An overview on Dengue Virus, its epidemiology, prevention, evolution, structure & components, transmission, life cycle, pathophysiology, coagulopathy, symptoms, diagnosis, antiviral drugs/vaccination. Performed by Catherine Duong, Diana Elborno, Zehraa Cheaib, Michael South, Veronica Nguyen & Zachary Jilesen at McMaster University, Virology, Fall of 2014.

1. DENGUE VIRUS

2. Case-study
17 year-old female admitted into Princeton Plainsborough Hospital. The
patient present the following symptoms:
• High Fever
• Severe nausea
• Mild nosebleeds
• Joint pain
• New appearance of skin rashes

3. Case-study

4. BACKGROUND INFORMATION
• Word origin may be derived from Swahili or Spanish
• 3 forms of dengue:
• Dengue fever (DF), dengue haemorrhagic fever (DHF), and
dengue shock syndrome (DSS)
• Arboviral infection transmitted by the Aedes aegypti mosquito
• Originates in African forests independent of humans  breeds in water
storage containers  slave and commerce trade brought it to South-East
Asia & “New World” in 17th-19th centuries  1800 in global tropical coasts
(Comprehensive Guidelines for Prevention and
Control of Dengue and Dengue Haemorrhagic
Fever, 2011)

5. GLOBAL DISTRIBUTION
(Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever, 2011)
• Found in tropical and subtropical regions of the world, 2.5 billion at risk
• Endemic in more than 100 countries in the WHO regions:
• Africa, Americas, Eastern Mediterranean, South-East Asia, and Western Pacific
• South-East Asia and Western Pacific regions are the most seriously affected

6. EPIDEMIOLOGY
• ~ 50-100 million infections occur worldwide, 0.5 million hospitalized for DHF
• ~ 90% are children under 5 years
• Classical DF more common in adults
•  frequency of epidemics observed, infection rates of the previously unexposed is 40% - 50%,
can reach 80% - 90%
• Needs coincidence of many vector mosquitoes, many people with no immunity to ¼ virus types, and
opportunity for contact
• Infection with any 1 of 4 serotypes  lifelong immunity to that 1 serotype
• Provides 1-3 years of cross-protection against other 3 serotypes
• Reoccurring infection with different serotypes  severe dengue
(DHF/DSS)
(CDC, 2014)

7. EPIDEMIOLOGY CONT’D
• Ae. aegypti strongly attracted to humans, domesticated, and is a nervous feeder
• Bites more than one host to complete blood meal and gonotropic cycle
• Breeds in safe clean water, feeds at dusk and dawn
• Results in multiple cases in cities
• Transmission usually occurs in rainy seasons when humidity
and temperature are conducive for survival and breeding
• transport, human contact, urbanization,
drinking water supply in rural areas brought DF to
urban and rural areas globally
(Comprehensive Guidelines for Prevention and Control of Dengue and Dengue
Haemorrhagic Fever, 2011)

8. PREVENTION
• Key is disease surveillance to detect epidemics
• Dengue-endemic regions should be educated about the virus,
recognize symptoms, and prevent transmission:
• Regularly remove sources of stagnant water to prevent breeding
• Use mosquito repellent, coils, and nets
• Wear long, loose clothing in the daytime
• Use nets and coils on those with DF to prevent mosquitoes
transmitting infections
• Stay in AC or well-screened housing
• Infected mosquitoes like to live in/around homes
with clean water

9. EVOLUTION
• Genus: Flavivirus
• Diverse
• 4 serotypes
• 1970: Central America
and Africa (DEN 1 & 2)
Southeast Asia (Den1-4)
• 2004: Worldwide (Den1-
4)
• 65% genome shared
• Clinical characteristics
conserved
(Holmes,E & Twiddy, S, 2003)

10. VIRUS STRUCTURE & COMPONENTS
Dengue
• Class IV: Positive Sense Single Stranded
RNA Virus
DNA Components
• Internal Structure: 10 genes (3 structural
and 7 non-structural )
Molecular Structure
• External Structure : icosahedral & 50
nm in diameter
(Kuhn, R.J. et al. , 2002)

11. TRANSMISSION
• Mosquito (female mosquito)
• Possibly through blood transfusions
• Human incubation period ~ 4 days
• Viremia lasts ~5 days
• During viremia biting mosquitos are
susceptible to infection
• Mosquitos incubation period ~8-12
days, which then it is infectious for life
• Virus resides in salivary glands of
infected mosquito (anti-clotting
factors, ect)
(Nishiura, H., & Halstead, 2007; Rodenhuis-Zybert, 2011).

12. THE DENGUE VIRUS
Envelope Proteins
Lipid Bilayer
Capsid
RNA Genome
DENGUE LIFE CYCLE
1. 1. Receptor-Mediated Endocytosis
2. 2. Fusion
3. 3. Nucleocapsid Release
4. 4. RNA Replication
5. 5. Translation
6. 6. Budding
7. 7. Furin Cleavage
8. 8. Progeny Release
(Martina, B. et al, 2009)

13. DENGUE LIFE CYCLE
Infected Mosquito Biting

14. DENGUE LIFE CYCLE
Extracellular Matrix

15. DENGUE LIFE CYCLE
Cognate receptor
Dengue virus
Extracellular Space
ERnecdeopctyotro Bsiisnding
Cell Membrane
Cytoplasm

16. DENGUE LIFE CYCLE
Endosome
Proton Pump
Fusion
H+
H+
CLoonwfoerrmeda tpioHnal Change
Cytoplasm

17. DENGUE LIFE CYCLE
Endosome
Proton Pump H+
H+
FCuosnifoonrmational Change
Cytoplasm

18. DENGUE LIFE CYCLE
NFuusciloenocapsid Release
Cytoplasm

19. DENGUE LIFE CYCLE
Nucleocapsid Release Cytoplasm

20. DENGUE LIFE CYCLE
Cytoplasm
Viral RNA
Viral RNA Translation
Ribosomes
Rough Endoplasmic Reticulum

21. DENGUE LIFE CYCLE
Cytoplasm
Viral RNA
Viral RNA Translation
Ribosomes
Rough Endoplasmic Reticulum

22. DENGUE LIFE CYCLE
Cytoplasm
Envelope Proteins
Viral RNA Translation
Capsid Proteins
RNA dependent
RNA Polymerase
Rough Endoplasmic Reticulum

23. DENGUE LIFE CYCLE
Cytoplasm
RdRp
Envelope Proteins
Viral RNA Translation
Capsid Proteins
RNA dependent
RNA Polymerase
Proteases
Peptidases
Cell Membrane
Rough Endoplasmic Reticulum

24. DENGUE LIFE CYCLE
RNA Replication
Cytoplasm
RdRp
RNA dependent RNA Polymerase
SDionugblel es tsrtarnanddeded R RNNAA

25. DENGUE LIFE CYCLE
RNA Transcription
Cytoplasm
Double stranded RNA
RdRp

26. DENGUE LIFE CYCLE
Cytoplasm
Viral RNA
Viral RNA Translation
Ribosomes
Rough Endoplasmic Reticulum

27. DENGUE LIFE CYCLE
Cytoplasm
Viral RNA
Viral RNA Translation
Ribosomes
Rough Endoplasmic Reticulum

28. R
DENGUE LIFE CYCLE
Cytoplasm
Envelope Proteins
Viral RNA Translation
Capsid Proteins
RdRp
Rough Endoplasmic Reticulum

29. DENGUE LIFE CYCLE
Cytoplasm
Viral RBNuAd dTirnagnslation
Cell Membrane
Rough Endoplasmic Reticulum

30. DENGUE LIFE CYCLE
RER
Golgi Apparatus

31. DENGUE LIFE CYCLE
Golgi Apparatus
Furin Cleavage
Furin

32. Golgi Apparatus Cytoplasm
Cognate receptor
Extracellular Space
Molecular Structure
• External Structure : icosahedral
Progeny Release

33. PATHOPHYSIOLOGY
Complement
Activation
ADE of virus
T-cell
Immunology
Plasma Leakage Bleeding Diathesis
Hypovolemic Shock
(Nishiura, H., & Halstead, 2007; Halstead & O’rourke, 1977) ).

34. PATHOPHYSIOLOGY- ADE Activation
Main Result: Increase in virus replication and numbers
Heterotypic antibodies
- Low concentration
- Partial neutralization
- Ie. prM
(Halstead et al. 2010)

35. PATHOPHYSIOLOGY- ADE Activation
Main Result: Increase in virus replication and numbers
Heterotypic antibodies
- Low concentration
- Partial neutralization
- Ie. prM
Virus uptake
- Delivery to Fcɣ receptor
cells
(Halstead et al. 2010)

36. PATHOPHYSIOLOGY- ADE Activation
Main Result: Increase in virus replication and numbers
Heterotypic antibodies
- Low concentration
- Partial neutralization
- Ie. prM
Virus uptake
- Delivery to Fcɣ receptor
cells
Replication
- Increased viral
replication compared
to virus alone
(Halstead et al. 2010)

37. PATHOPHYSIOLOGY- Complement Activation
• NS1- Glycoprotein secreted by dengue
infected cells
• Activation of innate immune system on
endothelial cell surface
Anaphylatoxins
1) Classical complement
2) Alternative complement
Result:
1) Apoptosis of endothelial cell
2) Inflammation = CYTOKINES!
(Guzman & Kouri, 2002)

38. PATHOPHYSIOLOGY- T-Lymphocyte Pathogenesis
Primary Infection
DENV-1
T- cell selection
(Dejnirattisai et al., 2010)

39. PATHOPHYSIOLOGY- T-Lymphocyte Pathogenesis
Primary Infection
DENV-1
Secondary Infection
T- cell selection
DENV-2 Clonal Expansion
Cross-Reaction
(Dejnirattisai et al., 2010)

40. PATHOPHYSIOLOGY- T-Lymphocyte Pathogenesis
Primary Infection
DENV-1
Secondary Infection
T- cell selection
DENV-2 Clonal Expansion
Cross-Reaction
TNF-α
IFN-ɣ
IL-2
CYTOKINE
STORM
(Dejnirattisai et al.,
2010)

41. ENDOTHELIAL CELL ACTIVATION
Cytokines induce changes in
endothelial morphology:
• Loss of vascular integrity
• Increased adhesion molecules
• Increased cytokine production
http://www.scielo.br/img/revistas/rb/v43n6/en_a13fig01.jpg
IMPLICATIONS: Plasma leakage
(Manson et al. 2003)
Edema Hypovolemic shock Thrombocytopenia

42. COAGULOPATHY
Loss of Plasma
Components
- Platelets
- Plasma
fibrinogen
Immune-mediated
Destruction of
Platelets
Glycocalyx
Damage
- Anti-coagulation
via heparan
sulphate
(Sellahewa, K, 2012)

43. SYMPTOMS
Dengue Fever and Dengue Hemorrhagic Fever
Febrile:
• Hallmark trait is fever
• Convulsions may occur due to high fever (DHF)
• Thrombocytopenia, leukopenia (levels more drastic
with DHF)
Critical
• Plasma leakage into pleural cavities, ascites (DHF)
• Subnormal temperatures, defervescence
• Varying degrees of hemorrhage (worsened for DHF)
Recovery
• Reabsorption of accumulated fluids
• Improved vital signs
• Important to monitor
(CDC, 2010)

44. Case-study
Back at Princeton Plainsborough, House has ordered his team to perform an
endoscopy to check for internal hemorrhaging …
New symptom: Vomiting blood, excessive bleeding in gastrointestinal tract…
Complications: Excessive loss of fluid, increased lymphatic return, overcompensation of
cardiovascular/adrenal/renal mechanisms, anoxia = DENGUE SHOCK SYNDROME

45. DIAGNOSIS
• Clinical symptoms
• Often misdiagnosed with influenza,
malaria, Typhoid fever, Leptospirosis
• Travel history
• Viral markers IgG, IgM, and NS1
• Laboratory confirmation:
• IgM Capture ELISA
• NS-1 specific assays
• Lateral flow test to detect IgM & IgG
antibody, NS1 antigen
(CDC, 2010; Mayo Clinic, 2013)

46. ANTIVIRAL THERAPY
AG129 Mouse Model
• deficient for interferon-α/β/ϒ receptors
• one of only models that permit infection by all 4 serotypes
• used to target virus entry, membrane fusion, RNA genome
DENVax
replication, assembly, & release from infected cell
• targets protein E to interfere with viral replication
7-deaza-2′- C-methyl-
adenosine
(Schul et al., 2007; Wilder-Smith et al., 2010)
N-nonyl-deoxynojirimycin
6-O-butanoyl
castanospermine
• treatment tested in viremia mouse model (AG129 mice) during acute phase
• block viral replication

47. LIVE-ATTENUATED VACCINATION
DENVax
DENVax
• based on the PDK53 DENV-2 backbone
• containing pre-membrane & E genes of serotypes 1-4
• tested in cynomolgus macaques
• Immunogenicity & efficacy results
LATV
(Durbin et al., 2013; Osorio et al., 2011)
• contains 30 nucleotide deletion; tetravalent
• tested in flavivirus-naïve adults
• no significant difference in adverse events between
vaccines and placebo-recepients
• Race factor influencing infectivity of LATV virus

48. CHIMERIC LIVE-ATTENUATED VACCINATION
ChimeriVax Vaccine (Sanofi Pasteur)
• uses 17D yellow fever vaccine virus
• Phase I, II, III
DENVax
• shows 57% overall efficacy
• reduction of hospitalization by 80%
• 89% reduction of dengue haemorrhagic fever
Mahidol
(Fink & Shi, 2014; Wilder-Smith et al., 2010)
• PDK-53 DEN-2 backbone
• Phase 1 trial (U.S. & Columbia)
• protective when administered in monkeys and
mice

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