3. Most common arbovirus
Named after Swahili word “dinga” meaning
fastidious or careful (gait of a person suffering from
bone pain of dengue fever)
Seen in tropics and sub-tropics
Break-bone fever –coined during the Philadelphia
epidemic in 1780
3
4. HISTORY
Oldest DENV-1 isolate, the Mochizuki strain, was
isolated in 1943 from Japan, with subsequent
DENV-1 activity reported in the America in 1977
and in Africa in 1984
DENV-2 diverged from the sylvatic ancestor
approximately 400–600 years ago. First reported in
1944 in Asia, 1964 in Africa and 1953 in America
4
5. DENV-3 was first reported in 1953 in Asia,1963 in
the America and during 1984–1985 in Africa
DENV-4 was reported for the first time in Asia in
1953 and in the Americas in 1981
5
6. ORIGIN
Originated in non-human primates (sylvatic DENV)
& forest-dwelling Aedes mosquito vectors in Africa
and Asia, with cross-species transfer to humans
Sylvatic DENVs emerged 1000 years ago, with
transmission in human established as recently as
the last few hundred years
World War II was responsible for spread of dengue
from Southeast Asia throughout the Pacific region
6
7. 7
DENV are only arbovirus that have fully adapted to a
human-mosquito-human cycle and no longer depend on
forest cycle for maintenance
8. GLOBAL SCENARIO
Dengue is endemic in more than 100 countries with
2.5 billion people at risk
No. of cases reported in 2019- 4.2 million
8
10. SITUATION IN INDIA
India is hyperendemic with all 4 serotypes
Initially confined to east coast of India
Epidemic in 1963 along with chikungunya virus
affected Culcutta and Madras
In 1990s- major epidemic in Delhi and Surat
Indian genotype was associated with DHF epidemic
in New Delhi, India in 1996
10
11. CLASSIFICATION
Taxonomic classification
Arenaviridae Bunyaviridae Filoviridae Flaviviridae Reoviridae Togaviridae
11
• Genus
Arenavirus
• Genus
Marburgvirus
• Genus
Ebolavirus
• Genus
Flavivirus
• Genus
Coltivirus
• Genus
Orbivirus
• Genus
Alphavirus
Genus:
•Orthobunyavirus
•Hantavirus
•Nairovirus
•Phlebovirus
Other families- Rhabdoviridae, orthomyxoviridae
13. STRUCTURE OF FLAVIVIRUS
Consists of about 70 viruses
Spherical
40–60 nm in diameter.
Genome: positive-sense, single-stranded RNA, 11
kb in size.
Lipid envelope covered densely with surface
projections
Inactivated by acid pH, heat, lipid solvents,
detergents, bleach, phenol, 70% alcohol, and
formaldehyde.
13
14. GENOME
3 structural proteins
Capsid ‘C’ protein
pre-membrane/membrane (prM/M)
protein
Envelope ‘E’ protein
7 non structural proteins
NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5
ORF encodes both structural and non-structural
genes
14
15. FUNCTIONS
Structural proteins form the components of the
DENV virion
The C protein is crucial for nucleocapsid formation
during the primary stages of DENV virion assembly
M protein plays an important role in the
arrangement and maturation of the DENV particle
15
16. E protein is composed of three domains (domain I–
III), with domain III responsible for receptor-binding
activity. It is crucial for virus binding and fusion to
host cell membrane
Non-structural proteins mainly NS1 is a 45 kDa N-
linked glycoprotein involved in the RNA replication
complex
16
17. SEROTYPES
DENV 1 to 4, DENV-5- discovered in 2013 in
Bangkok
Serotype• DENV-1: Three genotypes
• DENV-2: Six genotypes
• DENV-3: Four genotypes
• DENV-4: Four genotypes
Share approximately 65% amino acid sequence
similarity
17
18. RISK FACTORS
Infants and elderly
Obesity
Chronic diseases
Patients on steroids or NSAID treatment
Sex- females > males
Race- whites > blacks
Nutritional status- malnutrition is protective
Sequential infection
Travel to endemic area within the incubation period
for a given syndrome
Trade 18
19. VECTOR
Aedes aegypti & Aedes albopictus
A.aegypti- nervous feeder, discordant species,
strongly anthropophilic, hence is the most efficient
vector
A.albopictus (Asian origin)- aggressive and
concordant feeder, less efficient in transmission
19
Aedes
21. Aedes seen in fresh water sources such as water
jars, vases, discarded containers, coconut husks
and old tires, plants
Nondegradable tires and long-lived plastic
containers in trash repositories
Rainy season
Closed habitations with air-conditioning inhibit
transmission
21
22. Bites on day time
Temperature directly affects the replication rate of
virus in the mosquito
Aedes becomes infective only when it feeds on
viremic patients (from a day before to end of febrile
period i.e.,5 days)
22
23. INCUBATION PERIOD
Extrinsic incubation period- 8-10 days, once
infected, remains infective for lifetime
Intrinsic incubation period- 3 to 14 days
23
24. TRANSMISSION CYCLE
Principle reservoir- Man & Aedes mosquito
5th serotype follows sylvatic cycle
Transovarial transmission in mosquito is essential in
maintaining both human and sylvatic transmission
cycles during dry seasons or interepidemic periods.
Eradication is challenging
24
25. Non-vector transmission routes including blood
transfusion, bone marrow transplant, and
intrapartum and perinatal transmission have also
been reported
There is no evidence that DENV may be
transmitted via semen
Infection can be transmitted by accidental needle
stick injury
25
27. PATHOGENESIS
DENV- E protein binds to cell-surface attachment molecules
and receptors such as glycosaminoglycans, heat-shock
proteins, neolactotetraosylceramide, CD14, C-type lectins,
DC-SIGN and the mannose receptors (dentritic cells and
macrophages)
(Receptor mediated endocytosis)
Internalization
Low pH of the endosome triggers a conformational change
in the E protein
27
28. Fusion of the viral and cellular membranes
Disassembly of the DENV virion
DENV nucleocapsid is released into the cytoplasm
Virus uncoats and the DENV genome is released into
the cytoplasm
Translated into a polyprotein that is processed by viral
and cellular proteases
28
29. Positive-strand genome serves as mRNA for
translation into a single polyprotein and,
subsequently, as template for RNA synthesis
29
Polyprotein is then directed to
the endoplasmic reticulum and
is cleaved into the individual
structural and NS proteins by
host signalases and viral NS3
protein
Newly synthesized RNA can be
used for new rounds of
translation or for encapsidation
into new virions
30. Viral RNA and proteins are then assembled into
immature progeny virions at the endoplasmic
reticulum membrane
Transported to trans-Golgi network where the
immature (spiky) virion transforms to mature
(smooth) morphology
Precursor peptide is cleaved from prM mediated by a
host-encoded furin protease
30
32. IMMUNE RESPONSE
Innate immune system- does not provide a long-
term or specific response
Adaptive immune system- more specific and
involves cellular and humoral components
Both immune response results in either resolution
of infection or enhancement of disease severity
32
34. INNATE IMMUNITY
An important cytokine produced during DENV
infection is IFN-γ.
This cytokine controls the production of nitric oxide
that is essential for the control of DENV replication
and resistance to infection
Increased IFN-γ was associated with protection
against fever and reduce viral load, with higher
survival rates in DHF patients
In contrast, other proinflammatory cytokines seem
to play a pathologic role. 34
35. CELLULAR IMMUNITY
Principal target for both of CD4+ and CD8+ T cells
Original antigenic sin- dominance of T cell
responses mounted against a previously infecting
serotype, over the current infecting serotype
35
36. During a primary infection, T cells and cross-reactive memory T
cells are produced.
Upon secondary infection with a heterologous serotype, highly
cross-reactive CD8+ T cells with a high avidity for the secondary
DENV infection are activated massively and induce high
production of proinflammatory cytokines
Cross-reactive CD8+ T cells may lose their cytolytic activity
This may delay DENV clearance, prolong activation of cross-
reactive CD8+ T cells and induce a high level of proinflammatory
cytokines and other soluble factors
Together, these factors affect vascular permeability, leading to a
higher incidence of severe dengue
36
37. HUMORAL IMMUNITY
E protein induces the production of antibodies that
have pivotal roles in DENV neutralization
Incomplete cleavage of pre-M protein also induces
the pre-M protein-specific antibody that is highly
serotype cross-reactive and induces ADE
NSI protein activates antibody-dependent cellular
cytotoxicity and complement-dependent lysis of
infected cells
37
38. Neutralizing antibody- protective against infective
serotype (lasts lifelong) as well as other serotypes (lasts
for sometime)
Non-neutralising antibody- heterotypic in nature-
produced against other serotypes but not against
infective serotype
Inhibits bystander B cell activation
Promote recruitment of mononuclear cells
followed by release of cytokines, vasoactive
mediators and procoagulants leading to DIC
Antibody dependent enhancement (ADE)
38
39. Infection of DENV-1 followed by DENV-2 or DENV-3 was
associated with higher incidence of severe dengue
Longer the interval between two sequential DENV
infections (approximately beyond two years), the higher
the proportion of severe dengue
During sequential infection, only 2-4% of individuals
develop severe disease
Tertiary or quaternary infections are clinically silent or
very mild
39
40. Risk of hemorrhagic fever syndrome is about 0.2%
during first dengue infection but 10 fold higher
during second dengue infection
Ratio of inapparent to apparent infection is 15:1 in
primary infections; lower in secondary infections
Cross protection between DENV serotype lasts less
than 12 weeks
40
41. Infection of human haematopoietic cells
Alterations in megakaryocytopoieses
Impaired progenitor cell growth
Platelet dysfunction (platelet activation and aggregation),
increased destruction or consumption (peripheral
sequestration and consumption)
Thrombocytopenia
Haemorrhage / disseminated intravascular coagulation
41
DHF PATHOGENESIS
42. NS1 epitopes mimic those of endothelial cell
surface molecules
Antibodies to those shared epitopes induce
endothelial cell damage and elicit inflammatory
cytokines resulting in disturbances in capillary
permeability which causes plasma leakage
42
DHF PATHOGENESIS
45. FACTORS DETERMINING THE OUTCOME
Infecting serotype: Type 2 more dangerous than
other serotypes
Sequence of infection: serotype 1 followed by
serotype 2 – DHF & DSS
Age: children less than 12 years
45
46. CLINICAL PHASES IN DENGUE
Febrile phase- Dehydration; high fever may cause
neurological disturbances and febrile seizures in
young children
Critical phase- Shock from plasma leakage; severe
haemorrhage; organ impairment
Recovery phase- Hypervolemia (only if intravenous
fluid therapy has been excessive and/or has extended
into this period)
46
47. CLINICAL CLASSIFICATION
Traditional (1997) WHO classification:
1. Dengue fever
Biphasic/ breakbone/ saddleback fever (5-7 days)
Maculopapular rashes over chest and upperlimbs
Severe frontal headache
Muscle and joint pains
Lymphadenopathy
Retro-orbital pain
Loss of appetite, nausea and vomiting 47
48. Other features of dengue fever
Conjunctival/ scleral injection
Palatal vesicles
Altered (metallic) taste
48
49. 2. Dengue hemorrhagic fever (DHF):
High grade continuous fever
Hepatomegaly
Thrombocytopenia
Raised hematocrit by 20%
Evidence of hemorrhage- detected by positive
tourniquet test and spontaneous bleeding from skin,
nose, mouth and gums
49
54. ANTIGEN DETECTION
NS1-based assays have good diagnostic utility, for
both screening for and confirming DENV infection
ELISA and ICT
Detectable from day 1 of fever and remains positive
upto 18 days
Highly specific
54
55. Pitfalls of NS1 Ag detection:
Doesn’t differentiate between dengue serotypes
Sensitivity of NS1-based tests is lower during
secondary infections
Sensitivity is lower for DENV-4 and DENV-2
(compared to DENV-1)
55
56. ANTIBODY DETECTION
In primary infection: Slow and of low titre. IgM
appears after 5 days of fever and disappears within
90 days. IgG appears in 14-21 days and then
slowly increases
In secondary infections: IgG Ab titre rise rapidly.
Cross reactive with other flaviviruses and give false
positive result.
In past infection: low levels of IgG remain
detectable for over 60 years
56
57. IgM antibody cross react with other flaviviruses
IgG antibody doesn’t distinguish between current
and past infection
IgG antibody detection should be done in paired
serum sample
57
58. MAC-ELISA (IgM antibody capture ELISA)
Diagnosis is based on detecting dengue-specific
IgM antibodies in the test serum by capturing them
using anti-human IgM antibody previously bound on
a solid phase.
Sensitivity and specificity of approximately 90% and
98%, respectively but only when used five or more
days after onset of fever
58
60. Hemagglutination inhibition (HI) assay
Requires paired sera obtained upon hospital
admission (acute) and discharge (convalescent) or
paired sera with an interval of more than seven
days
HAI antibody titres in primary infection peak at
1:640 whereas titres of 1:1280 or greater are seen
in secondary infections
60
61. IgG specific ELISA
Samples with a negative IgG in the acute phase
and a positive IgG in the convalescent phase of the
infection are primary dengue infections.
Samples with a positive IgG in the acute phase and
a 4 fold rise in IgG titre in the convalescent phase
(with at least a 7 day interval between the two
samples) is a secondary dengue infection
61
62. IgM/IgG ratio
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.
62
63. Plaque reduction neutralizing test (PRNT)
Rapid tests
Other antibody detection assay
CFT
Neutralisation tests
IgG avidity test- on acute phase serum samples
Low-avidity antibodies are characteristic of
recent infections, whereas high-avidity IgG
antibodies are seen 6 months or more after
the onset of symptoms
63
OTHER ANTIBODY DETECTION TESTS
64. RAPID DIAGNOSTIC TESTS FOR DENGUE
Lateral flow ICT for dengue IgM antibodies or NS1
antigen- poor sensitivity and specificity
Government of India has passed an order in 2016,
that a positive RDT should be considered as
probable diagnosis; must be confirmed by ELISA
64
65. Probable Dengue fever: A case compatible with the clinical
description of dengue fever during the outbreak and/or Non
ELISA based NS1/IgM positive
Confirmed Dengue Fever: A case compatible with the
clinical descriptions of dengue fever with at least one of the
following:
• Demonstration of dengue virus antigen in a serum sample by
NS1-ELISA
• Demonstration of IgM antibody titre by ELISA in the single
serum sample
• IgG seroconversion in paired sera after 2 weeks with a fourfold
increase of IgG titre
• Detection of viral nucleic acid by PCR
• Isolation of the virus (virus culture positive) from serum, plasma
or leucocytes.
65
66. VIRUS ISOLATION
Dengue virus can be detected in blood from -1 to
+5 days of onset of symptoms
Isolated from blood, serum, plasma, liver, spleen,
lymph node and other tissues
66
67. Inoculation into mosquitoes (intrathoracic
inoculation of adult mosquitoes)
Inoculation into mosquito cell line, C6/36 (Ae.
albopictus) or onto mammalian cell lines such as
Vero (African green monkey kidney), LLCMK2
(Monkey Rhesus kidney) and BHK21 (baby
hamster kidney).
Intracerebral inoculation in brains of suckling mice
and hamsters
67
68. Clinical specimens used for viral isolation may be
whole blood, serum, plasma or homogenized tissue
(most often in fatal cases)
Some flavivirus strains produce CPE or form cell
syncytia in these lines, but most arboviruses do not
Immunofluorescence assay or reverse transcriptase
polymerase chain reaction (RT-PCR) is performed
from those cell lines
68
69. Limitations of cell culture:
Tedious and requires at least 7 days for incubation
and conformational testing
It requires well-established lab facilities with well-
trained personnel
The window period for sample collection is limited to
the acute phase of infection
Low level of DENV viremia is not suitable for virus
culture
69
70. MOLECULAR METHOD
Can detect DENV RNA in a clinical specimen within
24–48 h after infection
RT-PCR (nested, multiplex), Real time RT-PCR or
isothermal amplification methods such as nucleic
acid sequence-based amplification (NASBA)
Sensitivity- 80-100%
70
73. OTHER METHODS
Electron Microscopy
Detection of Viral Antigens by Use of Immuno
Histochemical staining or Immunofluorescence
73
74. NEWER DIAGNOSTIC TECHNIQUES
Microsphere-based immunoassays (MIAs)
Microarray technology
Nanodiagnostic tools, like nanosized materials
including liposomes, nanowires and nanopores,
coupled to conventional fluorescence potentiometry
Voltammetry methods
Western blot techniques
74
75. LAB FINDINGS
Leukopenia
Thrombocytopenia- below 1,00,000 per µL
Increased hematocrit by 20% or more
Elevated serum aminotransferases
75
76. TREATMENT
No specific antiviral therapy
Treatment is symptomatic and supportive
Replacement of plasma losses
Correction of electrolytes and metabolic
disturbances
Platelet transfusion if needed
Therapeutic antibodies to neutralise multiple
genotypes of dengue
Fatality rate can reach 15% but reduces to 1% with
proper treatment
76
79. TRIAL STUDIES ON DENGUE TREATMENT
Carbazochrome sodium sulfonate- for preventing
capillary leakage
Oral prednisolone as an anti-inflammatory agent
Lovastatin (statin) as an anti-DENV and anti-
inflammatory at the endothelium
79
80. Treatments to reduce severe bleeding or shorten
the time to cessation of bleeding, such as single
platelet donations or recombinant human (rh) IL-11,
have been tested in small-scale trials.
Other anti-DENV agents such as chloroquine,
balapiravir (nucleoside analogue and a polymerase
inhibitor) and celgosivir (glucosidase I inhibitor)
80
81. PREVENTION
VACCINATION:
Ideal dengue vaccine-
Rapid and highly protective
Long-term
Type- or cross-specific neutralizing antibodies
against all DENV serotypes regardless of individual
immune status and age of vaccination
81
82. 1. Dengue vaccine (CYD-TDV)- Chimeric Yellow
Fever Dengue, live attenuated Tetravalent Dengue
Vaccine- dengvaxia
Live attenuated yellow fever 17D virus as vaccine
vector in which target genes of all four serotypes
are integrated by recombinant technology
Indicated for 9-45 years
Schedule- 3 injections of 0.5ml subcutaneously at 6
month interval
82
83. Developed by Sanofi Pasteur Company
Available as lyophilized form
Efficacy- 60.8%
Not yet available in India
83
84. 2. Tetravalent dengue vaccine (TDV)
Previously named DENVax
Chimeric degue-2 PDK-53-based tetravalent
vaccine
Developed by Takeda Vaccines Inc
Efficacy 80.9%
84
85. 3. TV003/TV005 vaccine
Live attenuated tetravalent dengue vaccine
Produced by introducing a deletion in the
untranslated region (3’ UTR) into a wild type DENV
Still in phase-II trial
85
86. 4. Nucleic acid-based dengue vaccine, D1ME
Monovalent plasmid DNA vaccine expressing the
prM and envelope (E) genes of DENV-1 virus
In phase-I trial
86
87. 5. TDENV PIV (a tetravalent purified inactivated
vaccine) and V180
Recombinant subunit vaccine based on the DENV
wild type prM and truncated E protein (DEN-80E)
via expression in the Drosophila S2 cell expression
system,
Being evaluated in Phase I clinical trials
87
88. OBSTACLES IN DEVELOPING VACCINE
DENV evolution is rapid and unpredictable,
generating many strains within DENV serotypes
Great genetic variation either between the different
serotypes or between viral genotypes within each
serotype
88
89. MOSQUITO CONTROL MEASURES
Environmental interventions- mosquito control
Chemical control using insecticides and larvicides
Biological control- fish feed on mosquito larva
Individual protection against mosquitoes
89
90. REFERENCES
1. Jawetz,Melnick & Adelberg’s Medical Microbiology, 27th edition
2. Topley & Wilson’s Microbiology & Microbial infections, Virology volume 2, 10th
edition
3. Fields Virology, volume 1, seventh edition
4. Manual of Clinical Microbiology, Patrick R Murray, Ellem Jo Baron, James H.
Jorgensen, Marie Lousie Landry, Michael A. Pfaller, Volume 2, 9th edition
5. Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases,
8th edition
6. Harrison’s principle of internal medicine, 20th edition, volume 2
7. Anathanarayan & Paniker’s Textbook of Microbiology, 9th edition
8. Essentials of Medical Microbiology, Apurba Sastry, Sandhya Bhat, 2nd edition
9. Park’s textbook preventive and social medicine, K.Park, 23rd edition
10. Harapan H, Michie A, Sasmono RT, Imrie A. Dengue: A Minireview. Viruses.
2020 30;12(8).
11. Maheshwari M, Broor S. Recent Advances in Dengue Diagnosis. Indian J Health
Sci Care. 2015 Jan 1;2:135.
12. Dengue guidelines for diagnosis, treatment, prevention and control, New edition,
2009, WHO
13. National guideliness, Dengue case management during COVID-19 pandemic,
2020, NVBCP, MoHFW.
90
92. COINFECTION WITH COVID-19
Difficult to distinguish symptoms and signs
Both have an unpredictable clinical course
Most of the hospitals are busy with managing
COVID-19 at present
Most of the cases of COVID-19 and dengue are
asymptomatic
IV fluid therapy is challenging in coinfected patients
due to early development of ARDS/pulmonary
oedema
Treatment with LMWH for management of COVID-
19 may enhance bleeding in the presence of
dengue 92
