Dengue- WS on vector borne viral infection 2011


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

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Dengue- WS on vector borne viral infection 2011

  1. 1. Dengue virus infection <ul><li>Dr. Sunethra Gunasena </li></ul><ul><li>Consultant Virologist </li></ul><ul><li>Medical Research Institute </li></ul>
  2. 2. <ul><li>Dengue virus </li></ul>
  3. 3. Dengue Virus <ul><li>Family : Flaviviridae </li></ul><ul><li>Genus : Flavivirus </li></ul><ul><li>Serotypes : DV1, DV2, DV3, DV4 </li></ul><ul><li> multiple subtypes </li></ul><ul><li>Enveloped virus </li></ul><ul><li>3 major proteins </li></ul><ul><li>SS positive sense RNA </li></ul>
  4. 6. Laboratory Diagnosis <ul><li>Detection of Dengue viral antigen </li></ul><ul><li>Detection of the Dengue viral genome </li></ul><ul><li>Isolation of the Dengue virus </li></ul><ul><li>Detection of Dengue specific IgG, IgM </li></ul>
  5. 7. Dengue diagnosis experience in Sri Lanka <ul><li>Mostly for patients admitted to wards </li></ul><ul><li>Dengue IgM capture ELISA at National laboratory & at TH hospitals </li></ul><ul><li>ICT for dengue IgM / IgG at others </li></ul><ul><li>Dengue RT-PCR at National laboratory for surveillance & few selected samples </li></ul><ul><li>Virus isolation for PCR positives </li></ul><ul><li>ICT at private sector with limited use of RT-PCR & NS1 antigen assay </li></ul>
  6. 8. <ul><li>Specimens for virus isolation, genome detection </li></ul><ul><li>Plasma / serum / peripheral blood leucocytes from febrile patients (collected within first five days of illness) </li></ul><ul><li>Vector mosquitoes </li></ul><ul><li>Homogenized necropsy tissues (liver, spleen, lymph nodes, lung, thymus) </li></ul><ul><li>Infected host systems (cells / fluid / mosquitoes / mice) </li></ul>
  7. 9. <ul><li>Collection </li></ul><ul><li>Blood into sterile dry container </li></ul><ul><li>Necropsy specimens into VTM (no formalin) </li></ul><ul><li>Transport & storage </li></ul><ul><li>Prompt delivery to lab </li></ul><ul><li>Transport in ice </li></ul><ul><li>Short term storage at 4 o C, long term storage at - 70 o C </li></ul>
  8. 10. Dengue serology <ul><li>IgM detection (qualitative) </li></ul><ul><li>In a suspected case of dengue, presence of dengue IgM indicates recent infection </li></ul><ul><li>IgM capture ELISA (blood collected after 5th day) </li></ul><ul><li>IgG detection (quantitative) </li></ul><ul><li>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) </li></ul><ul><li>HI assay / ELISA / Neutralization assay </li></ul>
  9. 11. <ul><li>IgM & IgG detection </li></ul><ul><li>Semi-quantitative IgG & IgM detection (ELISA) also used in the classification of dengue cases to 1ry & 2ry infection. </li></ul><ul><li>Qualitative IgG & IgM detection immune-chromatographic assay. Use as a rapid assay </li></ul>
  10. 12. Laboratory diagnostic criteria One of the following: 1. PCR + 2. Virus culture + 3. IgM seroconversion in paired sera 4. IgG seroconversion in paired sera or fourfold IgG titer increase in paired sera One of the following: 1. IgM + in a single serum sample 2. IgG + in a single serum sample with a HI titre of 1280 or greater Confirmed Highly suggestive
  11. 14. 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)
  12. 15. 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
  13. 16. Role of cross reactive T cells <ul><li>Cross reactive T cells reacts with dengue virus of subsequent infection. Causes activation of these T cells </li></ul><ul><li>Activated cross 1. Are less effective </li></ul><ul><li>reacting T cells in eliminating the secondary infecting DV </li></ul><ul><li>2. T cell activation contribute to disease pathogenesis </li></ul>
  14. 17. 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
  15. 18. Thrombocytopenia <ul><li>Low production due to temporary bone marrow suppression (DV infection, effect of cytokines) </li></ul><ul><li>Increased consumption (activation of coagulation system, DIC) </li></ul><ul><li>Direct infection of platelets with the virus: kills platelets </li></ul><ul><li>Increased destruction of platelets by activated macrophages </li></ul>
  16. 19. Bleeding <ul><li>Thrombocytopenia </li></ul><ul><li>Activation of the coagulation system due to endothelial dysfunction, cytokines </li></ul><ul><li>Disseminated intravascular coagulation </li></ul><ul><li>Poor perfusion of GIT: can lead to mucosal bleeding </li></ul><ul><li>Drugs: Steroids, NSAIDS </li></ul>
  17. 20. Organ Involvement in Dengue <ul><li>Direct involvement - infection of hepatocytes or brain with the dengue virus </li></ul><ul><li>Circulatory failure - poor organ perfusion </li></ul><ul><li>Drugs – Paracetamol </li></ul>
  18. 21. <ul><li>Hepatitis that may lead to liver failure </li></ul><ul><li>Encephalitis </li></ul><ul><li>Encephalopathy: metabolic, hepatic etc.. </li></ul><ul><li>Disseminated intravascular coagulation </li></ul><ul><li>Myocarditis and cardiomyopathy </li></ul><ul><li>Acute renal failure </li></ul><ul><li>Hemolytic uraemic syndrome </li></ul>
  19. 22. Dengue Vaccine Candidates Approach Developer / Producer 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 isolates WRAIR / GSK 4 chimeras composed of yellow fever 17D virus non-structural genes + respective DENV 1,2,3 or 4 envelope genes Acambis/ Sanofi Pasteur
  20. 23. Status of Dengue Vaccines ? Panacea Q3-2010 Butantan ? Biological E NIH Mid-2009 Tetravalent Acambis 2009 SanofiPasteur WRAIR ? Glaxo SmithKline Process Development Developer Phase IIB-III Phase II Phase I Evaluation Producer CDC Q1-2010 tetravalent InViragen Hawaii Biotech Q3-2009 monovalent 2010 - tetravalent Hawaii Biotech
  21. 24. Surveillance <ul><li>Fever surveillance </li></ul><ul><li>LRH (active) </li></ul><ul><li>Other health institutions (passive ) </li></ul><ul><li>Detection of circulating dengue serotypes by RT-PCR </li></ul><ul><li>Dengue burden by serological diagnosis of suspected dengue cases </li></ul><ul><li>Data is shared with MOH, Epid unit </li></ul>
  22. 25. <ul><li>Facts for thoughts </li></ul>
  23. 26. <ul><li>Study of DV isolates from SL </li></ul><ul><li>Though all 4 serotypes circulating DV2 & DV3 was predominant before 1989. No DHF </li></ul><ul><li>During the first DHF outbreak in 1989, DV3 was isolated </li></ul><ul><li>DV3 also isolated from subsequent DHF outbreaks </li></ul><ul><li>Emergence and Global Spread of a Dengue Serotype 3, Subtype III Virus (2003) 9, 800 - 809 </li></ul>
  24. 27. <ul><li>Conclusion: </li></ul><ul><li>Genetic sequence comparison of isolates showed all DV 3 isolates (pre 1989 & post 1989) belonged to DV3 subtype III </li></ul><ul><li>All pre 1989 isolates that caused DF, belonged to DV3 subtype III group A </li></ul><ul><li>1989 & subsequent isolates caused DHF, belonged to DV3 subtype III group B </li></ul>
  25. 28. Phylogenetic analysis of DV3 isolates from SL
  26. 29. <ul><li>DV3 subtype III group B associated with DHF while same subtype group A associated with DF </li></ul><ul><li>Genetic changes in DV3 subtype III is associated with the change in disease severity from DF to DHF </li></ul>
  27. 30. Dengue serotypes (Source: Epid Unit & MRI) * MRI data Year Total Tested Total 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
  28. 31. 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
  29. 32. Summary <ul><li>Dengue situation </li></ul><ul><li>Dengue virus </li></ul><ul><li>Facts for thoughts </li></ul>