1. What are Viral Hemorrhagic Fevers (VHFs)? A group of illnesses that are caused by several distinctfamilies of viruses A severe multisystem syndrome (multiple organ systems inthe body are affected Vascular system damaged : SHOCK syndromes Bodys ability to regulate itself (Homeostasis) is impaired Many cause severe and life-threatening disease.

2. Viral hemorrhagic fever (contd) The prototypical viral hemorrhagic fever is Yellow Fever Not all viral hemorrhagic fevers are however arboviruses Hemorrhagic fever with Renal Syndrome (HFRS) arealso considered in relation to VHF HFRS Caused by: Hantaan Seoul Dobrava Puumala viruses (Ref: Mandell, Douglas and Bennetts Principles and Practice of Infectious Disease, 7th Ed) 3. Viral hemorrhagic fever (contd) Acute infection:fever, myalgia, malaise; progression to prostration Small vessel involvement:increased permeability, cellular damage Multisystem compromise (varies with pathogen) Hemorrhage may be small in volume(indicates small vessel involvement, thrombocytopenia) Poor prognosis associated with:shock, encephalopathy, extensive hemorrhage 4. Viral hemorrhagic fever (contd) Viruses of four distinct families Arenaviruses Filoviruses Bunyaviruses Flaviviruses RNA viruses Enveloped in lipid coating Survival dependent on an animal or insect host, for thenatural reservoir 5. Viral hemorrhagic fever (contd)ArenaviridaeBunyaviridaeFiloviridae Flaviviridae ClassificationJuninCrimean- CongoEbolaKyasanur HF Forest DiseaseMachupoHantavirusMarburgOmsk HFSabiaRift Valley feverYellow FeverGuanarito SFTS (China,Dengue 2011)LassaArgentine HF (In block Red:Diseases prevalent inINDIA)Venezuelan HF 6. Shapes of the above viruses 7. DengueDengue is the biggest Arbovirus problem in the world today with over 2 million cases per yearDengue is found in SE Asia, Africa and the Caribbean and South America.4 serotypes: DEN 1,2,3,4Human infections arise from a human- mosquito-human cycle 8. Dengue (.contd)Classically, dengue presents with a high fever, lymphadenopathy, myalgia, bone and joint pains, headache, and a maculopapular rash.Severecases may present with haemorrhagic fever and shock with a mortality of5-10%.{Dengue haemorrhagic fever (DHF)or Dengue shock syndrome (DSS)} 9. Global Occurrence of Dengue 10. Approximate actual and potential distribution of Aedes aegypti.The band between the 10 C isotherms represents potential distribution between 35 North 35 South(Ref: World Health Organization. Technical Guide for Diagnosis, Treatment,Surveillance, Prevention, and Control of Dengue Haemorrhagic Fever, 2nd ed.Geneva: World Health Organization; 1997.) 11. Magnitude of ProblemThe reasons for this dramatic global emergence of Dengue as a major public health problem are: Increased Air Travel Extensive vector infestations with declining vector control: effective mosquito control is virtually non existent in most Dengue endemic countries Unreliable Water supply and drainage systems Increasing non bio-degradable contaivers and poor solid Waste disposal Major global demographic changes: Urbanization with increasing population density in urban areas 12. Indian Scenario 13. Indian ScenarioThefirst recorded epidemic of clinically Dengue like illness occurred at Madras in 1780. Firstoutbreak in Indian subcontinent: 1812.First Dengue virus isolation- Kolkata in 1943 1944.First outbreak in India: 1963 in Kolkata.Ref: Jatanasen S and Thongcharoen P (1993) Dengue hemorrhagicfever in South East-Asian countries. Monograph on dengue/denguehaemorrhagic fever. NewDelhi: WHO 23-30. 14. Indian ScenarioRecent Dengue epidemic occurred in 1996, 2003 & 2006.In 2008, 12,419 Dengue cases and 80 deaths were reported.Delhi shares ~25% of dengue disease burden of country. 15. Indian Scenario.sharing experiences from our centre Primary Secondary Suspected Total Serologicallyinfection infectionsecondary Month Suspected Positive cases(IgM (IgM+ IgG infection (IgG cases(%)Positivity)Positivity) Positivity) August 123 (0.34%) 1 (0.5%) 1 (0.26%)1 (0.32%)September 157 68 (7.6%) 17 (8.6%)24 (6.3%)27 (8.6%) October982583 (65.3%) 126 (63.3%) 246 (64.57%) 211 (67.4%)November362 230 (25.76%) 49 (24.6%) 110 (28.87%) 71 (22.68%)December379 (1%) 6 (3%)0 (0%) 3 (1%)Total1550893 (57.36%) 199 (22.28%) 381 (42.67%) 313 (35.05%)Ref: AnitaChakravarti* and RajniKumaria: Virology Journal 2005, 2:3 16. Indian Scenario.sharing experiences fromour centre MonthDengue-specific Antibody Positive cases Children Adults (PositivityTotal (Positivity %)%)August303 (25%)A Clear SeptemberCut PEAK of incidence of (41.7%) 6818 (48.6%)50 newcases were seen in the (69.6%)October583133 Post-monsoon 450 (57%) November during the months of Octoberseason 23054 (44.3%)176 (83.8%)and November in cases ocurring8in or December 9 1 (11.1%)(28.6%)near Delhi 893 Total206 (56.4%)687 (58%)Ref: AnitaChakravarti* and RajniKumaria: Virology Journal2005, 2:3 17. Ref: J Infect Dev Countries 2011; 5(4):239-247The National figure also corroborates with the studyfrom our institute, carried out in Delhi 18. Ref: J Infect Dev Countries 2011; 5(4):239-247 19. Ref: J Infect Dev Countries 2011; 5(4):239-247 20. The Vector: Aedes mosquito Aedes(Stegomyia)aegypti Breeds in smallaccumulating standingwater Eggs resist drying Domesticated mosquito Found within or close-byhuman environments,often biting indoorsbiting is predominantlyby day 21. Dengue Transmission1. Mosquitoes transmitDengue virus to human dendriticcells. 12. Virus targets areaswith high WBC counts2(liver, spleen, lymphnodes, bone marrow,4And glands)333. Virus entersWBCs & lymphaticTissue4. Dengue virus enters bloodCirculation.http://phil.cdc.gov/PHIL_Images/08051999/00004/dengue_phf/sld006.htm 22. Steps required for any Flaviviruses infection andtransmission by a mosquito 23. Dengue - Virology Ref: Goodsell DS. RCSB Protein Data Bank. July, 2008.Dengue virus is a small virus that carries a single strand of RNA as its genome. Thegenome encodes only ten proteins. Three of these are structural proteins that formthe coat of the virus and deliver the RNA to target cells, and seven of them arenonstructural proteins that orchestrate the production of new viruses once the virusgets inside the cell. The outermost structural protein, termed the envelope protein, isshown here from PDB entry 1k4r 24. Dengue Virology ( Contd) The Deadly SwitchRef: Goodsell DS.RCSB Protein DataBank. July, 2008.When the virus is carried into the cell and into lysozomes, the acidic environmentcauses the protein to snap into a different shape, assembling into trimeric spike, asshown above from PDB entry 1ok8. Several hydrophobic amino acids at the tip of thisspike, colored bright red here, insert into the lysozomal membrane and cause the virusmembrane to fuse with lysozome. This releases the RNA into the cell and infectionstarts. 25. Dengue Virology ( Contd)Ref: Goodsell DS.RCSB Protein DataBank. July, 2008.Each of these enzymes performs a different part of the life cycle. The polymerasebuilds new RNA strands based on the viral RNA, the helicase helps to separate thesestrands, and the methyltransferase adds methyl groups to the end of them, protectingthe RNA strands and coaxing the cells ribosomes to create viral proteins based onthem. The viral proteins are created in one long polyprotein chain, which is finallyclipped into the functional units by the protease. The little chain coloured blue is aportion of another viral protein, NS2B, that assists with the protease activity. 26. Dengue Virology ( Contd) Ref: Goodsell DS. RCSB Protein Data Bank. July, 2008.The one shown here, from PDB entry 2r6p6, shows the envelopeprotein on the surface of the virus (in white) with many antibody Fabfragments (in blue) bound to the viral proteins. By looking carefully atthis structure, researchers have discovered that the antibodies distortthe arrangement of the envelope proteins, blocking their normalaction in infection. 27. Clinical Presentation Of Dengue Dengue Virus InfectionAsymptomatic SymptomaticDengue hemorrhagicUndifferentiated Dengue feverfever feversyndrome (plasma leakage)(viral syndrome)Without With unusual No shockDengue shockhemorrhagehemorrhagesyndromeDengue fever Dengue hemorrhagic FeverWHO 95629 28. Clinical spectrum, pathophysiology, andclassification of dengue hemorrhagic fever. At the top are key clinical findings; in the center, pathophysiologic mechanisms; and on the side, the World Health Organization classification of cases: Grade 1: Fever accompanied by nonspecific constitutional symptoms; the only hemorrhagic manifestations are a positive tourniquet test result, easy bruising, or both. Grade 2: Spontaneous bleeding in addition to the manifestations of grade 1, usually in the form of skin hemorrhages or other hemorrhages. Grade 3: Circulatory failure manifested by a rapid, weak pulse and narrowing of pulse pressure or hypotension, with the presence of cold, clammy skin and restlessness. Grade 4: Profound shock with undetectable blood pressure or pulse.(Ref: WHO. Technical Guide for Diagnosis, Treatment, Surveillance, Prevention, andControl of Dengue Haemorrhagic Fever, 2nd ed. Geneva: 1997.) 29. Dengue (cont) Dengue haemorrhagic fever and shock syndromeappear most often in patients previously infected by adifferent serotype of dengue, thus suggesting animmunopathological mechanism. Diagnosis is made by serology. No specific antiviral therapy is available. Prevention of dengue in endemic areas depends onmosquito eradication. The population should removeall containers from their premises which may serve asvessels for egg deposition. 30. Clinical Case Definition for Dengue Hemorrhagic Fever 4 Necessary Criteria:1. Fever, or recent history of acute fever2. Hemorrhagic manifestations3. Low platelet count (100,000/mm3 or less)4. Objective evidence of leaky capillaries : elevated hematocrit (20% or more over baseline) low albumin pleural or other serosal cavity effusions 31. Dengue Clinical SyndromesUndifferentiated feverClassic dengue feverDengue hemorrhagic feverDengue shock syndrome 32. Hemorrhagic Manifestationsof DengueSkin : petechiae, purpura, ecchymosesGingival bleedingNasal bleedingGastro-intestinal bleeding: hematemesis, melenaHematuriaIncreased menstrual flow 33. Four Grades of DHF Grade 1 Fever and nonspecific constitutional symptoms Positive tourniquet test is only hemorrhagicmanifestation Grade 2 Grade 1 manifestations + spontaneous bleeding Grade 3 Signs of circulatory failure (rapid/weak pulse, narrowpulse pressure, hypotension, cold/clammy skin) Grade 4 Profound shock (undetectable pulse and BP) 34. Laboratory Tests in Dengue Fever Clinical laboratory tests CBC--WBC, platelets, hematocrit Albumin Liver function tests Urine--check for microscopic hematuria Dengue-specific tests Virus isolation Serology 35. Laboratory Diagnosis of Dengue Fever: Virus detection Detection of virus by culture is obviously thedefinitive diagnostic test. By the time a person infected with Dengue developsfever, the infection is widely disseminated. The virus is found in serum or plasma, in circulatingblood cells and in selected tissues, especially thoseof the immune system, for approx. 2-7 days, roughlycorresponding to the period of fever. Detection of dengue RNA using specificoligonucleotide primers, reverse transcriptase andthermostable polymerase are Faster and are appliedin many Laboratories. 36. Laboratory Diagnosis of Dengue Fever: Virus detection Drawbacks and limitations of Viral isolation The period of illness when the dengue virus can besuccessfully detected is brief Within a day or 2 after subsidence of fever, therising level of antibody interfere with virus culture Dengue virus is heat-labile and special precautionsmust be taken against the thermal inactivation ofspecimens. Laboratories equipped and staffed to culture virusesare expensive to develop and maintain. 37. Laboratory Diagnosis of DengueFever: Virus detection 38. Laboratory Diagnosis of Dengue Fever:Virus detection Inoculation into mosquitos Most sensitive dengue viral culture technique Serum, Plasma, CSF, Pleural fluid, Peripheral bloodleucocytes & tissue homogenates can be used Toxorhynchites mosquitos generally used They are not hematophagus and their large size facilitatesinoculation Infection is detected by Immunofluorescence of a tissuesmear prepared from the crushed head of the mosquito(Head Squash) High sensitive culture requires 5-20 mosquitos per specimen adult male Aedes aegypti & Ae. Albopictus can also be used. 39. Laboratory Diagnosis of Dengue Fever: Virus detectionInoculation into mosquitosToxorhynchites Ae. aegypti & Ae. Albopictus Large, easy to inoculate Small, difficult to inoculate Raising is labourEasier to maintainintensive, as the larvae arecarnivorous & needs asecond mosquito specieslarvae as food source Non Hematophagus,Female spp cant be usedhence safe to handle due to ability to act as vector 40. Laboratory Diagnosis of Dengue Fever: Virus detection Inoculation into mosquito cell lines C6/36 and AP-61 cell lines can be used Less sensitive than direct inoculation into live mosquitoes Cell cultures to be screened for specific evidence of infection by an immunoassay as the cytopathic effects might be absent in many dengue virus isolates As mosquito cell lines are propagable in ambient tropical temperatures (25-34 C), it is easier to maintain and practice 41. Laboratory Diagnosis of Dengue Fever:Virus detectionInoculation into vertebrate cell lines VERO and LLC-MK2 cell lines can be used Least sensitive than other direct inoculation methodsAll cultures are examined using serotype-specific anti-Dengue monoclonal Abs tagged to a second labelled Ab.Positive control: Dengue-complex-reactibe MAb Intracerebral inoculation into newborn mice is also tried in certain laboratories : but have proven to be very less sensitive 42. Laboratory Diagnosis of Dengue Fever: Antigen detection in fixed tissueSample: Peripheral Blood Leukocyte Autopsy Lung, Liver specimen Less commonly: Autopsy Thymus, Spleen, Lymph node, Bone marrowMainlyfor epidemiologicalpurposeand confirmation of epidemic / outbreak.Immunohistochemistry examined using serotype- specific anti-Dengue monoclonal Abs tagged to a second labelled Ab. 43. Laboratory Diagnosis of Dengue Fever: Reverse transcriptase-PCR amplification of Dengue RNA Highpotential to detect dengue virus during convalescence, when circulating antibodies otherwise preclude its detection 2 step nested RT-PCR and 1 tube multiplex RT-PCR are among the most widely used methodsExperience at our centre have shown that the 1 tube multiplex RT-PCR is more sensitive and specific than the other available methods.(Ref: Kumaria R, Chakravarti A. Diagn Microbiol Infect Dis 2005) 44. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISA The IgM Capture or the MAC-ELISA is the most widely used serological test Serum, Saliva, dried blood sample collected in Filter paper and CSF can be used as sample Can even detect a rise in dengue-specific IgM in acute phase at 1-day to 2-day interval Specimens collected at an interval of 2-3 days spanning the day of defervescence are usually diagnostic 45. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISAExperience at our centre MAC-ELISA is regularly practiced at our centre IgM and IgG detection from non-invasiveSaliva samples were carried out at our centre which yielded wonderful reproducible results: Salivary IgM antibodies were detected in 100% of the serumIgM-positive samples and in 30% of the serum samples thatwere negative for IgM antibodies. Salivary IgG antibodieswere detected in 93.3% of the serum samples that werepositive for anti-dengue IgG antibodies and in none of theserum IgG-negative cases (Ref: Chakravarti A, Matlani M,Jain M. 2007, Curr Microbiol) IgM/IgG detection from reconstitution of dried blood samples from clinically suspected cases collected in filter paper are being carried out at present. 46. Laboratory Diagnosis of Dengue Fever: Serology: IgM capture ELISA The Interpretation of MAC-ELISA resultsRef: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.2nd edition. Geneva : World Health Organization 47. Laboratory Diagnosis of Dengue Fever: Rapid NS1 Antigen detection Extensive study taking place to establish rapiddiagnosis and shorten the window period of misdiagnosisby detecting the NS1 antigen of dengue virus Most of the studies have shown that a combinationrapid test comprising immunochromatographic assay fordetection of both the NS1 Antigen and the anti-dengueIgm together yields satisfactory clinical results,instead of sole NS1 antigen detection.(Ref: 1. Tontulawat P et al, Southeast Asian J Trop Med Public Health. May,2011.2. Fry SR et al, PLoS Negl Trop Dis. June, 2011.) 48. Laboratory Diagnosis of Dengue Fever: Serology: Haemagglutination-Inhibition test (HAI) Simple, sensitive and reproducible Reagents may be prepared locally Disadvantages: Pretreatment of serum samples reqd with acetone/kaolin and then adsorbed with type O human RBCs toremove non-specific inhibitors of agglutinin and non-specific agglutinins. Paired sera are required with a gap of at least 7 days. Cant reliably distinguish between closely relatedFlaviviruses: Between Dengue and Jap Encephalitis orWest Nile viruses 49. Laboratory Diagnosis of Dengue Fever: Serology: Haemagglutination-Inhibition test (HAI)The Interpretation of HAI resultsRef: Dengue haemorrhagic fever: diagnosis, treatment, prevention and control.2nd edition. Geneva : World Health Organization 50. Progress toward a Dengue Vaccine Control of dengue by widespread vaccination has been a priority of WHO for three decades (Ref:Brandt WE. J Infect Dis 1990) Background: Robust neutralising antibody responses develop afterdengue infection and are believed to provide lifelongprotection against reinfection with the same dengueserotype and short-lived protection, of severalmonths, against a heterologous dengue serotype. This naturally acquired immunity provides optimismfor the feasibility of a dengue vaccine. 51. Progress toward a Dengue VaccineVaccine development and the issue of ImmunopathogenesisThe fear:The pathogenesis of severe dengue results from a complex interaction between the virus, the host, and, at least in part, immune-mediated mechanisms. Vaccine development has been slowed by fears that immunisation might predispose individuals to the severe form of dengue infection.The assurance:Whatever the role of antibody-dependent enhancement, it seems that a vaccine inducing a long-lived neutralising antibody response against all four serotypes simultaneously should not induce any risk in this respect(Ref: 1. Guirakhoo F et al, Hum Vaccin 2006. 2. Sabchareon A et al, Am J Trop MedHyg 2002. 3. Sabchareon A et al, Pediatr Infect Dis J 2004.) 52. Progress toward a Dengue VaccineLEADING 53. Progress toward a Dengue Vaccine 54. Progress toward a Dengue Vaccine The leading candidate vaccine in clinical trials atpresent is the ChimeriVax dengue vaccine. Using a new technology, the premembrane andenvelope genes of yellow fever 17D virus arereplaced with those of each wild-type denguevirus serotype. ChimeriVax dengue vaccine virusesare then prepared by electroporation of Verocells with RNA transcripts prepared from viralcDNA.(Ref: 1. Guirakhoo F, Kitchener S et al. Hum Vaccin 20062. Webster DP, Farra J. Lancet Infect Dis 2009.) 55. Kyasanur forest disease Kyasanur forest disease is a tick-borne viralhemorrhagic fever endemic to South Asia The disease was first reported from KyasanurForest of Karnataka in India The disease was first manifested as anepizootic outbreak among monkeys killingseveral of them in the year 1957. Hence thedisease is also known as Monkey Disease. 56. Kyasanur forest disease (contd) The reservoir hosts for the disease are porcupines, rats and mice. The vector for disease transmission is Haemaphysalis spinigera, a forest tick. Humans contract infection from the bite of nymphs of the tick The disease has a high mortality rate of 10% The clinical manifestations of the disease in humans are: High fever Headache Hemorrhages from nasal cavity and throat Vomiting 57. Crimean Congo Hemorrhagic Fever: India affected Index case: Ameena Momin (Case A), 32yr old woman from Korat village in Sanand, 20 kms from Ahmedabad was admitted to Sterling Hospital on December 29 and later shifted to Shalby Hospital on January 1, expired on January 3rd 2011. Secondary cases: 42 yr old Dr Gaganjeet Sharma (Case B) treating the index case at Shalby, died January 13th. 25 yr old nurse Asha John (Case C), attending the index case, died January 18th. The husband of the index case (case D) also admitted to the same hospital on Jan 16, was positive for CCHF virus was treated with oral ribavarin and discharged after 10 days.(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagicfever in India. Lancet July, 2011) 58. Crimean Congo Hemorrhagic Fever: India affected(Ref: Mishra AC, Mehta M, Mourya DT, Gandhi S. Crimean-Congo haemorrhagicfever in India. Lancet July, 2011) 59. Crimean Congo Hemorrhagic Fever:India affectedDIAGNOSIS Only Nucleic acid Amplification Tests (eg. PCR, rt-PCR) are the most reliable method, along with RNA sequencing in case of outbreaks in previously unknown geographical areas High serum LDH, High serum Ferritin, and thrombocytopenia may lead to a strong suspicion Negative assays for locally prevalent diseases, that may lead to such fever (eg. Malaria, Leptospirosis, Dengue, Kyasanur Forest Disease for India) shall also lead to strong suspicion. 60. Crimean Congo Hemorrhagic Fever:India affectedIMPORTANCE: Many severely ill patients with CCHF require admission tointensive care facilities. To avoid infections in hospitalsettings, stringent infection control practices, proper airhandling in intensive-care units, isolation of patients, andcorrect handling of clinical specimens are essential. Tickbites and contact with infected animals are the main modesof infection to people. Disinfection of domestic animals and their accommodationcan help reduce the risk of human infection. Febrile patientswith haemorrhagic symptoms, who are negative for denguevirus, should be considered as possible cases of CCHF forthe purpose of hospital infection control and isolation ofpatients in India 61. Novel Bunyavirus identified in China (SFTS)Why do we discuss it here in context of India? RNA from SFTS bunyavirus was detected in roughly 5percent of ticks of the species Haemaphysalislongicornis recovered from animals in the area in whichaffected patients lived, and the authors propose that thistick may be a vector for SFTS bunyavirus. This tick is found throughout the Asia-Pacific regionincluding India Considering globalization, population migration and lackof SFTS surveillance, INDIA is at a high risk.Ref: Yu XJ, Liang MF, Zhang SY, et al: Fever with thrombocytopenia associated with anovel bunyavirus in China. N Engl J Med 2011; 364(16):1523-1532. 62. Novel bunyavirus identified in China (contd) Surveillance for infectious disease in China has advanced inrecent years. In 2009 and 2010, surveillance detected the emergence of a newviral pathogen that causes a clinical syndrome including feverand thrombocytopenia that has been termed severe fever withthrombocytopenia syndrome (SFTS). SFTS is characterized by Gastrointestinal symptoms Leukopenia Fever Thrombocytopenia 30% mortality rate. 63. Novel bunyavirus identified inChina(contd) In June 2009, a patient in Xinyang City in HenanProvince (central China) presented with SFTS Blood sample was obtained from this patient 1 week afteronset of symptoms. Multiple cell lines that were susceptible to both viral andrickettsial agents were inoculated with the patients blood. During the period June 2009 to March 2010, additionalcases of SFTS were identified in central and northeastChina, and blood samples were obtained from thesepatients as well. 64. Novel bunyavirus identified inChina(contd) In these cases, serum or white blood cells were inoculatedonto Vero cells. The virus isolated from the first patient was analyzed byRFLP assay, whereas samples from the second group ofpatients were analyzed using PCR. EM and neutralization assays were also performed. 65. Novel bunyavirus identified inChina(contd) The investigators isolated a novel pathogen, which theynamed SFTS bunyavirus. Analysis of viral RNA showed that the virus was a memberof the Bunyaviridae family in the genus Phlebovirus. EM confirmed bunyavirus morphology. Based onidentification of viral DNA or specific antibodies, or both,171 patients with SFTS were shown to have infection withSFTS bunyavirus. Immune response specific to the virus was shown in 100percent (35 of 35) of matched serum samples obtainedduring acute infection and convalescence. 66. All that are round and spiculated are not DengueThanks for your attention