Dengue Virus Overview

DENGUE VIRUS

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

Case-study

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)

GLOBAL DISTRIBUTION

• 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

(Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever, 2011)

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)

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)

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

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)

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)

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).

THE DENGUE VIRUS

RNA Genome

Capsid

Lipid Bilayer

Envelope Proteins

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)

DENGUE LIFE CYCLE

Infected Mosquito Biting

DENGUE LIFE CYCLEExtracellular Matrix

DENGUE LIFE CYCLE

Cognate receptor

Dengue virus

Receptor BindingEndocytosis

Cell Membrane

Cytoplasm

Extracellular Space

DENGUE LIFE CYCLE

Endosome

Proton Pump

Fusion

H+H+

Lowered pHConformational Change

Cytoplasm

DENGUE LIFE CYCLE

Endosome

Proton Pump H+H+

Conformational ChangeFusion

Cytoplasm

DENGUE LIFE CYCLE

FusionNucleocapsid Release

Cytoplasm

DENGUE LIFE CYCLE

Nucleocapsid Release Cytoplasm

DENGUE LIFE CYCLECytoplasm

Viral RNA

Viral RNA Translation

Ribosomes

Rough Endoplasmic Reticulum



Envelope Proteins

Capsid Proteins

RNA dependent RNA Polymerase




RdRp

Envelope Proteins

Capsid Proteins


Proteases

Peptidases

Cell Membrane


DENGUE LIFE CYCLE

RNA Replication

Cytoplasm

RdRp


Double stranded RNASingle stranded RNA

DENGUE LIFE CYCLE

RNA Transcription

Cytoplasm

Double stranded RNA

RdRp


Viral RNA


Ribosomes


R



Envelope Proteins

Capsid ProteinsRdRp


DENGUE LIFE CYCLE

Cytoplasm

Viral RNA TranslationBudding


Cell Membrane

DENGUE LIFE CYCLE

RER

Golgi Apparatus

Golgi Apparatus

Furin Cleavage

Furin

DENGUE LIFE CYCLE

Golgi Apparatus Cytoplasm

Extracellular Space

Cognate receptor

Progeny Release

Molecular Structure

• External Structure : icosahedral

PATHOPHYSIOLOGY

Complement Activation

ADE of virus

T-cell Immunology

Plasma Leakage Bleeding Diathesis

Hypovolemic Shock(Nishiura, H., & Halstead, 2007; Halstead & O’rourke, 1977) ).

PATHOPHYSIOLOGY- ADE Activation

Heterotypic antibodies

- Low concentration

- Partial neutralization

- Ie. prM

Main Result: Increase in virus replication and numbers

(Halstead et al. 2010)



- Low concentration


- Ie. prM

Virus uptake- Delivery to Fcɣ receptor cells





- Low concentration


- Ie. prM

Virus uptake- Delivery to Fcɣ receptor cells

Replication

- Increased viral

replication compared

to virus alone



PATHOPHYSIOLOGY- Complement Activation

Anaphylatoxins

• NS1- Glycoprotein secreted by dengue

infected cells

• Activation of innate immune system on

endothelial cell surface

1) Classical complement

2) Alternative complement

Result:1) Apoptosis of endothelial cell

2) Inflammation = CYTOKINES!

(Guzman & Kouri, 2002)

PATHOPHYSIOLOGY- T-Lymphocyte Pathogenesis

Primary Infection

DENV-1T- cell selection

(Dejnirattisai et al., 2010)


Primary Infection

Secondary Infection


DENV-2 Clonal Expansion

Cross-Reaction(Dejnirattisai et al., 2010)


Primary Infection

Secondary Infection


DENV-2 Clonal Expansion

Cross-Reaction

TNF-α

IFN-ɣ

IL-2

CYTOKINE

STORM

(Dejnirattisai et al.,

2010)

ENDOTHELIAL CELL ACTIVATION

http://www.scielo.br/img/revistas/rb/v43n6/en_a13fig01.jpg

Cytokines induce changes in

endothelial morphology:

• Loss of vascular integrity

• Increased adhesion molecules

• Increased cytokine production

IMPLICATIONS: Plasma leakage

Edema Hypovolemic shock Thrombocytopenia

(Manson et al. 2003)

COAGULOPATHY

Loss of Plasma Components

- Platelets

- Plasma fibrinogen

Immune-mediated

Destruction of Platelets

GlycocalyxDamage

- Anti-coagulation via heparansulphate

(Sellahewa, K, 2012)

SYMPTOMS

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

Dengue Fever and Dengue Hemorrhagic Fever

(CDC, 2010)

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

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)

ANTIVIRAL THERAPY

DENVax

AG129 Mouse Model

7-deaza-2′- C-methyl-adenosine

• 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

replication, assembly, & release from infected cell• targets protein E to interfere with viral replication

(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

LIVE-ATTENUATED VACCINATION

DENVax

DENVax

LATV

• based on the PDK53 DENV-2 backbone• containing pre-membrane & E genes of serotypes 1-4• tested in cynomolgus macaques• Immunogenicity & efficacy results

(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

CHIMERIC LIVE-ATTENUATED VACCINATION

DENVax

ChimeriVax Vaccine (Sanofi Pasteur)

Mahidol

• uses 17D yellow fever vaccine virus • Phase I, II, III

• shows 57% overall efficacy • reduction of hospitalization by 80%• 89% reduction of dengue haemorrhagic fever

(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

REFERENCES

Avirutnan, P., Zhang, L., Punyadee, N., Manuyakorn, A., Puttikhunt, C., Kasinrerk, W., Malasit, P., Atkinson, J., and Diamond, M.

(2007). Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin

sulfate E. Plos Pathogens 3, 183.

Avirutnan, P., Punyadee, N., Noisakran, S., Komoltri, C., Thiemmeca, S., Auethavornanan, K., Jairungsri, A., Kanlaya, R.,

Tangthawornchaikul, N., and Puttikhunt, C. et al. (2006). Vascular leakage in severe dengue virus infections: a potential role for

the nonstructural viral protein NS1 and complement. Journal Of Infectious Diseases 193, 1078-1088.

Centers for Disease Control and Prevention. (2010). Dengue. Retrieved from: http://www.cdc.gov/dengue/clinicallab/clinical.html

Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever: Revised and Expanded

Edition. (2011). Retrieved 13 October 2014, from http://apps.searo.who.int/pds_docs/B4751.pdf?ua=1

Dejnirattisai, W., Jumnainsong, A., Onsirisakul, N., Fitton, P., Vasanawathana, S., Limpitikul, W., Puttikhunt, C., Edwards, C.,

Duangchinda, T., and Supasa, S. et al. (2010). Cross-reacting antibodies enhance dengue virus infection in humans.

Science 328, 745-748.

Dejnirattisai, W., Jumnainsong, A., Onsirisakul, N., Fitton, P., Vasanawathana, S., Limpitikul, W., Puttikhunt, C., Edwards, C.,

Duangchinda, T., and Supasa, S. et al. (2010). Enhancing cross-reactive anti-prM dominates the human antibody response in

dengue infection. Science (New York, NY) 328.

Fink, M. (2005). Dengue. Textbook of critical care (Philadelphia: Elsevier Saunders).

Guzman, M., and Kouri, G. (2002). Dengue: an update. The Lancet Infectious Diseases 2, 33-42.

http://www.cdc.gov/dengue/clinicallab/clinical.html

REFERENCES

Halstead, S., and O'rourke, E. (1977). Dengue viruses and mononuclear phagocytes. I. Infection enhancement

by non-neutralizing antibody. The Journal Of Experimental Medicine 146, 201-217.

Halstead, S., Mahalingam, S., Marovich, M., Ubol, S., and Mosser, D. (2010). Intrinsic antibody-dependent

enhancement of microbial infection in macrophages: disease regulation by immune complexes. The Lancet

Infectious Diseases 10, 712-722.

Holmes, E. & Twiddy, S. (2003). The origin, emergence an evolutionary genetics of dengue virus. Infections,

Genetics and Evolution, 3(1), 1928.

In, L. (2003). Howard Hughes Medical Institute

Kuhn, R.J. et al. (2002). Implications for flavivirus organization, maturation, and fusion. Cell, 108, 717-725.

Manson, P., Cook, G., Zumla, A., and Manson, P. (2003). Manson's tropical diseases (London: Saunders).

Martina, B., Koraka, P., & Osterhaus, A. (2009). Dengue virus pathogenesis: an integrated view.Clinical

Microbiology Reviews, 22(4), 564--581.

Nature.com,. (2014). Retrieved 14 October 2014, from

http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/22400749/F2_dengue_2_1.jpg

Nimmannitya, S. (1999). Dengue hemorrhagic fever: disorders of hemostasis. 184-187.

REFERENCES

Nishiura, H., & Halstead, S. (2007). Natural History of Dengue Virus (DENV)—1 and DENV—4

Infections: Reanalysis of Classic Studies. Journal Of Infectious Diseases, 195(7), 1007--1013.

Rodenhuis-Zybert, I., Wilschut, J., & Smit, J. (2010). Dengue virus life cycle: viral and host factors

modulating infectivity. Cellular And Molecular Life Sciences, 67(16), 2773--2786.

Sellahewa, K. (2012). Pathogenesis of Dengue Haemorrhagic Fever and Its Impact on Case

Management. ISRN Infectious Diseases 2013.

Seynhaeve, A., Vermeulen, C., Eggermont, A., and ten Hagen, T. (2006). Cytokines and vascular

permeability. Cell Biochemistry And Biophysics 44, 157-169.

Srikiatkhachorn, A., and others, (2009). Plasma leakage in dengue haemorrhagic fever. Thromb

Haemost 102, 1042-9.

Srikiatkhachorn, A., and others, (2009). Plasma leakage in dengue haemorrhagic fever. Thromb

Haemost 102, 1042-9.

Stoermer, K., and Morrison, T. (2011). Complement and viral pathogenesis. Virology 411, 362-373.

REFERENCES

Zompi, S., and Harris, E. (2013). Original antigenic sin in dengue revisited. Proceedings Of The

National Academy Of Sciences 110, 8761-8762.

Dengue Virus Overview

Health & Medicine

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