Dengue virus

Dengue Virus

• Family : Flaviviridae

• Genus : Flavivirus

• Serotypes : DV1, DV2, DV3, DV4

multiple subtypes

• Enveloped virus

• 3 major proteins

• SS positive sense RNA

Laboratory Diagnosis

• Detection of Dengue viral antigen

• Detection of the Dengue viral genome

• Isolation of the Dengue virus

• Detection of Dengue specific IgG, IgM

Dengue diagnosisexperience in Sri Lanka

• Mostly for patients admitted to wards• Dengue IgM capture ELISA at National

laboratory & at TH hospitals• ICT for dengue IgM / IgG at others• Dengue RT-PCR at National laboratory for

surveillance & few selected samples• Virus isolation for PCR positives• ICT at private sector with limited use of RT-

PCR & NS1 antigen assay

Specimens for virus isolation, genome

detection

• Plasma / serum / peripheral blood leucocytes

from febrile patients (collected within

first five days of illness)

• Vector mosquitoes

• Homogenized necropsy tissues (liver, spleen,

lymph nodes, lung, thymus)

• Infected host systems (cells / fluid /

mosquitoes / mice)

• Collection

Blood into sterile dry container

Necropsy specimens into VTM (no formalin)

• Transport & storage

Prompt delivery to lab

Transport in ice

Short term storage at 4oC, long term

storage at - 70oC

Dengue serology

• IgM detection (qualitative)In a suspected case of dengue, presence of

dengue IgM indicates recent infection IgM capture ELISA (blood collected after

5th day)

• IgG detection (quantitative) Diagnostic sero-conversion is defined as a

four fold rise (or fall) in antibodies in paired sera (collected in the first 7 days & 10 – 14 days later)

HI assay / ELISA / Neutralization assay

• IgM & IgG detection

Semi-quantitative IgG & IgM detection (ELISA) also used in the classification of dengue cases to 1ry & 2ry infection.

Qualitative IgG & IgM detection immune-chromatographic assay. Use as a rapid assay

Laboratory diagnostic criteria

One of the following:1. PCR + 2. Virus culture +3. IgM seroconversion in

paired sera4. IgG seroconversion in paired

sera or fourfold IgG titer increase in paired sera

One of the following:1. IgM + in a single serum

sample2. IgG + in a single serum

sample with a HI titre of 1280 or greater

ConfirmedHighly suggestive

IgG antibody - specific to the initial infecting DV serotype + cross reacting antibody

IgM antibody to the secondary infecting DV serotype

Following primary infection –

Specific antibody response + CMI (memory T cells)

Cross reactive antibody response + CMI (memory T cells)

Pathogenesis – Role of cross reactive DV antibodies

Cross reactive antibody binds to the infecting virus

Form v- ab complexes. V- ab complexes attach to cells bearing receptors for the Fc

portion of the ab

Facilitates entry of the virus into these cells and the viral replication. Therefore, more cells are infected

Increased immune response & release of cytokines

Role of cross reactive T cells

Cross reactive T cells reacts with dengue virus of subsequent infection. Causes activation of these T cells

Activated cross 1. Are less effectivereacting T cells in eliminating the

secondary infecting DV

2. T cell activation contribute to

disease pathogenesis

Cytokines secreted from infected macrophages and endothelial cells

Cytokines secreted from activated T cells

Exaggerated Cytokine response

Endothelial dysfunction

DV specific antibody interact with the endothelium

DV infects endothelium and kills cells

Thrombocytopenia

• Low production due to temporary bone marrow suppression (DV infection, effect of cytokines)

• Increased consumption (activation of coagulation system, DIC)

• Direct infection of platelets with the virus: kills platelets

• Increased destruction of platelets by activated macrophages

Bleeding

• Thrombocytopenia

• Activation of the coagulation system due to endothelial dysfunction, cytokines

• Disseminated intravascular coagulation

• Poor perfusion of GIT: can lead to mucosal bleeding

• Drugs: Steroids, NSAIDS

Organ Involvement in Dengue

• Direct involvement - infection of hepatocytes or brain with the dengue virus

• Circulatory failure - poor organ perfusion

• Drugs – Paracetamol

• Hepatitis that may lead to liver failure• Encephalitis• Encephalopathy: metabolic, hepatic etc..• Disseminated intravascular coagulation• Myocarditis and cardiomyopathy• Acute renal failure• Hemolytic uraemic syndrome

Dengue Vaccine Candidates

Subunit - 80% preM expressed in Drosophila S2 cell lines, +/- NS1, alum adjuvant Hawaii Biotech

Genetically engineered, stable mutations in 3’ non-coding region of DENV-1, 2, 4 vaccine candidates. DENV-3 candidates = DENV-4/DEN-3 chimeras

NIAID Laboratory of

Infectious Diseases

DENV-2 attenuated virus + 3 chimeras composed of DENV-2 non-structural genes + respective DENV 1,3, or 4 envelope genes

CDC/InViragen

Cell culture passage of clinical isolatesWRAIR / GSK

4 chimeras composed of yellow fever 17D virus non-structural genes + respective DENV 1,2,3 or 4 envelope genes

Acambis/ Sanofi Pasteur

ApproachDeveloper / Producer

?Panacea

Q3-2010

Butantan

?Biological E

NIH

Mid-2009 Tetravalent

Acambis 2009 SanofiPasteur

WRAIR ? Glaxo

SmithKline

Process Develop

ment Developer Phase

IIB-III

Phase

II Phase

I

Evaluation Producer

Status of Dengue Vaccines

CDC Q1-2010 tetravalent InViragen

Hawaii Biotech

Q3-2009 monovalent

2010 - tetravalent Hawaii Biotech

Surveillance

• Fever surveillance

LRH (active)

Other health institutions (passive )

• Detection of circulating dengue serotypes by RT-PCR

• Dengue burden by serological diagnosis of suspected dengue cases

• Data is shared with MOH, Epid unit

Facts for thoughts

Study of DV isolates from SL

• Though all 4 serotypes circulating DV2 & DV3 was predominant before 1989. No DHF

• During the first DHF outbreak in 1989, DV3 was isolated

• DV3 also isolated from subsequent DHF outbreaks

Emergence and Global Spread of a Dengue Serotype 3, Subtype III Virus (2003) 9, 800 - 809

Conclusion:

Genetic sequence comparison of isolates showed all DV 3 isolates (pre 1989 & post 1989) belonged to DV3 subtype III

All pre 1989 isolates that caused DF, belonged to DV3 subtype III group A

1989 & subsequent isolates caused DHF, belonged to DV3 subtype III group B

Phylogenetic analysis of DV3 isolates from SL

• DV3 subtype III group B associated with DHF while same subtype group A associated with DF

• Genetic changes in DV3 subtype III is associated with the change in disease severity from DF to DHF

Dengue serotypes (Source: Epid Unit & MRI)

YearTotal

TestedTotal

Positive D1 (%) D2 (%) D3 (%) D4 (%)

2006 1795 287 20 121 126 14

2007 461 56 01 26 20 00

2008 305 33 00 16 09 00

2009 264 49 19 10 15 00

*2010 510 33 33 00 00 00

*2011 166 10 10 00 00 00

* MRI data

Distribution of A. albopictus

Presence of A. albopictus before 1980

Areas invaded by A. albopictus since 1980

A. albopictus may become important in spreading the infection given it’s extensive distribution

Summary

Dengue situation

Dengue virus

Facts for thoughts