1. VIROLOGY

2. 1796:Edward Jenner (1749-1823) used cowpox to vaccinate against smallpox was the first person to deliberately vaccinate against any infectious disease used a preparation to elicit an immune response. Variolation infecting people with smallpox to protect them from the worst type of the disease 3. 1885: Louis Pasteur (1822-1895) experimented with rabiesvaccination using the term "virus" to describethe agent "virus" and "vaccination" (in honorof Jenner) 4. 1886: John Buist Scottish pathologist stained lymph from skin lesions of asmallpox "elementary bodies" he thought were the spores ofmicrococci smallpox virus particles 5. 1892: Dmiti Iwanowski (1864-1920) described the first "filterable"infectious agent tobacco mosaic virus (TMV) smaller than any known bacteria first to discriminate betweenviruses and other infectiousagents 6. 1898: Martinus Beijerinick (1851-1931) extended Iwanowskis work with TMV developed the concept of the virus as a distinct entity 7. Freidrich Loeffler (1852-1915)and Paul Frosch (1860-1928) demonstrated that foot and mouth disease first to prove that viruses could infect animals as well as plants 8. 1900: Walter Reed (1851-1902) demonstrated that yellow fever isspread by mosquitoes first to show that viruses could bespread by insect vectors such asmosquitoes1908: Karl Landsteiner (1868-1943) and Erwin Popper proved that poliomyelitis: virus proved that viruses could infecthumans as well as animals 9. 1911: Francis Peyton Rous (1879-1970) demonstrated that a virus (Roussarcoma virus) can cause cancer inchickens first person to show that a viruscould cause cancer 10. 1915: Frederick Twort (1877-1950) discovered viruses infecting bacteria1917: Felix dHerelle (1873-1949) independently discovered viruses ofbacteria coins the term bacteriophage 11. 1938: Max Theiler (1899-1972) developed a live attenuated vaccineagainst yellow fever safe and effective that it is still inuse today!1940: Helmuth Ruska (1908-1973) used an electron microscope: virusparticles direct visualization of virions 12. 1941: George Hirst demonstrated that influenza virusagglutinates red blood cells viruses could be counted1945: Salvador Luria (1912-1991)and Alfred Hershey (1908-1997) demonstrated thatbacteriophages mutate antigenic variation in viruses. 13. 1957: Alick Isaacs and JeanLindemann discovered interferon first cytokines to be studied indetailCarleton Gajdusek proposed: a "slow virus" isresponsible for the prion diseasekuru kuru is similar to that of scrapie kuru can be transmitted to 14. 1961: Sydney Brenner, FrancoisJacob, and Matthew Meselson demonstrated that bacteriophageT4 uses host cell ribosomes to direct virus protein synthesis fundamental molecular mechanism of protein translation. 15. 1963: Baruch Blumberg discovered hepatitis B virus (HBV) developed the first vaccine againstthe HBV first vaccine against cancer1989: Hepatitis C virus (HCV) nonA, nonB hepatitis first infectious agent to be identifiedby molecular cloning of the genome 16. Sending Specimens to theLaboratoryRight specimenTaken at the right timeStored and transportedimmediately 17. Specimens for viral isolation or AgdetectionRespiratory Infection Nasal or throat swabs, postnasal washingsGastrointestinal infxn FecesVesicular rashVesicular fluid, throatswab, fecesHepatitis Serum, fecesCNS CSF, throat swab,fecesAIDSUnclotted blood 18. SPECIMENS: Blood serum syringe vacutainer Swabs skin lesions throat transport medium antibacterial antifungal 19. Vesicle fluid tip of a scalpel blade large needle spread on a slide CSF dry sterile container Feces dry sterile container rectal swab 20. Storage: secure plastic bags labeled transported ASAP 4oC: overnight Request forms: date on onset of disease clinical signs suspected diagnosis 21. Laboratory Diagnosis forviral Infections 22. 3 Basic categories of methodsused in the Diagnosis of virusinfections Direct Examination Indirect examination Serology 23. Direct ExaminationClinicalspecimen examined directly for presence of virus particles, virus antigens, viral nucleic acids and virus-induced histological changes 24. Direct ExaminationClinical specimen virus particles virus antigens viral nucleic acids virus-induced histologicalchanges 25. Direct ExaminationMain advantage: short length of time required for resultResults available the same dayspecific antiviral chemotherapy 26. Direct ExaminationAutomatedmolecularbiology techniquesavailable: 1. PCR-based amplicor system 2. Abbott LCR system 3. Chiron branched DNA system 27. Direct Examination Wave of the future:DNA chip technology detect viruses from clinical specimens tell viral load determine antiviral agent the virus is sensitive to 28. Indirect ExaminationVirus amplification:growing in tissue culture, eggs or animalsGrowing virus: changes cytopathic effect (CPE) ability to hemadsorbIdentity of virus confirmed by: virus neutralization Immunofluorescence complement fixation electron microscopy 29. Indirect ExaminationDisadvantage of virus culture: long time required for CPE ability to hemadsorb to become apparentMay take a few days to a fewweeksVirus culture sensitivity: low anddepends on quality of clinicalspecimen receivedNot applicable to viruses that aredifficult or can not be cultivated 30. SEROLOGYRemains the bulk of work carriedout by a routine diagnosticlaboratory Complement fixation test hemagglutination-inhibition enzyme linked immunoassay Radioimmunoassay particle agglutination Immunofluorescence single radial hemolysis western blot 31. SEROLOGYSensitivity and specificity rates varyMost techniques will detect allclasses of antibodySome assays like RIA, EIA and IFcan be made to detect one specificclass IgM, IgG, IgAVirus infections usually diagnosedby serology:* hepatitis A, B and C 32. DIFFICULT VIRUSESViral infections of the Centralnervous system infection in immunocompetenthosts infection caused by HIV and opportunistic infectionsViral pneumoniasViral skin infectionsEmerging viral infections 33. Viral infections of the CNS Mostcases of acute encephalitis inimmunocompetent hosts due to: * HSV-1 and HSV-2 * EBV * HHV-6 and HHV-7 Among immunocompromisedindividuals: * CMV * VZV 34. Viral infections of the CNSDiagnosis revolutionized by availability of cerebrospinal fluid (CSF) polymerase chain reaction analysisPCR allows rapid, specific and sensitive diagnosis 35. Viral infections of the CNS CSF-PCR instead of brain biopsy Dx standard for HSV encephalitis mild or atypical cases 16-25% of cases HSVPCR analysis of CSF monitoring adequacy of therapy 14day treatment with acyclovir negative PCR Prognosis: Determination of number of viralDNA copies 36. Viral infections of the CNS EBVsemi-quantitative PCRsignificantly higher: active EBV infection latently infected patients seropositive: LOWER positive EBV PCR in CSFsensitive and specificprimary CNS lymphoma in patients with AIDSCNS mass lesions 37. Viral infections of the CNS HHV6 and HHV 7 Almost all children PCR review of CSF specimens frompatients initially suspected to haveHSV encephalitis found HHV 6 DNAin ~ 7% of specimens suggests HHV 6 may be animportant cause of sporadic focalencephalitis 38. Viral infections of the CNS UsingCSF RT-PCR analysis most cases (85-95%) of acuteviral meningitis enteroviruses Signs and symptoms indicative ofencephalitis rather thanmeningitis develop in ~ 3% ofpatients Patients withagammaglobulinemia are at riskfor chronic enteroviralmeningoencephalitis 39. Viral Pneumonias Causes in adults:* Adenovirus * Influenza A & B* CMV* Measles* HSV* Parainfluenzavirus* RSV * VZV Causes in children* Influenza A & B * Measles* Parainfluenza * RSV 40. Diagnostic techniques for herpesvirus infectionsVirus Cytologic ViralAntigen Geneevaluation culture detection ampli- ficationHSV Cowdry CPE IFA PCRType A SVA ELISAbodiesVZV Cowdry CPE IFA MRT-PCRType AbodiesCMV owls CPE IFA MRT-PCReye cells SVA ELISA 41. Diagnostic techniques for herpesvirus infectionsVirus Cytologic Viral Antigen Geneevaluation culturedetection ampli-ficationHighlyCPE IFA MRT-PCRRSV eosinophilicintracytoplas SVA ELISAmicinclusionsParaLarge cells HAIFAMRT-PCRwith singleinfluenza nucleus & SVA ELISAmultiplesmalleosinophilicinclusionsMeasles HAIFAELISA 42. Diagnostic techniques for viral infectionsInfluenz HAIFAa virusSVA ELISAAdeno Intra- CPE IFA MRT-virus nuclear SVAELISA PCRinclusions 43. Herpes virus skin infectionDiagnosis Tzanckpreparation :used to rapidly determine presenceof HSV or VZVdoes not distinguish between these2 viruses Stains : Giemsa,Wrights, methylene blue Characteristic multinucleated giantcells inexpensive, efficient provisional diagnosis 44. Herpes virus skin infectionDiagnosis HSVtissue culture: usingmonoclonal antibodies, requiresonly 24 hourssensitive test but expensive Polymerase chain reactionexpensive Serology not very useful general population has antibodies toherpes simplex 45. Emerging Viral Infections Acute Hemorrhagic fever syndromes: Ebola and Marburg v Hantavirus Arena virus Other viral encephalitis agents: Nipah virus (previously unknownparamyxovirus) 46. Labtesting currently available only at CDC: antigen detection IgM antibody detection isolation in cell culture visualization by electronmicroscopy immunohistochemicaltechniques RT-PCR 47. Emerging Viral Infections Manyof these emerging viralinfections are thought to pose aserious risk as biologic weapons Rapid diagnostics should bemade available worldwide inorder to detect as rapidly aspossible both justified andunjustified suspects of HF andattacks by other biologicalweapons 48. Emerging Viral Infections SARSSevere acute respiratory syndromeKiller pneumonia Cause has yet to be identified Laboratory tests found two types ofvirus:paramyxoviruscorona virus A rapid diagnostic test should comesoon 49. SUMMARYDespite enhancedlaboratory techniques suchas viral culture, rapidantigen detection and geneamplification, a confidentdiagnosis of viralpneumonia continues to bea CHALLENGE 50. SUMMARYThe non-specific nature of clinical characteristics & the extreme sensitivity of lab techniques make the diagnosis difficult, even when a viral agent is detected 51. SUMMARYUnderstanding the limitations of these technological advances and the use of histopathological techniques can greatly enhance a skilled clinicians ability to make an accurate diagnosis 52. ADENOVIRUS 53. ADENOVIRUSDNA viruses first isolatedfrom adenoidal tissue in1953 54. ADENOVIRUSFamily AdenoviridaeGenusMastadenovirus 55. Transmission electron micrograph of adenovirus Source- PHIL, CDC 56. ADENOVIRUS - Classification Subdivided into 6 subgroups based on hemagglutination (A-F) Human pathogens belong to 49 serotypes Common serotypes:- 1-8, 11, 21, 35, 37, 40 Enteric Adenoviruses belong to subgroup F 57. ADENOVIRUS - Structure Non-enveloped DNA virus 70-90 nm in size Linear ds DNA genome with core proteins 58. ADENOVIRUS EM APPEARANCE 59. ADENOVIRUS - Ultrastructure Icosahedral capsid with 252 capsomeres(12 pentons at vertices and 240 hexons) Each penton has a fibers with terminalknob projecting from it 60. Adenovirus- 3 D structure 61. Adenovirus - EM appearanceNote- projecting fibers and terminal knob 62. ADENOVIRUS-Ultrastructure 63. ADENOVIRUS STRUCTURE 64. Structure 65. Pathogenesis and Replication Infects mucoepithelial cells of respiratory, GI and GU tracts Enter via epithelium, replicate and spread to lymphoid tissue Viremia occurs Secondaryinvolvement of 66. Pathogenesis and Replication (contd.) Fiber protein determines target cell specificity and attachment Viral DNA enters host cell nucleus Virus replicates in cytoplasm 67. Adenovirus- replication 68. Replication (contd.) Early and late phases ofreplication Errror-prone process Inclusion bodies in nucleus 69. ADENOVIRAL INCLUSIONBODIES 70. Types of infection Lytic Latent/occult Oncogenic Transformation 71. Types of infection Lytic Results in cell death; seen in mucoepithelical cells Latent/occult Virus remains in host cell; seen in lymphoid tissue, Groups B and C OncogenicTransformation Uncontrolled cell growth and replication occur; seen with Group A viruses in hamsters 72. Adenovirus Used as VECTORS to transfer desiredgenetic material into cells e Viral genome is relatively easilymanipulated in vitro Efficient expression of inserted DNA inrecipient cell 73. Adenovirus- Properties Stable in the environment Relatively resistant to disinfection (Alcohol, chlorhexidine, detergents) Stable in GI tract- can withstand low pH, bile acids and proteolytic enzymes 74. Time-course of infection Incubationperiod- 2-14 days Infective period continues for weeks Intermittent and prolonged rectalshedding Secondary attack rate within families upto 50% 75. Timecourse - Respiratory infectionSource- Medical Microbiology- Murray, Rosenthal, Kobayshi and Pfaller 76. EPIDEMIOLOGYEndemic, epidemic and sporadic infectionsMany infections are subclinical 77. EPIDEMIOLOGY-contd. Tip of the iceberg phenomenon Classical disease presentationMild clinical disease Asymptomatic infection but infectivity (+) 78. Epidemiology of AdenoviralInfections (source-centers for disease control and prevention) 79. EPIDEMIOLOGYOutbreaks noted in military recruits,swimming pool users, hospitals,residential institutions, day care settings 80. EPIDEMIOLOGY-transmission Prolonged infective period (weeks) Intermittent and prolonged rectal shedding Stable in the environment 81. TRANSMISSION Droplets Fecal-oral route Direct and through poorly chlorinated water Fomites 82. CLINICALSYNDROMES Respiratory Eye Genitourinary Gastrointestinal Others 83. Acute Respiratory Disease(LRI) Fever Tracheobronchitis Pneumonia Childrenand adults Epidemics in military recruits Types 4 and 7 most frequently 84. Acute Respiratory Disease 85. Pharyngoconjunctival fever Headache, fever, malaise Conjunctivitis and Pharyngitis Cervical adenopathy, rash and diarrheaalso Main adenovirus types: 3, 4, 7, 14 Epidemics in summer months Contaminated water in swimming pools,fomites 86. Adenoviral Infections of the eye Epidemic Keratoconjunctivitis (EKC) Acute follicular conjunctivitis Pharyngoconjunctival fever 87. Adenoviral Infections of theeye 88. Epidemic Keratoconjunctivitis Incidence in summer Conjunctivitis usually followed bykeratitis Headache Pre-auricular lymphadenopathy Types 8, 19, 37 Nosocomial spread by fomites, hands,ophthalmologic equipment, medications 89. Gastrointestinal Infections Types 40, 41 Age50% of HIV-infected persons haveprogressed to AIDSThere is NO strong evidence there is any other infectiousagent involved than HIV 113. HIV - The Virus RetrovirusMembrane: host derived Three genes GAG POL ENV 114. HIV - The VirusRetrovirusTwo glycoproteins: gp160gp120 and gp41gp41 is fusogen that spans the membranesugarsvaccine problem ENV gene 115. HIV - The VirusRetrovirusGroup-Specific Antigensp17: inner surface - myristoylatedp24: nucleocapsidp9: nucleocapsid associated with RNAGAG gene Polyprotein 116. HIV - The VirusEnzymes Retrovirus Polymerase (reversetranscriptase RNAdependent DNApolymerase) Integrase POL gene Protease (cuts Polyproteinpolyproteins) 117. The Genome of HIVThree structural genesLTRsExtra open reading frames are clue to latency 118. HIV - The Virus 119. HIV - The VirusLife HistoryA retrovirus Latency Specific destruction ofCD4+ cells 120. HIV - Life History Fusion at ambient pH No need for entry into lysosomes Profound significance Syncytiafor AIDS progression:Spread from cell tocell Profound significance 121. HIV - Life History Entry into the cell T4 (CD4+) cells are major target Human HeLa Human Cell transfected HeLa Cell with CD4 antigenNOT INFECTED INFECTED But NOT the whole answer since thisdoes not happen if CD4 is transfected in 122. HIV - Life HistoryWhy do CD4-transfected human cellbut CD4-transfected mouse cells doHuman cells must possess a co-factor for infection that mouCo-ReceptorsCD8+ CellsMIP-1 alpha MIP-1 beta RANTESChemokines 123. HIV - Life HistoryHIV chemoki MutantCD4neCCR5 CCR5CCR5 CD4CD4 macrophageChemokine receptors are necessary co-recep 124. HIV and AIDSSome people do not get AIDSLong term survivorsExposed uninfected personsThe chemokine receptor story 125. HIV and AIDSCo-receptors and HIV infection CCR5 is a chemokine receptor Cells with homozygous mutant CCR5 molecules are notinfected by HIV1 in 100 CaucasiansNo Africans Persons with heterozygous mutant CCR5 moleculesprogress to AIDS more slowly 126. HIV and AIDSCo-receptors 25% of long term survivors are CCR5 or CCR2mutants (deletions) The same CCR5 mutation (called delta 32) isthought to be the mutation that rendered somepeople immune to the plague in the middleages Many other chemokine receptors 127. HIV and AIDSLong term non-progressers People who have been infected withHIV for more than seven years thathave stable CD4+ cell counts above600 per cu mm with no symptoms andno chemotherapyMany have produced a very goodimmune response to the virus 128. HIV and AIDS Nairobi prostitutesClient infection rate more than 25% Rare HLA antigens Associations between resistance to infection and theirclass I and class II MHC (HLA) haplotypes 129. HIV - Life HistoryHIV is a retrovirusIt carries with it: Reverse transcriptase HIV genes Integrase GAG POL ProteaseENV tRNA primerHIV has no oncogene but could still be oncogenicvaccine problem 130. HIV - Life HistoryLatency Cellular The problem of memory T4 cells Only activated T4 cells can replicate virus Most infected T4 cells are rapidly lyzed but are replaced Some T4 cells revert to resting state as memory cells wh Memory T4 cells cannot replicate the virus unless they b Clinical LatencyHIV infection is not manifested as disease for years 131. Dynamics of CD4 T cells in anHIV infection Cell death Chronically- apoptosis etcinfectedmemory T Return tocells withInfectionrestingUninfected stateprovirusactivated LongT cellReactivation lived!UninfectedCell deathLongunactivatedmemoryimmunelived! Adapted from Saag and KilbyT cell pool destructionNat Med 5: 609, 1999 132. Long tern latent HIV Immune responseT4 resting T4 activatedIt may be impossible to cure the patient oEven if combination therapy stops HIV re HIV 133. Inexorable decline of CD4+T4 cellsWhy do allof the T4cellsAt earlydisappear?stages ofinfectiononly 1 in10,000 cellsis infected Of great importance to therapeutic strategy 134. Virus destroys the cell as a result ofbudding But few cells are infected: Early stage of infection 1:10,000 Late 1:40 Why do all T4 cells1. PUNCTURED disappear?MEMBRANE 135. Why do all T4 cellsdisappear? - 2But syncytianot commonInfected CD4 CellscellMost T4 cells FuseGp120 are not HIV+positiveCouldsweep up UninfectedKilling of CD4 cells CD4 cell uninfected2. Syncytium Gp120cellsFormationnegative 136. Why do all T4 cells disappear? Cytotox ic T cellKilling of CD4 cells3. Cytotoxic T cell-BUT: Most mediated lysiscells are 137. Killing ofCD4+ cells4. Binding of freeGp120 to CD4antigen makesuninfected T4 celllook like aninfected cellComplement-mediated lysis 138. Why do all T4 cellsdisappear? Induction of apoptosis CD8 gp120MacrophagHIVcell e(no CD4 antigen)chemokine CXCR 4G proteinsignal ? ?Binding toBinding toCXCR4CXCR4 results results inin expression expression ofof TNF-alphaTNF-alpha on 139. Why do all T4 cellsdisappear? Induction of apoptosis CD8 cellCXCR4 Macrophag Deat e h CD8 T cell 140. Macrophages may be infected by two routesHIV gp120CD4 HIV gp120 binds to macrophage CD4 antigen Virus is opsonized by anti gp120 antibodies whichFc receptorbind to macrophage Fc receptors - anAnti-gp120vaccine problem enhancing antibodyHIV 141. Overview: Hepatitis Virology Transmission Epidemiology Pathogenesis Symptoms Diagnosis Management Prevention 142. Viral Hepatitis - Overview Types of Hepatitis AB C DESource of Feces Blood Blood Blood Feces Blood-derived Blood-derived Blood-derivedVirus body fluids body fluids body fluidsRoute ofFecal-Oral PercutaneousPercutaneousPercutaneous Fecal-Trans-PermucosalPermucosalPermucosal OralmissionChronic No Yes Yes YesNoInfectionPrimary Pre/Post-Pre/Post-Blood DonorPre/Post- Ensure safeExposure ExposureScreening Exposure drinkingPrevent- ImmunizationwaterImmunization ImmunizationionRisk BehaviorHandwashingRisk Behavior ModificationRisk Behavior ModificationModification 143. Hepatitis A, B, and C at a GlanceVirus SexIDUTrans- Fecal- Occu-Course of Infection Does Vaccinefusion Oral pational Protective Available Immunity Develop?A High Low* LowHigh None Acute -> Resolved YesYesB High High LowNone High Acute -> ChronicYesYes 90% of infants 30% in children aged 1-5 10% of older children and adults.C LowHigh LowNone LowAcute -> ChronicNo No in 75%-85% of adults. 144. Age at Infection Viral Hepatitis Perinatal ChildhoodAdolescent AdultHAV - ++++++ +++HBV++++ +++ +++++HCV + -++++++ 145. REPORTED CASES OF SELECTED NOTIFIABLE DISEASES PREVENTABLE BY VACCINATION,UNITED STATES, 2001 Hepatitis A 10,609 Hepatitis B7,843 Pertussis7,580 Meningococcal disease2,333 H. influenzae, invasive1,597 Mumps266 Measles116 Source: NNDSS, CDC 146. HEPATITIS A VIRUS 147. Hepatitis A Structure 148. Hepatitis A Virus: Structure and Classification RNA Picornavirus Separate genus because of differenceswith other enteroviruses Naked icosahedral capsid SS RNA (740 nucleotides) Single serotype worldwide Humans only reservoir 149. HEPATITIS A VIRUS TRANSMISSION Fecal-oral transmission Close personal contact (e.g., household contact, sex contact, child day-care centers) Contaminated food, water (e.g., infected food handlers, contaminated raw oysters) Blood exposure (rare