, Culiseta, and Anopheles lay their eggs on the water surface while many Aedes and Ochlerotatus lay their eggs on damp soil that
will be flooded by water.
There is a 24–48-hour time lag between emergence and mating. Mating is not needed for egg development and maturation, but in
most species eggs can only be deposited when insemination has occurred.
 As a rule, female mosquitoes mate before taking a first blood meal, but in several anophelines a large proportion of virgins may blood-
feed prior to mating. Such a blood meal is essential for the development of a metabolic energy reservoir.
 Many females may feed on nectar or other carbohydrate sources prior to mating to acquire an energy reservoir for flying and mate
finding.
Transmission cycle for vector borne disease
Infectivity Period (about 7 days)
Both symptomatic and asymptomatic persons are viremic and can transmit DENV to mosquitoes that
bite them during this approximately 7-day period. This viremic period is known as the "period of
infectivity". In sick persons, viremia typically coincides with the presence of fever.

Extrinsic incubation period in mosquitoes (8-12 days)

Vector-borne pathogens typically enter midgut, nerve tissue, body fat and ovaries before invading
the salivary glands. The pathogens will continue replicating in the salivary glands until the end of the
mosquito’s lifespan.
Following an 8–12 day extrinsic incubation period, the DENV-infected female mosquito is able to  
during its one-month lifespan

Intrinsic Incubation Period (3-14 days)

Once bitten, DENV replicates in the human for 3–14 days. After incubation, the human can become
ill.
 Biological Approaches to Targeting the Vector
1.Impair pathogen development
•Impairing pathogen development (vector-borne pathogens) was proposed by Laven H.
et al as early as 1967
•The use of Wolbachia pipentis, a intracellular insect bacterium which was isolated in
1924 in the ovaries of Culex pipens.
•This strategy involves embryonic introduction of strains of the obligate intracellular
bacterium wolbachia into A. aegypti. Wolbachia-infected A. aegypti are partially
resistant to dengue virus infection and can invade natural A. aegypti populations,
suggesting the possibility of induction of widespread biologic resistance to dengue
viruses in A. Aegypti populations
•It confers 4 different phenotypes:
Male killing: males are killed during larval development
Feminization: infected males develop as either females or infertile pseudo-females
Parthenogenesis: reproduction of infected females without the need for male
Cytoplasmic incompatibility: inability of infected males to mate with uninfected
females or females who are infected with another Wolbachia strain.
Wolbachia-infected male mosquitoes mates with an uninfected female mosquito .
Wolbachia-infected females produce infected progeny in all matings allowing the
infection to rapidly spread through mosquito population.
Sterile Insect Technique (SIT)
• Invented by Edward F Kipling
• By releasing sterile males to mate with wild females, reducing their reproductive potential and
ultimately, if enough sterile males are released, it would bring about eradication of the pest
population.
• Progeny of GM insects with wild-type insects are targeted to possess the following traits:
1. Reduced competition in mating
2. Sterile progeny
3. Progeny with development defects
4. Reduced lifespan
5. Flightless phenotypes etc
• Traditional SIT involves mass rearing of mosquitoes
to produce equal numbers of the 2 sexes.
• Females are generally separated and discarded
before release. They are not thought to help control
efforts and may be detrimental.
• Various mechanical and genetic sexing methods were
employed but fairly yield single sex population.
• Radiation induced translocations to the Y
chromosome as dominant selectable markers.

Pupal mass sorting – females generally have larger

mass
Time of eclosion – females generally emerge later
than males
 Release of Insects Carrying a Dominant Lethal Gene (RIDL)
•RIDL (Release of Insects Carrying a Dominant Lethal Gene) system consists of introducing a lethal dominant gene that
could be under control of a female-specific promoter, such as that of vitellogenin gene.
•Expression of the lethal gene could be inactivated by treatment with tetracycline, allowing a colony to be maintained.
•When male and female separation is required, tetracycline is removed from the system, causing the death of all females.
•Insects engineered to carry a female-specific lethal gene could be used to remove females prior to release. A system
based on a lethal gene (RIDL) that acts late in development would prevent mosquitoes from becoming adults, the only
harmful life stage, yet enable them to survive and compete at the larval stage, when density-dependent competition
occurs.
•F1 progeny of RIDL males and wild females inherit a dominant female-specific lethal gene; the F1 females die, thereby
reducing the reproductive potential of the wild population, but the F1 males are viable and fertile. This provides a genetic
sexing mechanism facilitating male only release, either by employing the female-lethal version of RIDL and withdrawing
the repressor from the generation prior to release, or by combining a bisex-lethal system with female lethality (with an
independent means of repressing or inducing lethality) to permit male only release of bisex-lethal strains designed to kill
progeny of both sexes in the field.
•When preparing mosquitoes for release, the repressor is inactivated and the lethal gene is expressed, causing the death
of all females. When mating with wild females, males homozygous for the lethal gene will produce heterozygous
progenies, of which only males will survive.
 Transmission to humans
Transmission to humans is through the bite of an infected mosquito, A. aegypti.
This mosquito lays its eggs in containers found around the home. The adult mosquitoes
feed on humans during daylight hours.
There are two peaks of biting activity, early morning for 2 to 3 hours after daybreak and
in the afternoon for several hours before dark.
 A. aegyptii females often feed on several persons during a single blood meal hence
increasing the transmission rate.
The incubation period is 3 - 14 days (average 4 to 7 days). There follows an acute febrile
period of 2 – 10 days accompanied by nonspecific symptoms. During this period dengue
viruses circulate in the peripheral blood. During this viraemic stage other biting
mosquitoes become infected.
Clinical Manifestations of Dengue
 Clinical features
Dengue fever is mostly occurs in children and young adults. Clinical features vary with the age of
the patient although clinically occult infection occurs in about 80%.
There are four presentations:
a. Non-specific febrile illness
b. Classical dengue fever
c. Dengue haemorrhagic fever
d. Dengue haemorrhagic fever with dengue shock syndrome, encephalopathy and  liver failure
 Non-specific febrile illness
A maculopapular rash(characterized by flat, red area on skin that is confluent bumps) occurs mostly in
young children. Upper respiratory features, especially pharyngitis, are common.

Classical dengue fever (DF)

Classical dengue fever (DF) is primarily a disease of older children and adults.

.
Febrile Phase
The initial phase is characterized by:-
 high temperature (≥38.5°C)
 Headache
 vomiting
 myalgia
 joint pain
 transient macular rash
Children have high fever but are generally less symptomatic than adults during this phase of the illness.
 Mild hemorrhagic manifestations like petechiae and bruising, particularly palpable liver
 Mild-to-moderate thrombocytopenia and leukopenia, with a moderate elevation of hepatic
aminotransferase levels.
This phase lasts for 3 to 7 days, after which most patients recover without complications
Critical Phase
• In a small proportion of patients, typically in children and young adults, a systemic vascular leak syndrome becomes apparent
around the time of defervescence, evidenced by increasing hemoconcentration, hypoproteinemia, pleural effusions, and ascites.
• Initially, physiological compensatory mechanisms are up-regulated in an attempt to maintain adequate circulation to critical
organs, resulting in narrowing of the pulse pressure when loss of plasma volume becomes critical. If the pulse pressure narrows to
20 mm Hg or less, accompanied by signs of peripheral vascular collapse, dengue shock syndrome is diagnosed.
• Systolic pressure may remain normal or even elevated at this time, and the patient may appear deceptively well, but once
hypotension develops, systolic pressure decreases rapidly and irreversible shock and death may follow.
• During the transition from the febrile to the critical phase, between days 4 and 7 of the illness warning signs that clinically
significant vascular leakage may be developing in the patient.
These signs include
persistent vomiting
severe abdominal pain
tender hepatomegaly
a high or increasing hematocrit level that is concurrent with a rapid decrease in the platelet count
serosal effusions
mucosal bleeding
lethargy or restlessness.
• Hemorrhagic manifestations are common during critical period. In children, clinically significant bleeding occurs
only rarely, usually in association with profound and prolonged shock.
• Major skin bleeding, mucosal bleeding (gastrointestinal or vaginal), or both may occur in adults with no risk
factors and only minor plasma leakage.
• Moderate to-severe thrombocytopenia is common, with nadir platelet counts below 20×109 per liter observed
during the critical phase, followed by rapid improvement during the recovery phase. A transient increase in the
activated partial thromboplastin time and a decrease in fibrinogen levels is observed.
• Other severe manifestations includes liver failure, myocarditis, and encephalopathy, occur with minimal
associated plasma leakage.

Recovery Phase

• The altered vascular permeability is short-lived, reverting spontaneously to a normal level after approximately
48 to 72 hours, and is concurrent with rapid improvement in the patient’s symptoms.
• A second rash may appear during the recovery phase, ranging from a mild maculopapular rash to a severe,
itchy lesion suggesting leukocytoclastic vasculitis that resolves with desquamation over a period of 1 to 2
weeks. Adults may have profound fatigue for several weeks after recovery.
Fig:- Petechial rash in an infant with dengue Fig:- Minor bleeding around injection sites, a
very common feature in dengue.
Fig:- Hematoma in a patient with severe dengue. Coagulation
disorders and the combination of increased vascular fragility, platelet
dysfunction, and thrombocytopenia are believed to explain the
hemorrhagic manifestations in dengue.
Fig:- Diffuse macular rash that appears after recovery
from the acute illness in an adult patient with dengue.
The rash may appear between 3 and 6 days after the
onset of fever. “Islands of white” of normal skin are
surrounded by an erythematous rash.
Dengue haemorrhagic fever (DHF) is primarily a disease of children under 15 years in hyper endemic areas.
 It usually follows a secondary dengue infection and is characterized by
a. high fever
b. haemorrhages
c. circulatory failure
d. hepatomegaly
 Patients either recover or progress to the plasma leakage stage remaining ill despite normalisation of
temperature.
 Plasma leakage is characterized by tachycardia and hypotension with sweating, restlessness and cold
extremities.
 Most patients recover, but in severe cases patients may develop circulatory shock. Mortality may reach 10
– 20% without early appropriate treatment but can be reduced to less than 1% with aggressive fluid
replacement therapy.
 Leucopaenia and thrombocytopenia (less than 100,000/mm3) are usually found between days 3 and 8.
Elevated liver enzymes are common.
Dengue shock syndrome (DSS) is associated with almost 50% mortality.
Warning signs include
a. abdominal pain
b. Vomiting
c. irritability
d. fall in body temperature
e. decrease in platelet count
 Patients die from multi-organ failure and disseminated intravascular coagulation.
 DSS may be accompanied by encephalopathy caused by metabolic and electrolyte disturbances.
 Haemostatic disturbances result from vascular changes, thrombocytopenia and coagulation disorders arising
from, for example,. a decreased fibrinogen level, and increased level of fibrinogen degradation products. Other
reported complications include liver failure, disseminated intravascular coagulation, myocarditis and acute renal
failure.
 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).
• Because dengue virus is heat-labile, specimens awaiting transport to the laboratory should be kept in a
refrigerator or packed in wet ice.
• For storage up to 24 hours, specimens should be kept at between +4 °C and +8 °C.
• For longer storage, specimens should be frozen at -70 °C in a deep-freezer or stored in a liquid nitrogen
container. Storage even for short periods at –20 °C is not recommended.
• Cell culture is used for dengue virus isolation. The mosquito cell line C6/36 (cloned from Aedes
albopictus) or AP61 (cell line from Aedes pseudoscutellaris) are the host cells of choice for routine
isolation of dengue virus. Since not all wild type dengue viruses induce a cytopathic effect in mosquito cell
lines (because of essential roles of the N-terminal half of NS2A in RNA replication and virus-induced
Cytopathic effect), cell cultures must be screened for specific evidence of infection by an antigen
detection immuno-fluorescence assay using serotype-specific monoclonal antibodies and flavivirus group-
reactive or dengue complex-reactive monoclonal antibodies.
• Virus isolation followed by an immuno-fluorescence assay for confirmation generally requires 1–2
weeks and is possible only if the specimen is properly transported and stored to preserve the
viability of the virus in it.
• When no other methods are available, clinical specimens may also be inoculated by intracranial
route in suckling mice or intra thoracic inoculation of mosquitoes. Newborn animals can develop
encephalitis symptoms but with some dengue 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.
Diagnostic Tests
 Diagnostic Tests

Nuclei acid Detection of haemagglutination

Serological test Hematological Test
detection antigen -inhibition Test
 Diagnostic Tests
• Laboratory diagnosis of dengue is established directly by detection of viral components in serum or
indirectly by serologic means. The sensitivity of each approach is influenced by the duration of the
patient’s illness.
• During the febrile phase, detection of viral nucleic acid in serum by means of reverse-transcriptase–
polymerase-chain reaction (RT-PCR) assay or detection of the virus expressed soluble non structural
protein 1 (NS1) by means of ELISA or the lateral-flow rapid test is sufficient for a confirmatory
diagnosis.
• For primary infections in persons who have not been infected previously the diagnostic sensitivity of
NS1 detection in the febrile phase can exceed 90%, and antigenemia may persist for several days after
the resolution of fever. The sensitivity of NS1 detection in the febrile phase is lower in secondary
infections (60 to 80%), reflecting an anamnestic serologic response due to a previous dengue virus or
related flavivirus infection
• Serologic diagnosis of dengue relies on the detection of high levels of serum IgM that bind dengue virus
antigens in an ELISA or a lateral-flow rapid test; IgM can be detected as early as 4 days after the onset of
fever. IgM seroconversion between paired samples is considered a confirmatory finding, whereas
detection of IgM in a single specimen obtained from a patient with a clinical syndrome that is consistent
with dengue is used to establish a presumptive diagnosis.
• Patients with secondary infections mount rapid anamnestic antibody responses in which dengue virus–
reactive IgG may predominate over IgM.
Laboratory Diagnostic Options in a Patient
with Suspected Dengue Infection

Detection of viral nucleic acid, non-

structural protein 1 (NS1), or IgM
seroconversion is a confirmatory finding in
patients in whom dengue is a possible
diagnosis. Day 0 is the first day when the
patient noted any symptom during this
illness. ELISA denotes enzyme-linked
immunosorbent assay, and RT-PCR reverse-
transcriptase polymerase chain reaction.
 Nucleic acid detection
• RT-PCR assays have sensitivity for virus isolation with rapid turn around time.
• All nucleic acid detection assays involve 3 basic steps: nucleic acid extraction and purification,
amplification of the nucleic acid, and detection and characterization of the amplified product.
• RT-PCR assay used universal dengue primers targeting the C/prM region of the genome for an initial
reverse transcription and amplification step, followed by a nested PCR amplification that is serotype-
specific. A combination of the 4 serotype-specific oligonucleotide primers in a single reaction tube
(one-step multiplex RT-PCR) used.
• The products of these reactions are separated by electrophoresis on an agarose gel, and the
amplification products are visualized as bands of different molecular weights in the agarose gel using
ethidium bromide dye, and compared with standard molecular weight markers.
• In this assay design, dengue serotypes are identified by the size of their bands.
Real-time RT-PCR
• Real-time RT-PCR assay is a one step assay system used to quantitate viral RNA and using primer pairs
and probes that are specific to each dengue serotype.
• The use of a fluorescent probe enables the detection of the reaction products in real time, in a
specialized PCR machine, without the need for electrophoresis.

Isothermal amplification methods

• The NASBA (Nucleic acid sequence based amplification) assay is an isothermal RNA specific
amplification assay that does not require thermal cycling instrumentation.

• The initial stage is a reverse transcription in which the single-stranded RNA target is copied into a
double-stranded DNA molecule that serves as a template for RNA transcription.

• Detection of the amplified RNA is accomplished either by electro-chemiluminescence or in real-time

with fluorescent-labelled molecular beacon probes.

• NASBA has been adapted to dengue virus detection with sensitivity near that of virus isolation in cell
cultures and may be a useful method for studying dengue infections in field studies.
For more info on this topic:-http://loopamp.eiken.co.jp/e/lamp/principle.html
https://www.youtube.com/watch?v=L5zi2P4lggw
Detection of antigens
• Until recently, detection of dengue antigens in acute-phase serum was rare in patients with secondary
infections because such patients had pre-existing virus-IgG antibody immuno complexes.
• New developments in ELISA and dot blot assays directed to the envelop/membrane (E/M) antigen and
the non-structural protein 1 (NS1) demonstrated that 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 9 days after the onset of illness.
• NS1 glycoprotein is produced by all flaviviruses and is secreted from mammalian cells. NS1 produces a
very strong humoral response. NS1 antibodies can recognize host factors by molecular mimicry to cause
tissue damage and impair physiological functions. The notion that anti-NS1 antibodies do participate in
dengue pathogenesis resulted from experiments by Falconar, who showed that anti-NS1 monoclonal
antibodies cross-react with human fibrinogen, thrombocytes and endothelial cells and produce
hemorrhage in a mouse model. Many studies have been directed at using the detection of NS1 to make
an early diagnosis of dengue virus infection.
• Fluorescent antibody, immuno peroxidase and avidin-biotin enzyme assays allow detection of dengue
virus antigen in acetone-fixed leucocytes and in snap-frozen or formalin-fixed tissues collected at autopsy
 Serological tests
MAC-ELISA
• For the IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) total IgM in
patients’ sera is captured by anti-µ chain specific Ab (specific to human IgM) coated onto a
microplate.
• Dengue-specific antigens, from one to 4 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. The optical density is measured by spectrophotometer.
• Serum, blood and saliva, but not urine, can be used for detection of IgM if samples are taken
within the appropriate time frame (5 days or more after the onset of fever).
• MAC-ELISA has good sensitivity and specificity but only when used five or more days after the
onset of fever.
• It is not possible to use IgM assays to identify dengue serotypes as these antibodies are broadly
cross-reactive even following primary infections.
Fig:-Principle of a MAC-ELISA test
 IgG ELISA
• The IgG ELISA is used for the detection of recent or past dengue infections.

• 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.

• An ELISA inhibition method (EIM) to detect IgG dengue antibodies is also used for the serological diagnosis. This
system is based in the competition for the antigen sites by IgG dengue antibodies in the sample and the conjugated
human IgG anti-dengue (FC Specific produced in goat). This method can be used to detect IgG antibodies in serum or
plasma and permits identification of a case as a primary or secondary dengue infection.

• In general, IgG ELISA lacks specificity within the flavivirus serocomplex groups. Following viral infections, newly
produced antibodies are less avid than antibodies produced months or years after infection.

• Antibody avidity is used in a few labs to discriminate primary and secondary dengue infections.
IgM/IgG ratio
• A dengue virus E/M protein-specific IgM/IgG ratio can be used to distinguish primary from secondary
dengue virus infections.
• IgM capture and IgG capture ELISA 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.

IgA
• Positive detection for serum anti-dengue IgA as measured by anti-dengue virus IgA capture ELISA (AAC-
ELISA) often occurs one day after that for IgM. The IgA titre peaks around day 8 after onset of fever and
decreases rapidly until it is undetectable by day 40.
• No differences in IgA titres were found in patients with primary or secondary infections.
 Haemagglutination-inhibition test
• The haemagglutination-inhibition (HI) test is based on the ability of dengue antigens to agglutinate red
blood cells (RBC).
• Anti-dengue Ab in sera can inhibit this agglutination and the potency of this inhibition is measured in an
HI test.
• Serum samples are treated with acetone or kaolin to remove non-specific inhibitors of
haemagglutination, and then adsorbed with human RBC to remove non-specific agglutinins. Each batch
of antigens and RBC is optimized. PH optima of each dengue haemagglutinin requires the use of
multiple different pH buffers for each serotype.
• Optimally the HI test requires paired sera obtained upon hospital admission (acute) and discharge
(convalescent) or paired sera with an interval of more than seven days.
• The assay does not discriminate between infections by closely related flaviviruses (e.g. between
dengue virus and Japanese encephalitis virus or West Nile virus) nor between immunoglobulin isotypes.
• The response to a primary infection is characterized by the low level of Ab in the acute-phase serum
drawn before day 5 and a slow elevation of HI antibody titres thereafter. During secondary dengue
infections HI antibody titres rise rapidly, usually exceeding 1:1280.
Fig:-Haemagglutination-inhibition assay
 Haematological tests
• Platelets and haematocrit values are measured during the acute stages of dengue infection. A
drop of the platelet count below 100 000 per µL may be observed in dengue fever but it is a
constant feature of DHF. DENV infection induces platelet consumption due to disseminated
intravascular coagulation (small blood clot in blood steam), platelet destruction due to increased
apoptosis, lysis by the complement system and by the involvement of antiplatelet antibodies
• Thrombocytopaenia ( in the number of platelets) is observed in the period between day 3 and
day 8 following the onset of illness. The mechanisms involved in thrombocytopenia and bleeding
during DENV infection are not fully understood.
• Haemoconcentration ( blood viscosity.), as estimated by an increase in haematocrit (% of RBC in
blood) of 20% or more compared with convalescent values, is suggestive of hypovolaemia
(condition in which the liquid portion of the blood (plasma) is too low) due to vascular
permeability and plasma leakage.
Cure
 Vaccines
•As primary dengue virus infection does not give long term protection to re-infection with the other three viral serotypes,
it has been generally held that a vaccine will need to induce protective responses against all four serotypes, mandating a
tetravalent formulation.

Dengvaxia

• Sanofi Pasteur vaccine synthesized the tetravalent dengue vaccine named as Dengvaxia (CYD-TDV-chimeric yellow fever
dengue tetravalent dengue vaccine).
•This is a chimaera vaccine using the related yellow fever 17D vaccine strain as a backbone, with dengue virus prM
protein and E protein genes replacing those from yellow fever virus. The vaccine contains a mixture of 4 recombinant
viruses representing each serotype (termed CYD1 to CYD4).
• WHO has not qualified the use of this vaccine in general and also it was observed to have low efficacy against serotype
1 and 2. It cannot protect against serotype 5 of DENV which has been discovered in 2013.
•This vaccine is given in a 3-dose schedule at an interval of 6 months to human beings who live in dengue-endemic areas
with the age of 9-45 years.

.
]
Tetravalent dengue vaccine
(TDV)
• TDV contains DENV-2 strain followed by 3 chimeric viruses which have precursor membrane and
envelope protein of DENV-1, 3 and 4 strains and then these are genetically combined into
attenuated TDV 2 genome backbone.
• Regardless of patient exposed to dengue previously, TDV induces a humoral and cellular immune
response in adult and children.
• It exhibited neutralizing antibody response to all type of DENV serotypes.
• TDV is also effective against wild type dengue viruses in mouse and non-human primate animal
model.
• Humoral immunogenicity was more against DENV-2 followed by DENV 1, 3 and 4 respectively.
TDV has shown acceptable tolerability and safety profile in ad.ult and children
 Antiviral agents
• Antiviral agents are under development for the treatment of dengue fever. These agents generally act
on RNA genome of dengue virus. The genome of the dengue virus is single-stranded RNA which has
three structural proteins and five non-structural proteins.
• Antiviral agents are under development on the basis of the protein they act. Some antiviral agents act
on proteins like NS5, capsid, and NS3, so on this basis antiviral agents are classified as follows:-
Mechanism of action of different therapies
on dengue virus
 REFERENCE
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http://www.apjtm.org/article.asp?issn=1995-7645;year=2019;volume=12;issue=4;spage=147;epag
e=152;aulast=Rozera
https://library.wur.nl/frontis/disease_vectors/17_takken.pdf
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0036-46652012000500009
https://www.slideshare.net/QingYuanPang/genetically-modified-mosquitoes/5
https://www.cdc.gov/dengue/training/cme/ccm/page51935.html
https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue
https://www.cdc.gov/dengue/index.html
https://www.ncbi.nlm.nih.gov/pubmed/21760609
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/en/
https://www.hindawi.com/journals/mi/2015/313842/
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https://www.ncbi.nlm.nih.gov/pubmed/26603900
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https://www.nejm.org/doi/full/10.1056/NEJMra1110265
https://www.worldmosquitoprogram.org/en/work/wolbachia-method/how-it-works
http://www.apjtm.org/article.asp?issn=1995-7645;year=2019;volume=12;issue=4;spage=147;epage=
152;aulast=Rozera
https://en.wikipedia.org/wiki/Dengue_fever
https://www.niaid.nih.gov/diseases-conditions/dengue-fever
https://talk.ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rna-viruses/w/flaviviridae/3
60/genus-flavivirus
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https://www.researchgate.net/figure/TDV-vaccine-candidate-The-Takeda-tetravalent-vaccine-TDV-has
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https://books.google.co.in/books?id=WAnpCgAAQBAJ&pg=RA1-PA233&dq=International+Encycloped
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https://www.medsci.org/v14p1342.htm
Thank you