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A lecture by the Srilankan team on dengue, vector borne viral infection.
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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