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  1. 1. VIROLOGY
  2. 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. 3. 1885: Louis Pasteur (1822-1895) experimented with rabies vaccination using the term "virus" to describe the agent "virus" and "vaccination" (in honor of Jenner)
  4. 4. 1886: John Buist Scottish pathologist stained lymph from skin lesions of a smallpox – "elementary bodies" – he thought were the spores of micrococci – smallpox virus particles
  5. 5. 1892: Dmiti Iwanowski (1864- 1920) described the first "filterable" infectious agent – tobacco mosaic virus (TMV)  smaller than any known bacteria  first to discriminate between viruses and other infectious agents
  6. 6. 1898: Martinus Beijerinick (1851- 1931) – extended Iwanowskis work with TMV – developed the concept of the virus as a distinct entity
  7. 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. 8. 1900: Walter Reed (1851-1902) demonstrated that yellow fever is spread by mosquitoes first to show that viruses could be spread by insect vectors such as mosquitoes1908: Karl Landsteiner (1868- 1943) and Erwin Popper proved that poliomyelitis: virus proved that viruses could infect humans as well as animals
  9. 9. 1911: Francis Peyton Rous (1879- 1970) demonstrated that a virus (Rous sarcoma virus) can cause cancer in chickens first person to show that a virus could cause cancer
  10. 10. 1915: Frederick Twort (1877-1950) discovered viruses infecting bacteria1917: Felix dHerelle (1873-1949) – independently discovered viruses of bacteria – coins the term bacteriophage
  11. 11. 1938: Max Theiler (1899-1972) developed a live attenuated vaccine against yellow fever safe and effective that it is still in use today!1940: Helmuth Ruska (1908-1973) used an electron microscope: virus particles direct visualization of virions
  12. 12. 1941: George Hirst demonstrated that influenza virus agglutinates red blood cells viruses could be counted1945: Salvador Luria (1912-1991) and Alfred Hershey (1908-1997) demonstrated that bacteriophages mutate antigenic variation in viruses.
  13. 13. 1957: Alick Isaacs and Jean Lindemann discovered interferon – first cytokines to be studied in detailCarleton Gajdusek – proposed: a "slow virus" is responsible for the prion disease kuru – kuru is similar to that of scrapie – kuru can be transmitted to
  14. 14. 1961: Sydney Brenner, Francois Jacob, and Matthew Meselson demonstrated that bacteriophage T4 uses host cell ribosomes – to direct virus protein synthesis – fundamental molecular mechanism of protein translation.
  15. 15. 1963: Baruch Blumberg discovered hepatitis B virus (HBV) developed the first vaccine against the HBV – first vaccine against cancer1989: Hepatitis C virus (HCV) nonA, nonB hepatitis first infectious agent to be identified by molecular cloning of the genome
  16. 16.  Sending Specimens to the Laboratory –Right specimen –Taken at the right time –Stored and transported immediately
  17. 17. Specimens for viral isolation or Ag detectionRespiratory Infection Nasal or throat  swabs, postnasal washingsGastrointestinal infxn FecesVesicular rash Vesicular fluid, throat swab, fecesHepatitis Serum, fecesCNS CSF, throat swab, fecesAIDS Unclotted blood
  18. 18. SPECIMENS: Blood serum – syringe – vacutainer Swabs – skin lesions – throat – transport medium  antibacterial  antifungal
  19. 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. 20.  Storage: – secure plastic bags – labeled – transported ASAP – 4oC: overnight Request forms: – date on onset of disease – clinical signs – suspected diagnosis
  21. 21. Laboratory Diagnosis for viral Infections
  22. 22. 3 Basic categories of methodsused in the Diagnosis of virus infections Direct Examination Indirect examination Serology
  23. 23. Direct ExaminationClinicalspecimen examined directly for presence of virus particles, virus antigens, viral nucleic acids and virus-induced histological changes
  24. 24. Direct ExaminationClinical specimen –virus particles –virus antigens –viral nucleic acids –virus-induced histological changes
  25. 25. Direct ExaminationMain advantage: short length of time required for resultResults available the same dayspecific antiviral chemotherapy
  26. 26. Direct ExaminationAutomated molecularbiology techniquesavailable: 1. PCR-based amplicor system 2. Abbott LCR system 3. Chiron branched DNA system
  27. 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. 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. 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. 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. 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. 32. DIFFICULT VIRUSESViral infections of the Centralnervous system infection in immunocompetent hosts infection caused by HIV and opportunistic infectionsViral pneumoniasViral skin infectionsEmerging viral infections
  33. 33. Viral infections of the CNS Most cases of acute encephalitis in immunocompetent hosts due to: * HSV-1 and HSV-2 * EBV * HHV-6 and HHV-7 Among immunocompromised individuals: * CMV * VZV
  34. 34. Viral infections of the CNSDiagnosis revolutionized by availability of cerebrospinal fluid (CSF) polymerase chain reaction analysisPCR allows rapid, specific and sensitive diagnosis
  35. 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 HSV PCR analysis of CSF –monitoring adequacy of therapy –14day treatment with acyclovir  negative PCR Prognosis: – Determination of number of viral DNA copies
  36. 36. Viral infections of the CNS EBV semi-quantitative PCR –significantly higher: active EBV infection – latently infected patients seropositive: LOWER positive EBV PCR in CSF –sensitive and specific primary CNS lymphoma in patients with AIDS CNS mass lesions
  37. 37. Viral infections of the CNS HHV 6 and HHV 7 –Almost all children –PCR review of CSF specimens from patients initially suspected to have HSV encephalitis found HHV 6 DNA in ~ 7% of specimens – suggests HHV 6 may be an important cause of sporadic focal encephalitis
  38. 38. Viral infections of the CNS Using CSF RT-PCR analysis –most cases (85-95%) of acute viral meningitis  enteroviruses Signs and symptoms indicative of encephalitis rather than meningitis develop in ~ 3% of patients Patients with agammaglobulinemia are at risk for chronic enteroviral meningoencephalitis
  39. 39. Viral Pneumonias Causes in adults: * Adenovirus * Influenza A & B * CMV * Measles * HSV * Parainfluenza virus * RSV * VZV Causes in children * Influenza A & B * Measles * Parainfluenza * RSV
  40. 40. Diagnostic techniques for herpesvirus infectionsVirus Cytologic Viral Antigen Gene evaluation culture detection ampli- ficationHSV Cowdry CPE IFA PCR Type A SVA ELISA bodiesVZV Cowdry CPE IFA MRT-PCR Type A bodiesCMV “owl’s CPE IFA MRT-PCR eye” cells SVA ELISA
  41. 41. Diagnostic techniques for herpesvirus infectionsVirus Cytologic Viral Antigen Gene evaluation culture detection ampli- fication Highly CPE IFA MRT-PCRRSV eosinophilic intracytoplas SVA ELISA mic inclusionsPara Large cells HA IFA MRT-PCR with singleinfluenza nucleus & SVA ELISA multiple small eosinophilic inclusionsMeasles HA IFA ELISA
  42. 42. Diagnostic techniques for viral infectionsInfluenz HA IFAa virus SVA ELISAAdeno Intra- CPE IFA MRT-virus nuclear SVA ELISA PCR inclusions
  43. 43. Herpes virus skin infection Diagnosis Tzanck preparation : –used to rapidly determine presence of HSV or VZV –does not distinguish between these 2 viruses Stains : Giemsa,Wright’s, methylene blue Characteristic multinucleated giant cells – inexpensive, efficient provisional diagnosis
  44. 44. Herpes virus skin infection Diagnosis HSV tissue culture: using monoclonal antibodies, requires only 24 hours –sensitive test but expensive Polymerase chain reaction –expensive Serology not very useful – general population has antibodies to herpes simplex
  45. 45. Emerging Viral Infections Acute Hemorrhagic fever syndromes: – Ebola and Marburg v – Hantavirus – Arena virus Other viral encephalitis agents: – Nipah virus (previously unknown paramyxovirus)
  46. 46.  Labtesting currently available only at CDC: –antigen detection – IgM antibody detection –isolation in cell culture –visualization by electron microscopy – immunohistochemical techniques –RT-PCR
  47. 47. Emerging Viral Infections Many of these “emerging” viral infections are thought to pose a serious risk as biologic weapons Rapid diagnostics should be made available worldwide in order to detect as rapidly as possible both justified and unjustified suspects of HF and attacks by other “biological weapons”
  48. 48. Emerging Viral Infections SARS –Severe acute respiratory syndrome –Killer pneumonia Cause has yet to be identified Laboratory tests found two types of virus: –paramyxovirus –corona virus A rapid diagnostic test should come soon
  49. 49. SUMMARYDespite enhancedlaboratory techniques suchas viral culture, rapidantigen detection and geneamplification, a confidentdiagnosis of viralpneumonia continues to bea CHALLENGE
  50. 50. SUMMARYThe non-specific nature of clinical characteristics & the extreme sensitivity of lab techniques make the diagnosis difficult, even when a viral agent is detected
  51. 51. SUMMARYUnderstanding the limitations of these technological advances and the use of histopathological techniques can greatly enhance a skilled clinician’s ability to make an accurate diagnosis
  52. 52. ADENOVIRUS
  53. 53. ADENOVIRUSDNA viruses first isolatedfrom adenoidal tissue in1953
  54. 54. ADENOVIRUSFamily AdenoviridaeGenus Mastadenovirus
  55. 55. Transmission electron micrograph of adenovirus Source- PHIL, CDC
  56. 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. 57. ADENOVIRUS - Structure Non-enveloped DNA virus 70-90 nm in size Linear ds DNA genome with core proteins
  59. 59. ADENOVIRUS - Ultrastructure Icosahedral capsid with 252 capsomeres (12 pentons at vertices and 240 hexons) Each penton has a fibers with terminal knob projecting from it
  60. 60. Adenovirus- 3 D structure
  61. 61. Adenovirus - EM appearanceNote- projecting fibers and terminal knob
  62. 62. ADENOVIRUS-Ultrastructure
  64. 64. Structure
  65. 65. Pathogenesis and Replication• Infects mucoepithelial cells of respiratory, GI and GU tracts• Enter via epithelium, replicate and spread to lymphoid tissue• Viremia occurs• Secondary involvement of
  66. 66. Pathogenesis and Replication (contd.) Fiber protein determines target cell specificity and attachment Viral DNA enters host cell nucleus Virus replicates in cytoplasm
  67. 67. Adenovirus- replication
  68. 68. Replication (contd.)• Early and late phases ofreplication• Errror-prone process• Inclusion bodies in nucleus
  70. 70. Types of infection Lytic Latent/occult Oncogenic Transformation
  71. 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 Oncogenic Transformation Uncontrolled cell growth and replication occur; seen with Group A viruses in hamsters
  72. 72. Adenovirus Used as VECTORS to transfer desired genetic material into cells e Viral genome is relatively easily manipulated in vitro Efficient expression of inserted DNA in recipient cell
  73. 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. 74. Time-course of infection Incubation period- 2-14 days Infective period continues for weeks Intermittent and prolonged rectal shedding Secondary attack rate within families up to 50%
  75. 75. Timecourse - Respiratory infection Source- Medical Microbiology- Murray, Rosenthal, Kobayshi and Pfaller
  76. 76. EPIDEMIOLOGYEndemic, epidemic and sporadic infectionsMany infections are subclinical
  77. 77. EPIDEMIOLOGY-contd. ”Tip of the iceberg phenomenon” Classical disease presentation Mild clinical disease Asymptomatic infection but infectivity (+)
  78. 78. Epidemiology of Adenoviral Infections (source-centers for disease control and prevention)
  79. 79. EPIDEMIOLOGYOutbreaks noted in military recruits,swimming pool users, hospitals,residential institutions, day care settings
  80. 80. EPIDEMIOLOGY-transmission Prolonged infective period (weeks) Intermittent and prolonged rectal shedding Stable in the environment
  81. 81. TRANSMISSION• Droplets• Fecal-oral route• Direct and through poorly chlorinated water• Fomites
  82. 82. CLINICAL SYNDROMES• Respiratory• Eye• Genitourinary• Gastrointestinal• Others
  83. 83. Acute Respiratory Disease (LRI) Fever Tracheobronchitis Pneumonia Childrenand adults Epidemics in military recruits Types 4 and 7 most frequently
  84. 84. Acute Respiratory Disease
  85. 85. Pharyngoconjunctival fever Headache, fever, malaise Conjunctivitis and Pharyngitis Cervical adenopathy, rash and diarrhea also Main adenovirus types: 3, 4, 7, 14 Epidemics in summer months Contaminated water in swimming pools, fomites
  86. 86. Adenoviral Infections of the eye Epidemic Keratoconjunctivitis (EKC) Acute follicular conjunctivitis Pharyngoconjunctival fever
  87. 87. Adenoviral Infections of the eye
  88. 88. Epidemic Keratoconjunctivitis Incidence in summer Conjunctivitis usually followed by keratitis Headache Pre-auricular lymphadenopathy Types 8, 19, 37 Nosocomial spread by fomites, hands, ophthalmologic equipment, medications
  89. 89. Gastrointestinal Infections Types 40, 41 Age <4 years Spread via fecal-oral route Year round
  90. 90. Gastrointestinal Infections- (contd.) Incubation period 3-10 days Diarrhea lasts for 10-14 days Fever Also, intussusception, mesenteric adenitis, appendicitis
  92. 92. ADENOVIRAL INFECTIONS- Genitourinary systemAcute hemorrhagic cystitis fever, dysuria, hematuria Types 11, 7, 4, 21, 1 More common in boysOthers Orchitis, nephritis, cervicitis with ulcerated vesicular lesions Types 2, 19, 37
  93. 93. Other Infections due to Adenovirus•Myocarditis•Pericarditis•Meningitis•Rash•Arthritis
  94. 94. Adenovirus infections in Immunocompromised hosts Disseminated, severe and often fatal infections Due to new infection or reactivation of latent virus Prolonged infections with prolonged viremia and viral shedding Necrotizing pneumonia, hepatitis, rash, DIC, CNS involvement
  95. 95. Adenovirus infection in the immunocompromised
  96. 96. DIAGNOSIS Variety of clinical specimens depending on clinical syndrome-NP, conjunctival, stool, urine, tissue, etc. Transport in viral transport media Isolation from pharyngeal site correlates better with current clinical infection
  97. 97. Methods for diagnosis Culture in HeLa, HEK cell lines Shell vial cell culture DFA PCR, nucleic acid probes EM and Immune EM
  98. 98. Diagnosis- Enteric adenoviruses Isolation requires special media- Graham 293 ELISA for rapid detection is available
  99. 99. Prevention Good handwashing Contact precautions Chlorination of water Disinfection or sterilization of ophthalmologic equipment Use of single dose vials Oral vaccine- restricted
  100. 100. HIV and AIDSThe cellular and immunological picture - The course ofthe disease
  101. 101. HIV and AIDSThe cellular and immunological picture - The courseof the disease
  102. 102. HIV and AIDSThe cellular and immunological pictureThe course of the disease1. Acute Infection • High virus titer • Mild symptoms • Fall in CD4+ cells but recovers • Rise in CD8+ cells but recovers • A high virus titer (up to 10 million Macrophages bring HIV into the body if se viruses per ml blood)
  103. 103. HIV and AIDS 2. A strong immune responseVirus almost disappears from circulation• Good cytoxic T cell response• Soluble antibodies appear later against bothsurface and internal proteins• Most virus at this stage comes from recentlyactivated (dividing) and infected CD4+ cells
  104. 104. HIV and AIDS 3. A latent stateLatency of virus and of symptoms• Virus persists in extra-vasculartissues• Lymph node dendritic cells• Resting CD4+ memory cells (last averylong time - a very stable population ofcells) carry provirus
  105. 105. HIV and AIDS• 10 billion HIV particles per day• Virus half life 5.7 hours• 100-10 million virions per ml blood (set point)• Small minority of T4 cells are infected• Virus found in lymph nodes
  106. 106. HIV and AIDS 4. The beginning of diseaseMassive loss of CD4+ cells• CD4+ cells are the targets of the virus• Cells that proliferate to respond to the virus are killed by it• Dendritic cells present antigen and virus to CD4 cells• Epitope variation allows more and moreHIV toescape from immune response just as
  107. 107. HIV and AIDS 5. Advanced disease - AIDSCD8+ cells destroy moreCD4+ cells• CD4 cell loss means virus andinfected cells no longer controlled• As CD4+ cells fall below 200per cu mmvirus titer rises rapidly andremaining immune response
  108. 108. HIV and AIDS Good correlation betw number of HIV particle measured by PCR an progression to disease
  109. 109. HIV and AIDS Viral load predicts survival time
  110. 110. HIV and AIDS CD4 cell count is not good predictor of progression to diseas
  111. 111. HIV and AIDS CofactorsNot all cases of Kaposi’s are associated with HIVNot all HIV infected persons suffer from Kaposi’s20% of homosexual HIV+ males get Kaposi’sFew IV drug users or hemophiliacs get Kaposi’s Kaposi’s sarcoma associated herpes virus Human herpes virus-8
  112. 112. HIV and AIDS Three Views of AIDS Gallo: Infection by HIV is sufficient to cause AIDSMontagnier: HIV may be harmless in the absence of other co-factorsDuesberg / Mullis: HIV is too silent to be the etiologic agent ofAIDS. It is a much maligned by-standerSo far it seems that >50% of HIV-infected persons haveprogressed to AIDSThere is NO strong evidence there is any other infectiousagent involved than HIV
  113. 113. HIV - The Virus RetrovirusMembrane: host derived Three genes GAG – POL – ENV
  114. 114. HIV - The Virus RetrovirusTwo glycoproteins: gp160 gp120 and gp41gp41 is fusogen that spans the membranesugars vaccine problem ENV gene
  115. 115. HIV - The Virus Retrovirus Group-Specific Antigensp17: inner surface - myristoylatedp24: nucleocapsidp9: nucleocapsid associated with RNAGAG gene Polyprotein
  116. 116. HIV - The Virus Enzymes Retrovirus• Polymerase (reversetranscriptase – RNAdependent DNApolymerase)• Integrase • POL gene• Protease (cuts Polyproteinpolyproteins)
  117. 117. The Genome of HIVThree structural genesLTRsExtra open reading frames are clue to latency
  118. 118. HIV - The Virus
  119. 119. HIV - The Virus Life History A retrovirus • Latency • Specific destruction of CD4+ cells
  120. 120. HIV - Life History • Fusion at ambient pH • No need for entry into lysosomes •Profound significance Syncytia for AIDS progression: Spread from cell to cell Profound significance
  121. 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 this does not happen if CD4 is transfected in
  122. 122. HIV - Life History Why do CD4-transfected human cell but CD4-transfected mouse cells do Human cells must possess a co-factor for infection that mouCo-ReceptorsCD8+ CellsMIP-1 alpha MIP-1 beta RANTESChemokines
  123. 123. HIV - Life History HIV chemoki Mutant CD4 ne CCR5 CCR5 CCR5 CD4 CD4 macrophageChemokine receptors are necessary co-recep
  124. 124. HIV and AIDSSome people do not get AIDSLong term survivorsExposed uninfected persons The chemokine receptor story
  125. 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. 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. 127. HIV and AIDS Long term non-progressers People who have been infected with HIV for more than seven years that have stable CD4+ cell counts above 600 per cu mm with no symptoms and no chemotherapy Many have produced a very good immune response to the virus
  128. 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. 129. HIV - Life HistoryHIV is a retrovirusIt carries with it: • Reverse transcriptase HIV genes • Integrase GAG POL • Protease ENV • tRNA primer HIV has no oncogene but could still be oncogenic vaccine problem
  130. 130. HIV - Life History Latency – 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. 131. Dynamics of CD4 T cells in an HIV infection Cell death Chronically- apoptosis etc infected memory T Return to cells with Infection restingUninfected state provirusactivated LongT cell Reactivation lived! Uninfected Cell death Long unactivated memory immune lived! Adapted from Saag and Kilby T cell pool destruction Nat Med 5: 609, 1999
  132. 132. Long tern latent HIV Immune responseT4 resting T4 activated It may be impossible to cure the patient o Even if combination therapy stops HIV re HIV
  133. 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. 134. Virus destroys the cell as a result of budding But few cells are infected: Early stage of infection 1:10,000 Late 1:40 Why do all T4 cells1. PUNCTURED disappear?MEMBRANE
  135. 135. Why do all T4 cells disappear? - 2 But syncytia not commonInfected CD4 Cellscell Most T4 cells FuseGp120 are not HIV+positive Could “sweep up” UninfectedKilling of CD4 cells CD4 cell uninfected2. Syncytium Gp120 cellsFormation negative
  136. 136. Why do all T4 cells disappear? Cytotox ic T cell Killing of CD4 cells 3. Cytotoxic T cell-BUT: Most mediated lysiscells are
  137. 137. Killing ofCD4+ cells4. Binding of freeGp120 to CD4antigen makesuninfected T4 celllook like aninfected cellComplement-mediated lysis
  138. 138. Why do all T4 cells disappear? Induction of apoptosis CD8 gp120 MacrophagHIVcell e (no CD4 antigen) chemokin e CXCR 4 G protein signal ? ? Binding to Binding to CXCR4 CXCR4 results results in in expression expression of of TNF-alpha TNF-alpha on
  139. 139. Why do all T4 cellsdisappear? Induction of apoptosis CD8 cell CXCR 4 Macrophag Deat e h CD8 T cell
  140. 140. Macrophages may be infected by two routesHIV gp120 CD4 HIV gp120 binds to macrophage CD4 antigen Virus is opsonized by anti gp120 antibodies which Fc receptor bind to macrophage Fc receptors - an Anti-gp120 vaccine problem enhancing antibody HIV
  141. 141. Overview: Hepatitis Virology Transmission Epidemiology Pathogenesis Symptoms Diagnosis Management Prevention
  142. 142. Viral Hepatitis - Overview Types of Hepatitis A B C D ESource of Feces Blood Blood Blood Feces Blood-derived Blood-derived Blood-derivedVirus body fluids body fluids body fluidsRoute of Fecal-Oral Percutaneous Percutaneous Percutaneous Fecal-Trans- Permucosal Permucosal Permucosal OralmissionChronic No Yes Yes Yes NoInfectionPrimary Pre/Post- Pre/Post- Blood Donor Pre/Post- Ensure safe Exposure Exposure Screening Exposure drinkingPrevent- Immunization water Immunization Immunizationion Risk Behavior Handwashing Risk Behavior Modification Risk Behavior Modification Modification
  143. 143. Hepatitis A, B, and C at a GlanceVirus Sex IDU Trans- Fecal- Occu- Course of Infection Does Vaccine fusion Oral pational Protective Available Immunity Develop?A High Low* Low High None Acute -> Resolved Yes YesB High High Low None High Acute -> Chronic Yes Yes 90% of infants 30% in children aged 1-5 10% of older children and adults.C Low High Low None Low Acute -> Chronic No No in 75%-85% of adults.
  144. 144. Age at Infection – Viral Hepatitis Perinatal Childhood Adolescent AdultHAV - ++++ ++ +++HBV ++ ++ +++ +++++HCV + - + +++++
  145. 145. REPORTED CASES OF SELECTED NOTIFIABLE DISEASES PREVENTABLE BY VACCINATION, UNITED STATES, 2001 Hepatitis A 10,609 Hepatitis B 7,843 Pertussis 7,580 Meningococcal disease 2,333 H. influenzae, invasive 1,597 Mumps 266 Measles 116 Source: NNDSS, CDC
  147. 147. Hepatitis A Structure
  148. 148. Hepatitis A Virus: Structure and Classification RNA Picornavirus • Separate genus because of differences with other enteroviruses • Naked icosahedral capsid • SS RNA (740 nucleotides) • Single serotype worldwide • Humans only reservoir
  149. 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 <<<1%) (e.g., injection drug use, rarely by transfusion)
  150. 150. HEPATITIS A, UNITED STATES Most disease occurs in the context of community- wide outbreaks Infection transmitted from person to person in households and extended family settings - facilitated by asymptomatic infection among children Some groups at increased risk – specific factor varies – do not account for majority of cases – Children are the most frequently infected group No risk factor identified for 40%-50% of cases
  151. 151. DISEASE BURDEN FROM HEPATITIS A UNITED STATES, 2001Number of acute clinical 10,609cases reportedEstimated number of acute 45,000clinical casesEstimated number of 93,000new infectionsPercent ever infected 31.3%
  153. 153. NUMBER OF YEARS REPORTED INCIDENCE OFHEPATITIS A EXCEEDED 10 CASES PER 100,000, BY COUNTY, 1987-1997 0-1 2-3 4-5 6-7 8-11
  154. 154. Hepatitis A: Pathogenesis Incubation 4 weeks (range 2-6 weeks) Oral cavityGI tractliver via blood Replicates in hepatocytes (little damage to cells) released via bile to intestines 7-10 days prior to clinical symptoms Liver damage and clinical syndrome result of immune response and not direct effect of virus
  155. 155. Hepatitis A: Clinical Features– An acute illness with:  discrete onset of symptoms (e.g. fatigue, abdominal pain, loss of appetite, intermittent nausea, vomiting)  jaundiceor elevated serum aminotransferase levels, dark urine, light stool  Adults usually more symptomatic  Patients are infective while they are shedding the virus in the stool- usually before the onset of symptoms  Mostcases resolve spontaneously in 2-4 weeks  Complete recovery 99%
  156. 156. HEPATITIS A - CLINICAL FEATURES•Jaundice by <6 yrs <10% age group: 6-14 yrs40%-50% >14 yrs70%-80%•Rare complications: Fulminant hepatitis Cholestatichepatitis Relapsinghepatitis•Incubation period: Average 30 days Range 15-50 days
  157. 157. EVENTS IN HEPATITIS A VIRUS INFECTION Clinical illness Infection ALT IgM IgGResponse Viremia HAV in stool 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Week
  158. 158. Hepatitis A Diagnosis Detection of IgM antibody IgG positive 1-3 weeks later; suggests prior infection or vaccination.
  159. 159. Hepatitis A Treatment Supportive- no specific role of antiviral therapy Lifelong immunity likely after infection or vaccination
  160. 160. PREVENTING HEPATITIS A• Hygiene (e.g., hand washing)• Sanitation (e.g., clean water sources)• Hepatitis A vaccine (pre-exposure)• Immune globulin (pre- and post- exposure)
  161. 161. HEPATITIS A VACCINES• Inactivated vaccine• Highly immunogenic •97%-100% of children, adolescents, and adults have protective levels of antibody within 1 month of receiving first dose; essentially 100% have protective levels after second dose• Highly efficacious •In published studies, 94%-100% of children protected against clinical hepatitis A after equivalent of one dose
  162. 162. HEPATITIS A VACCINES1st dose at time 02nd dose 6-12 months afterwards
  163. 163. POST-VACCINATION TESTINGNot recommended:  High response rate among vaccinees  Commercially available assay not sensitive enough to detect lower (protective) levels of vaccine-induced antibody
  164. 164. DURATION OF PROTECTION AFTER HEPATITIS A VACCINATION Protection begins 4 weeks after vaccine Persistence of antibody • At least 5-8 years among adults and children Efficacy – No cases in vaccinated children at 5-6 years of follow-up Mathematical models of antibody decline suggest protective antibody levels persist for at least 20 years Other mechanisms, such as cellular memory, may contribute
  165. 165. Hepatitis A Vaccine Recommendations Vaccine is recommended for the following persons 2 years of age and older: – Travelers to areas with increased rates of hepatitis A – Men who have sex with men – Injecting and non-injecting drug users – Persons with clotting-factor disorders (e.g. hemophilia) – Persons with chronic liver disease – Children living in areas with increased rates of hepatitis A during the baseline period from 1987- 1997- mainly West Coast.
  166. 166. Hep A : Passive Immunization Hepatitis A immune globulin can be given up to 2 weeks after an exposure Immunity temporary (4-5 months) Also given in travelers leaving for endemic area on short notice (ie not enough time for the vaccine to be effective)
  167. 167. Hepatitis A Surveillance & Response Urgently reportable condition in S.C. – Acute HAV infection must be reported by phone to DHEC within 24 hours. Investigation of a case of hepatitis A must be initiated by CO DADE and district epi staff within 24 hours of notification. All cases must be reported to CDC.
  168. 168. Hepatitis B Virus
  169. 169. Hepatitis B: Structure Member of the hepadnavirus group Virion also referred to as Dane particle 42nm enveloped virus Core antigens located in the center (nucleocapsid) – Core antigen (HbcAg) – e antigen (HBeAg)- an indicator of transmissibility (minor component of the core- antigenically distinct from HBcAg) 22nm spheres and filaments other forms- no DNA in these forms so they are not infectious (composed of surface antigen)- these forms outnumber the actual virions
  170. 170. Structure and Replication Circular partially double stranded DNA of virus Initial replication to complete circular DNA with subsequent transcription to make several mRNAs some of which are translated into viral proteins One of the mRNAs is replicated with a reverse transcriptase making the DNA that will eventually be the core of the progeny virion Some DNA integrates into host genome causing carrier state Virus stable and resist many stresses making them more infectious
  171. 171. Hepatitis B virus particles
  172. 172. Epidemiology - United States1  100,000 new infections per year  8,000 - 32,000 chronic infections/year  5,000 - 6,000 deaths/year  1.25 million Americans with chronic HBV infection – 15 to 25% of chronically infected patients will die from chronic liver disease1. Center for Disease Control
  173. 173. Geographic Distribution of Chronic HBV Infection HBsAg Prevalence 8% - High 2-7% - Intermediate <2% - Low
  174. 174. High Prevalence of CHBV in AsianAmerican Communities is Often Overlooked1 US prevalence for chronic HBV is < 2% However, chronic HBV prevalence of 10- 15% in Asian American communities has been reported In Asian American men living in California, HCC ranks as a leading cause death – #2 in Vietnamese and Cambodian Americans – #4 in Chinese and Korean Americans 50% of children born to mothers with chronic HBV in the US are Asian American1.
  175. 175. Hepatitis B VirusModes of Transmission • Sexual • Parenteral • Perinatal
  176. 176. Concentration of Hepatitis B Virus in Various Body Fluids Low/Not High Moderate Detectable blood semen urine serum vaginal fluid feceswound exudates saliva sweat tears breastmilk
  177. 177. Risk Factors for Acute Hepatitis B United States, 1992-1993 Heterosexual* (41%) Injecting Drug Use Homosexual Activity (15%) (9%) Household Contact (2%) Health Care Employment (1%) Unknown (31%) Other (1%)* Includes sexual contact with acute cases, carriers, and multiple partners. Source: CDC Sentinel Counties Study of Viral Hepatitis
  178. 178. HBV Pathogenesis Virus enters hepatocytes via blood Immune response (cytotoxic T cell) to viral antigens expressed on hepatocyte cell surface responsible for clinical syndrome 5 % become chronic carriers (HBsAg> 6 months) Higher rate of hepatocellular ca in chronic carriers, especially those who are “e” antigen positive Hepatitis B surface antibody likely confers lifelong immunity Hepatitis B e Ab indicates low
  179. 179. Hepatitis B - Clinical Features• Incubation period: Average 60-90 days Range 45-180 days• Clinical illness (jaundice): <5 yrs, <10% 5 yrs, 30%-50% 1/3 adults-no symptoms• Acute case-fatality rate: 0.5%-1%• Chronic infection: <5 yrs, 30%-90% 5 yrs, 2%-10% • More likely in asymptomatic infections• Premature mortality from chronic liver disease: 15%-25%
  180. 180. Hepatitis B Clinical Features Incubation Period: 6 weeks to 6 months (av. 120 days) Preicteric or Prodromal phase from initial symptoms to onset of jaundice usually lasts from 3 to 10 days: Non-specific, insidious onset of malaise, anorexia, n/v, RUQ pain, fever, headache, myalgias, skin rashes, arthralgias, arthritis, and dark urine beginning 1-2 days before onset of jaundice.
  181. 181. Hepatitis B Clinical Features Icteric phase usually lasts from 1-3 weeks: jaundice, light or gray stools, hepatic tenderness, hepatomegaly. Convalescence phase may persist for weeks or months: Malaise, fatigue. Jaundice, anorexia, and other symptoms disappear.
  182. 182. Hepatitis B Symptoms About 50%-60% of adults with HBV infection have no signs or symptoms. Those who do have symptoms might experience:  Jaundice  Fatigue  Abdominal pain  Loss of appetite  Nausea, vomiting  Joint pain
  183. 183. Outcome of Hepatitis B Virus Infection 100 by Age at Infection 100 Symptomatic Infection (%) 80 80Chronic Infection 60 60 Chronic Infection 40 40(%) 20 20 Symptomatic Infection 0 0 Birth 1-6 months 7-12 months 1-4 years Older Children and Adults Age at Infection
  184. 184. Possible Outcomes of HBV Infection Acute hepatitis B 95% of infection 3-5% of infant- Chronic HBV adult- acquiredinfection acquired infectionsChronic infections 12-25% in hepatitis 6-15% in 5 Cirrhosi years 5 20-23% in years Hepatocell s 5 years Liver ular failureDeath carcinoma Liver Death
  185. 185. Acute Hepatitis B Virus Infection with Recovery Typical Serologic Course Symptoms HBeAg anti-HBe Total anti-HBcTiter HBsAg IgM anti-HBc anti-HBs 0 4 8 12 16 20 24 28 32 36 52 100 Weeks after Exposure
  186. 186. Progression to Chronic Hepatitis B Virus Infection Typical Serologic Course Acute Chronic (6 months) (Years) HBeAg anti-HBe HBsAg Total anti- HBcTiter IgM anti- HBc 0 4 8 1 1 2 2 2 3 3 5 Years Weeks 6 0 4 8 2 6 2 after Exposure 2
  187. 187. INTERPRETATION OF THE HEPATITIS B PANELTests Results InterpretationHBsAg Negative SusceptibleAnti-HBc NegativeAnti-HBs NegativeHBsAg Negative ImmuneAnti-HBc Negative or PositiveAnti-HBs PositiveHBsAg PositiveAnti-HBc Positive Acute InfectionIgM Anti-HBc PositiveAnti-HBs NegativeHBsAg Positive Chronic InfectionAnti-HBc PositiveIgM Anti-HBc NegativeAnti-HBs NegativeHBsAg Negative Four possible interpretationsAnti-HBc PositiveAnti-HBs Negative
  188. 188. Interpretation HBsAg negative HBcAb positive HBsAb negative1. May be recovering from acute infection.2. May be distantly immune and test is not sensitive enough to detect very low level of HBsAb in serum.3. May be susceptible with a false positive HBcAb.4. May be undetectable level of HBsAg present in the serum and the person is actually a carrier.
  189. 189. Current Treatment Options for HBV Pegylated Interferon alfa (Intron A) Lamivudine (Epivir HBV) Adefovir dipivoxil (Hepsera)
  190. 190. Elimination of Hepatitis B Virus Transmission United States Strategy• Prevent perinatal HBV transmission• Routine vaccination of all infants• Vaccination of children in high-risk groups• Vaccination of adolescents – all unvaccinated children at 11-12 years of age – “high-risk” adolescents at all ages
  191. 191. Hepatitis B Vaccine Infants: several options that depend on status of the mother – If mother HepBsAg negative: birth, 1-2m,6- 18m – If mother HepBsAg positive: vaccine and Hep B immune globulin within 12 hours of birth, 1-2m, <6m Adults – 0, 1, 6 months Vaccine recommended in – All those aged 0-18 – Those at high risk
  192. 192. Hepatitis B High Risk Groups Persons with multiple sex partners or diagnosis of a sexually transmitted disease Men who have sex with men Sex contacts of infected persons Injection drug users Household contacts of chronically infected persons Infants born to infected mothers Infants/children of immigrants from areas with high rates of HBV infection Health care and public safety workers Hemodialysis patients
  193. 193. Estimated Incidence of Acute Hepatitis B United States, 1978-1995 80 HBsAg screening Infant Vaccine of pregnant immunization 70 licensed women recommende recommended d 60 OSHA Rule 50 enactedPopulationCases per Adolescent100,000 40 immunization recommende d 30 20 * Decline among Decline 10 among homosexual men & injecting 0 HCWs drug users 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95* Provisional date Year
  194. 194. Hepatitis B: Passive Immunization Infants of surface antigen positive mothers Exposures to infected blood or infected body fluids in individuals who are unvaccinated, unknown vaccination, or known non- responders. – Ideally within 24 hours – Probably not effective >7days post exposure
  195. 195. Hepatitis B Surveillance Acute Hepatitis B is an urgently reportable condition. It must be reported by phone to DHEC within 24 hours. Chronic Hepatitis B is a reportable condition and must be reported to DHEC within 7 days. Perinatal Hepatitis B is a reportable condition and must be reported to
  196. 196. Hepatitis B Surveillance Acute,chronic, and perinatal hepatitis B must be reported to CDC. CDC and S.C. conducts enhanced surveillance on acute and perinatal hepatitis B cases.
  197. 197. Hepatitis D (Delta) Virusantigen HBsAg RNA
  198. 198. Hepatitis D Structure Defective virus that requires co- infection with hepatitis B for replication Enveloped with SS RNA genome Onlyantigen encoded in the delta antigen
  199. 199. Hepatitis D virus genome
  200. 200. Hepatitis D Virus Modes of Transmission• Percutaneous exposures – injecting drug use• Permucosal exposures – sex contact
  201. 201. Geographic Distribution of HDV Infection Taiwan Pacific IslandsHDV Prevalence High Intermediat e Low Very Low No Data
  202. 202. Hepatitis D:Pathogenesis Pathogenesis – Immune mediated – Co-infection- infection with B at the same time (more severe) – Superinfection: acquisition of Hep D in chronically Hep B
  203. 203. Sequelae of Hepatitis D virus
  204. 204. Hepatitis D - Clinical Features• Coinfection –severe acute disease –low risk of chronic infection• Superinfection –usually develop chronic HDV infection –high risk of severe chronic liver disease
  205. 205. HBV - HDV Coinfection Typical Serologic Course Symptoms ALT Elevated anti-Titer IgM anti- HBs HDV HDV RNA HBs Ag Total anti-HDV Time after Exposure
  206. 206. HBV - HDV Superinfection Typical Serologic Course Jaundice Symptoms Total anti-HDV ALTTiter HDV RNA HBsAg IgM anti-HDV Time after Exposure
  207. 207. Hepatitis C Virus
  208. 208. Hepatitis D - Prevention• HBV-HDV Coinfection Pre or postexposure prophylaxis to prevent HBV infection• HBV-HDV Superinfection Education to reduce risk behaviors among persons with chronic HBV infection• Alpha interferon may help reduce hepatocellular damage
  209. 209. Hepatitis C viruses particles and genome
  210. 210. Hepatitis C Structure and Classification Member of the flavivirus family (other members yellow fever and dengue) Enveloped single stranded RNA virus Humans and chimpanzees only known reservoirs 6 serotypes (genotypes) and multiple subtypes based on high variability of envelope glycoproteins
  211. 211. Hepatitis C Virus Infection, United States New infections per year 1985-89 242,000 2001 25,000 Deaths from acute liver failure Rare Persons ever infected (1.8%) 3.9 million (3.1-4.8)* Persons with chronic infection 2.7 million (2.4-3.0)* HCV-related chronic liver disease 40% - 60% Deaths from chronic disease/year 8,000-10,000 *95% Confidence Interval
  212. 212. Estimated Incidence of Acute HCV Infection United States, 1960-2001 140New Infections/100,000 120 100 Decline in injection 80 drug users 60 40 Decline in transfusion recipients 20 0 1960 1965 1970 1975 1980 1985 1989 1992 1995 1998 2001 Year Source: Hepatology 2000;31:777-82; Hepatology 1997;26:62S-65S; CDC, unpublished data
  213. 213. Exposures Known to Be Associated With HCV Infection in the United States Injecting drug use Transfusion, transplant from infected donor Occupational exposure to blood – Mostly needle sticks Iatrogenic (unsafe injections) Birth to HCV-infected mother Sex with infected partner – Multiple sex partners
  214. 214. Sources of Infection for Persons With Hepatitis C Injecting drug use 60% Sexual 15% Transfusion 10% (before screening) Occupational 4% Other 1%* Unknown 10% * Nosocomial; iatrogenic; perinatalSource: Centers for Disease Control and Prevention
  215. 215. Posttransfusion Hepatitis C 30 All volunteer donors HBsAg % of Recipients Infected 25 20 15 Donor Screening for HIV Risk Factors Anti-HIV 10 ALT/Anti-HBc Anti-HCV 5 Improved HCV Tests 0 1965 1970 1975 1980 1985 1990 1995 2000 YearAdapted from HJ Alter and Tobler and Busch, Clin Chem 1997
  216. 216. Injecting Drug Use and HCV Transmission Highly efficient – Contamination of drug paraphernalia, not just needles and syringes Rapidly acquired after initiation – 30% prevalence after 3 years – >50% after 5 years Four times more common than HIV
  217. 217. Occupational Transmission of HCV Inefficient by occupational exposures Average incidence 1.8% following needle stick from HCV-positive source – Associated with hollow-bore needles Case reports of transmission from blood splash to eye; one from exposure to non-intact skin Prevalence 1-2% among health care workers – Lower than adults in the general population – 10 times lower than for HBV infection
  218. 218. Perinatal Transmission of HCV Transmission only from women HCV- RNA positive at delivery – Average rate of infection 6% – Higher (17%) if woman co-infected with HIV – Role of viral titer unclear No association with – Delivery method – Breastfeeding Infected infants do well – Severe hepatitis is rare
  219. 219. Sexual Transmission of HCV Occurs, but efficiency is low – Rare between long-term steady partners – Factors that facilitate transmission between partners unknown (e.g., viral titer) Accounts for 15-20% of acute and chronic infections in the United States Partner studies – Low prevalence (1.5%) among long-term partners  infections might be due to common percutaneous exposures (e.g., drug use), BUT – Male to female transmission more efficient  more indicative of sexual transmission
  220. 220. Household Transmission of HCV Rare but not absent Could occur through percutaneous/mucosal exposures to blood – Contaminated equipment used for home therapies  IV therapy, injections – Theoretically through sharing of contaminated personal articles (razors, toothbrushes)
  221. 221. Other Potential Exposures to Blood No or insufficient data showing increased risk – intranasal cocaine use, tattooing, body piercing, acupuncture, military service No associations in acute case-control or population-based studies Cross-sectional studies in highly selected groups with inconsistent results – Temporal relationship between exposure and infection usually unknown – Biologically plausible, but association or causal relationship not established
  222. 222. Hep C: Pathogenesis Blood-borne pathogen that infects hepatocytes Much like Hep A and B, liver damage and clinical illness due more to elicited immune response as opposed to direct cytopathic effect of the virus Likely cytotoxic T cells that mediate most of the damage Like other chronic liver diseases (Hep B and chronic alcoholism), can cause hepatocellular ca (HCC) Some genotypes more amenable to therapy- i.e. 3a
  223. 223. Features of Hepatitis C Virus InfectionIncubation period Average 6-7 weeks Range 2-26 weeksAcute illness (jaundice) Mild (<20%)Case fatality rate LowChronic infection 60%-85%Chronic hepatitis 10%-70% (most asx) Age-Cirrhosis related <5%-20%Mortality from CLD 1%-5%
  224. 224. Hepatitis C: Clinical Features Acuteinfection asymptomatic in over 80% of patients, when present, acute illness usually mild – Acute symptoms include jaundice, nausea, abdominal pain, loss of appetite, dark urine
  225. 225. Hepatitis C: Extrahepatic Manifestations  Seen with chronic infection  ? Due to immune complexes  Extrahepatic manifestations – Essential mixed cryoglobulinemia (vasculitis, skin rash, fatigue) – Porphyria cutanea tarda – Membranoproliferative glomerulonephritis – ?Diabetes mellitus – Other autoimmune disease – ?Lymphoma
  226. 226. Porphyria cutanea tarda
  227. 227. Chronic Hepatitis C Factors Promoting Progression or Severity Increased alcohol intake Age > 40 years at time of infection HIV co-infection Other – Male gender – Chronic HBV co-infection
  228. 228. Serologic Pattern of Acute HCV Infection with Recovery anti- Symptoms +/- HCV HCV RNATiter ALT Norm 0 1 2 3 al 5 6 1 2 3 4 4 Mont Year Time after Exposure hs s
  229. 229. Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection anti- Symptoms +/- HCV HCV RNA Titer ALT Norm 0 1 2 3 4 al 6 1 2 3 4 5 Mont Year Time after Exposure hs s
  230. 230. Hepatitis C: Diagnosis ELISA-a serological test which is usually. positive within 2-5 months after infection – 3rd generation assays now 99% specific and sensitive Confirmatory testing – PCR (positive 1-2 weeks post infection) both quantitative and qualitative (I.e. ye/no) available – RIBA (recombinant immunoblot assay)- looks for 2 or more antibodies to HCV viral antigens Genotype testing done when treatment anticipated
  231. 231. HCV Testing Routinely RecommendedBased on increased risk for infection Ever injected illegal drugs Received clotting factors made before 1987 Received blood/organs before July 1992 Ever on chronic hemodialysis Evidence of for exposure managementBased on need liver disease Healthcare, emergency, public safety workers after needle stick/mucosal exposures to HCV-positive blood Children born to HCV-positive women
  232. 232. HCV Infection Testing Algorithm for Diagnosis of Asymptomatic Persons Screening Test for Negative STOP Anti-HCV Positive OR RIBA for Negative NAT for HCV Anti-HCV RNA Negative Indeterminate Positive Positive Additional Laboratory Medical STOP Evaluation (e.g. PCR, ALT) Evaluation Negative PCR, Positive PCR, Normal ALT Abnormal ALTSource: MMWR 1998;47 (No. RR 19)
  233. 233. Medical Evaluation and Management for Chronic HCV Infection Assess for biochemical evidence of CLD Assess for severity of disease and possible treatment, according to current practice guidelines – 40-50% sustained response to antiviral combination therapy (peg interferon, ribavirin) – Vaccinate against hepatitis A Counsel to reduce further harm to liver – Limit or abstain from alcohol
  234. 234. Hepatitis C Therapy Standard of care is pegylated interferon alpha and ribavirin Many barriers to treatment as the above regimen is difficult to take and has many systemic side effects (fatigue, myalgias, depression, anemia to name a few) Overall response rate to treatment
  235. 235. Postexposure Management for HCV IG, antivirals not recommended for prophylaxis Follow-up after needlesticks, sharps, or mucosal exposures to HCV-positive blood – Test source for anti-HCV – Test worker if source anti-HCV positive  Anti-HCV and ALT at baseline and 4-6 months later  For earlier diagnosis, HCV RNA at 4-6 weeks – Confirm all anti-HCV results with RIBA Refer infected worker to specialist for medical evaluation and management
  236. 236. Hepatitis E Virus
  237. 237. Hepatitis E Non-enveloped single stranded RNA virus Resembles calicivirus or Norwalk agent Similarillness to Hep A except high mortality in pregnant women
  238. 238. Geographic Distribution of Hepatitis EOutbreaks or Confirmed Infection in >25% of Sporadic Non-ABC Hepatitis
  239. 239. Hepatitis E - Epidemiologic Features• Most outbreaks associated with fecally contaminated drinking water• Minimal person-to-person transmission• U.S. cases usually have history of travel to HEV-endemic areas
  240. 240. Hepatitis E - Clinical Features• Incubation period: Average 40 days Range 15-60 days• Case-fatality rate: Overall, 1%-3% Pregnant women, 15%-25%• Illness severity: Increased with age• Chronic sequelae: None identified
  241. 241. Hepatitis E Virus Infection Typical Serologic Course Symptoms ALT IgG anti- HEV IgM anti-Titer HEV Virus in stool 0 1 2 3 4 5 6 7 8 9 1 1 1 1 Weeks after Exposure 0 1 2 3
  242. 242. Prevention and Control Measures for Travelers to HEV-Endemic Regions• Avoid drinking water (and beverages with ice) of unknown purity, uncooked shellfish, and uncooked fruit/vegetables not peeled or prepared by traveler• IG prepared from donors in Western countries does not prevent infection• Unknown efficacy of IG prepared from donors in endemic areas• Vaccine?
  244. 244. „FLU‟ True influenza – influenza virus A or influenza virus B (or influenza virus C infections - much milder) Febrilerespiratory disease with systemic symptoms caused by a variety of other organisms often inaccurately called „flu‟
  245. 245. South Carolina 1996-1997 DHEC bulletin malathia influenzae per le stel no virus CULTURE RESULTS influenza A influenza B
  246. 246. THE IMPACT OF INFLUENZA PANDEMICSDeaths: 1918-19 Spanish flu 500,000 US 20,000,000 world 1957-58 Asian flu 70,000 US
  247. 247. THE IMPACT OF INFLUENZA  In the United States, on average:  36,000 deaths per year  114,000 hospitalizations per yearCDC: MMWR 53:8-11, 2004
  248. 248. THE IMPACT OF INFLUENZA recently some increase in morbidity and mortality - possible factors? – more elderly people – CF patients live longer – more high risk neonates – more immunosuppressed patients
  249. 249. ORTHOMYXOVIRUSESpleomorphicinfluenza types A,B,Cfebrile, respiratory illness with systemic symptoms
  250. 250. ORTHOMYXOVIRUSES HA - hemagglutinin NA - neuraminidase helical nucleocapsid (RNA plu NP protein) lipid bilayer membrane polymerase complex M1 proteintype A, B, C : NP, M1 proteinsub-types: HA or NA protein
  252. 252. NORMAL TRACHEAL MUCOSA 3 DAYS POST-INFECTION 7 DAYS POST-INFECTIONLycke and Norrby Textbook of Medical Virology 1983
  253. 253.  DECREASED CLEARANCE RISK BACTERIAL INFECTION VIREMIA RARE Lycke and Norrby Textbook of Medical Virology 1983
  255. 255. An immunological diversion INTERFERON
  256. 256. INTERFERONtimecourse of virus production will vary fro
  257. 257. INTERFERON
  258. 258. INTERFERON antiviral stateantiviral state antiviral state antiviral state
  259. 259. INTERFERON antiviral stateantiviral state antiviral state antiviral state
  260. 260. INTERFERON antiviral stateantiviral state antiviral state antiviral state
  262. 262. TYPES OF INTERFERONTYPE I Interferon-alpha (leukocyte interferon, about 20 related proteins) - leukocytes, etc Interferon-beta (fibroblast interferon) - fibroblasts, epithelial cells, etc
  263. 263. INDUCTION OF INTERFERONinterferon-alpha and interferon-beta - viral infection (especially RNA viruses), double stranded RNA, certain bacterial components - strong anti-viral propertiesinterferon-gamma - antigens, mitogenic stimulation lymphocytes
  264. 264. INTERFERON induces variety of proteins in target cells manyconsequences, not all fully understood
  266. 266. interferon-alpha, interferon-beta interferon receptor induction of induction of induction of2’5’oligo A synthase ribonuclease L protein kinase R (PKR) ds RNA 2’5’oligo A ds RNA activated activated activated2’5’oligo A synthase ribonuclease L protein kinase R ATP ATP phosphorylated 2’5’oligo A initiation factor (eIF- 2) mRNA degraded inhibition of protein synthesi
  267. 267. interferons only made when needed
  270. 270. THERAPEUTIC USES OF INTERFERONS ANTI-VIRAL – e.g. interferon-alpha is currently approved for certain cases of acute and chronic HCV and chronic HBV MACROPHAGE ACTIVATION – interferon-gamma has been tried for e.g. lepromatous leprosy, leishmaniasis, toxoplasmosis ANTI-TUMOR – have been used in e.g. melanoma, Kaposi‟s sarcoma, CML MULTIPLE SCLEROSIS
  271. 271. Viral response to host immune systemViruses may : block interferon binding inhibit function of interferon-induced proteins inhibit NK function interfere with MHC I or MHC II expression block complement activation inhibit apoptosis etc!
  274. 274. PROTECTION AGAINST RE-INFECTION IgG and IgA – IgG less efficient but lasts longer antibodies to both HA and NA important – antibody to HA more important (can neutralize)
  277. 277. PULMONARY COMPLICATIONS CROUP (YOUNG CHILDREN) PRIMARY INFLUENZA VIRUS PNEUMONIA SECONDARY BACTERIAL INFECTION – Streptococcus pneumoniae – Staphlyococcus aureus – Hemophilus influenzae
  278. 278. NON-PULMONARY COMPLICATIONS myositis (rare, > in children, > with type B) cardiac complications recent studies report encephalopathy – 2002/2003 season studies of patients younger than 21 yrs in Michigan - 8 cases (2 deaths) liver and CNS – Reye‟s syndrome peripheral nervous system – Guillian-Barré syndrome
  279. 279. Reye‟s syndrome liver - fatty deposits brain - edema vomiting, lethargy, coma risk factors – youth – certain viral infections (influenza, chicken pox) – aspirin
  280. 280. Guillian-Barré syndrome 1976/77 swine flu vaccine – 35,000,000 doses  354 cases of GBS  28 GBS-associated deaths  recent vaccines much lower risk
  282. 282. DIAGNOSIS ISOLATION – NOSE, THROAT SWAB – TISSUE CULTURE OR EGGS SEROLOGY PCR RAPID TESTS provisional - clinical picture + outbreak
  283. 283. ANTIGENIC DRIFT HA and NA accumulate mutations – RNA virus immune response no longer protects fully sporadic outbreaks, limited epidemics
  284. 284. ANTIGENIC SHIFT “new” HA or NA proteins pre-existing antibodies do not protect may get pandemics
  285. 285. INFLUENZA A PANDEMICSRyan et al., in Sherris MedicalMicrobiology
  286. 286. where do “new” HA and NA come from?13 types HA 9 types NA – all circulate in birds pigs – avian and human
  287. 287. Where do “new” HA and NA come from?
  288. 288. Where do “new” HA and NA come from- can „new‟ bird flu directly infect humans? Bird flu H5N1?
  289. 289. why do we not have influenza B pandemics? so far no shifts have been recorded no animal reservoir known
  290. 290. SURVEILLANCECDC/Katherine Lord
  291. 291. actual percentage of deaths(CDC MMWR July 1, 2005 / 54(25);631-634)
  292. 292. % typed cases100 90 80 70 60 H1N1 50 H3N2 40 B 30 20 10 0 00/01 01/02 02/03 03/04 04/05 influenza
  293. 293. VACCINE „BEST GUESS‟ OF MAIN ANTIGENIC TYPES – CURRENTLY  type A - H1N1  type A - H3N2  type B  each year choose which variant of each subtype is the best to use for optimal protection
  294. 294. VACCINE inactivated egg grown some formulations licensed for children reassortant live vaccine approved 2003 – for healthy persons (those not at risk for complications from influenza infection) ages 5-49 years
  295. 295. CDC
  298. 298. VIRAL AGENTS CAUSING GASTROENTERITIS1. Rotavirus2. Enteric adenoviruses3. Calicivirus4. Astrovirus
  299. 299. ROTAVIRUSFamily ReoviridaeGenus Rotavirus
  300. 300. ROTAVIRUS First isolated in 1973 from children with diarrhea EM identification from duodenal biopsies Human and animal strains
  303. 303. STRUCTURAL FEATURES OF ROTAVIRUS 60-80nm in size Non-enveloped virus EM appearance of a wheel with radiating spokes Icosahedral symmetry Double capsid Double stranded (ds) RNA in 11 segments
  304. 304. STRUCTURE Double capsid (outer and innercapsid) Core with genome Capsid is cleaved by trypsin toform ISVP- infective sub-viral particle
  306. 306. ROTAVIRUS- ultrastructure
  308. 308. VIRAL STRUCTURAL PROTEINS (VP)  Outer structural proteins - VP7 and VP4 VP7 - Glycoprotein VP4 - protease-cleaved, P protein, viral hemagglutinin; forms spikes from the surface  Inner core structural proteins VP 1, 2, 3, 6  VP6 is an important antigenic
  309. 309. STRUCTURE
  310. 310. CLASSIFICATION Groups- 7 Groups (A through G) based on VP6 differences Group A is the most common and has 2subgroups
  311. 311. CLASSIFICATION (contd.) Serotypes based on viral capsid proteins 14 G serotypes based on G protein (VP 7) differences 20 P serotypes based on P protein (VP4) Common PG combinations are:- P8G1, P8G2, P4G2, P8G4
  312. 312. CLASSIFICATION (contd.) Electropherotypes are based on the mobility of RNA segments by PAGE Useful in epidemiologic studies
  313. 313. ROTAVIRUS- PROPERTIES Virus is stable in the environment Relatively resistant to handwashing agents Susceptible to disinfection with 95% ethanol, „Lysol‟, formalin
  314. 314. PATHOGENESIS Targeted host cells - mature enterocytes lining the tips of intestinal villi Intermediate/infective sub-viral particle (ISVP) produced through proteolysis Enter host cell by endocytosis Virus replicates in the host cell cytoplasm
  315. 315. REPLICATION mRNA transcription with viral RNA polymerase Capsid proteins formed mRNA segments formed, assembled into immature capsid mRNA replicated to form double stranded RNA genome
  316. 316. HISTOPATHOLOGY Mature enterocytes lining the tips of intestinal villi are affected Villous atrophy and blunting Death of the mature enterocytes
  317. 317. HISTOPATHOLOGY Infiltration of lamina propria with mononuclear cells Repopulation of the villous tips with immature secretory cells [crypt hyperplasia]
  319. 319. EPIDEMIOLOGYA major cause of diarrhea- associated hospitalizations and deaths Seroprevalence studies show that antibody is present in most by age 3y.
  321. 321. WORLDWIDE DISTRIBUTION OF ROTAVIRUS (Source- Centers for Disease Control and Prevention)
  322. 322. EPIDEMIOLOGY - USA 2.7- 3.5 million affected each year Physician visits ~ 500,000/year 50,000-70,000 hospitalizations/year 20-40 deaths/year Cases with dehydration ~ 1-2.5% Economic impact
  323. 323. EPIDEMIOLOGY Age- 4mo - 2 years Protection of younger infants through through transplacental antibody transfer Asymptomatic infections are common, especially in adults Nosocomial infections Outbreaks Severe Disease young, immunocompromised
  324. 324. Epidemiology (contd.) Seasonality Winter months (Nov. through May in US) Gradual spread W to E Year-round in the tropics Incubation period - thought to be <4 days
  325. 325. EPIDEMIOLOGY(Source- Centers for Disease Control and Prevention)
  326. 326. SEASONAL SPREAD - U.S. (Source- CDC)
  327. 327. TRANSMISSION Mainly person to person via fecal-oral route Fomites Foodand water-borne spread is possible Spreadvia respiratory route is speculated
  328. 328. EPIDEMIOLOGY - spread Contagious from before onset of diarrhea to a few days after end of diarrhea Large amounts of viral particles are shed in diarrheal stools Infective dose is only 10-100 pfu
  329. 329. EPIDEMIOLOGY Differences in Groups Group A infections are most common Group B has been associated with outbreaks in adults in China Group C is responsible for sporadic cases of diarrhea in infants around the world
  330. 330. CLINICAL CASE A 22 month old female is admitted to the pediatric ward for cough and fever up to 103 F. Chest X ray shows left lower lobe pneumonia. She is being treated with intravenous Ceftriaxone and her fever is gradually improving. On hospital day #5, she develops diarrhea with 4 watery stools and a fever of 102 F. Stool studies showed no traces of blood and no fecal leukocytes. Further studies are pending.
  331. 331. CLINICAL FEATURES Incubation period - thought to be <4 days Fever- can be high grade (>102 F in 30%) Vomiting, nausea precede diarrhea Diarrhea • usually watery (no blood or leukocytes) • lasts 3-9 days • longer in malnourished and immune deficient individuals. • NEC and hemorrhagic GE seen in neonates
  332. 332. MECHANISM OF DIARRHEA Watery diarrhea due to net secretion of intestinal fluid Activation of the enteric nervous system Role of NSP4 peptide regions as an enterotoxin
  333. 333. CLINICAL FEATURES (contd.) Dehydration is the main contributor to mortality. Secondary malabsorption of lactose and fat, and chronic diarrhea are possible
  334. 334. DIAGNOSIS Antigen detection in stool by ELISA, LA (for Group A rotavirus) EM- non-Group A viruses also Culture- Group A rotaviruses can be cultured in monkey kidney cells Serology for epidemiologic studies
  335. 335. TREATMENT AND PREVENTION Treatment Supportive- oral, IV rehydration Prevention Handwashing and disinfection of surfaces
  336. 336. VACCINE Live tetravalent rhesus-human reassortant vaccine (Rotashield) Licensed for use in August 1998 Removed from the market in October 1999 due to risk of intussusception Cases were seen 3-20 days after vaccination Approx. 15 cases/1.5 million doses
  337. 337. NEWER VACCINES Rotarix Rota Teq Monovalent G1P8 oral  Pentavalent Human- vaccine bovine reassortant vaccine Efficacy for prevention  Efficacy for prevention of severe disease = of severe disease = 85% 100%
  339. 339. GASTROENTERITIS DUE TO ADENOVIRUS  Types 40, 41  Belong to serogroup F
  340. 340. Diarrhea due toEnteric Adenovirus Age <4 years Year round Spread via fecal-oral route
  341. 341. CLINICAL FEATURES- Adenovirus gastroenteritis Incubation period 3 -10 days Diarrhea lasts for 10 -14 days Can also cause intussusception, mesenteric adenitis, appendicitis
  342. 342. DIAGNOSIS Enteric adenoviruses Isolation requires special media- Graham 293 ELISA for rapid detection is available
  344. 344. HUMAN CALICIVIRUSES (HuCV)• Family Caliciviridae• Non-enveloped RNA viruses with ss RNA• 27-35 nm in size• Contain a single capsid protein