1. Dr. Jing Qian Dept. of Medical Microbiology and Parasitology Medical school, Zhejiang university Email: jingqian@zju.edu.cnjingqian@zju.edu.cn 2014.5.29 Lectures for biomedical students Hepatitis Virus, Hemorrhagic fever viruses, Arbovirus 肝炎病毒、出血热病毒和虫媒 病毒

2. 肝炎和肝炎病毒 Hepatitis & Hepatitis Virus

3. Hepatitis- Inflammation of the liver, destruction of hepatocytes Acute Infection-Icteric ( 黄疸的 ) phase Chronic Infection- May progress to: Hepatic fibrosis, cirrhosis, liver failure Increased risk of hepatocellular carcinoma Definitions and Introduction

4. Icteric phase/Jaundice * Skin, sclera ( 巩膜 ), underneath tongue

5. A “Infectious” “Serum” Viral hepatitis Enterically transmitted Parenterally transmitted F, G, TTV ? other E NANB BD C Viral Hepatitis - Historical Perspectives

8. Focus on the viruses – Viral structure, biology – Transmission and epidemiology – Pathogenesis and immunity – Clinical findings – Laboratory diagnosis – Treatment and Prevention HAV HBV HCV HDV HEV

9. Hepatitis A Virus (HAV) “ Epidemic jaundice ” “ Infectious hepatitis ” (1912) Hepatitis A

10. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Extremely stable capsid mature particle Immature particle http ： //www.ncbi.nlm.nih.gov/books

11. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Positive-sense, single-stranded RNA genome, 7500 nt NCR: 非编码区 IRES: 内部核糖体进入位点 3B-VPg/primer protein 病毒基因组连接蛋白

12. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Positive-sense, single-stranded RNA genome Resistance: Stronger than enterovirus, resistant to detergents, acid (pH 1.0 for 2h), 60 ℃ for 1h ， survive for months in fresh water and salt water one serotype

13. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Positive-sense, single-stranded RNA genome Extremely stable capsid Resistance: Stronger than enterovirus, resistant to detergents, acid (pH 1.0 for 2h), 60 ℃ for 1h ， survive for months in fresh water and salt water 1 serotype and 7 genotypes 1 个血清型 主要抗原决定簇 VP1 7 个基因型（ VP1 ）

14. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Positive-sense, single-stranded RNA genome Extremely stable capsid Resistance: Stronger than enterovirus, resistant to detergents, acid (pH 1.0 for 2h), 60 ℃ for 1h ， survive for months in fresh water and salt water 1 serotype and 7 genotypes Animal model and cell culture

15. Biological properties Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus 27 nm naked (non-enveloped) icosahedral capsid Positive-sense, single-stranded RNA genome Extremely stable capsid Resistance: Stronger than enterovirus, resistant to detergents, acid (pH 1.0 for 2h), 60 ℃ for 1h ， survive for months in fresh water and salt water 1 serotype and 7 genotypes Animal model and cell culture

16.  Fecal-Oral spread  contaminated water or food ( shellfish, green onions)  Risk factors: poor sanitation and hygiene, overcrowding, daycare Transmission of HAV

17. Epidemiology

19.  Enters bloodstream through gastrointestinal epithelium  Replicates in hepatocytes and Kupffer cells  Released by exocytosis, not cell lysis in cultured cells  It is likely that attack by cytotoxic T cells causes the demage to the hepatocytes  Goes into bile, intestine, excreted in feces  Shedding of virus for 10 days prior to any symptoms Pathogenesis Not fully understood

20. Immunity 无论显性感染或隐性感染 均能产生抗 -HAV 的 IgM 和 IgG 抗体 抗 -HAV 的 IgM 在急性期和恢复早期出现 阳性可作为甲肝的确诊依据 抗 -HAV 的 IgG 在恢复后期出现 有保护作用，维持终身

21.  May be mild to asymptomatic in children  Abrupt onset of disease in adults  “Self-limited”-controlled by immune system  Low overall mortality from fulminant ( 暴发性的 ) hepaptitis  Higher risk with simultaneous liver disease such as cirrhosis due to alcohol or chronic Hepatitis B or C Clinical findings Acute Hepatitis A Infection

23. Clinical syndrome Detection of anti-HAV specific antibodies IgM titer in acute infection, positive for 4-6 months IgG titer present for decades Research testing -Virus feces by electron microscopy (no cell culture available) RNA PCR Diagnosis of HAV

24. No antiviral therapy is available Treatment and Prevention of HAV Passive immunization Polyclonal anti-HAV antibodies that persist for 6 months Only effective for 2 weeks prior to exposure Expensive, painful, IM injection site reactions Active immunization a killed HAV vaccine a live attenuated HAV vaccine Sanitation Avoidance of questionable food and water in endemic regions

25. Hepatitis B virus (HBV) Baruch Blumberg, 1963: ‘ Australian antigen - Au‘. 1968: Au was a viral antigen = HBsAg (surface antigen)HAA Dane, 1970: Discovered 42nm 'Dane particles ‘ HBcAg (core antigen). 1973: HBeAg discovered (endogenous antigen = a truncated version of HBcAg). 1983: members of Hepadnaviridae

26. Biological properties

27. Electron microscopy of hepatitis B virus-positive serum reveals 3 morphologically distinct forms of particles spherical particle (HBsAg) Dane particle (complete virion ) tubular particle

28. (capsid) detergent HBeAg ( also found in the soluble forms in virus-positive sera) Dane particle-structure

29. Dane particle-antigen HBsAg surface (coat) protein “ a ” antigenic determinant ： 124- 147aa 4 phenotypes adw, adr, ayw, ayr HBcAg inner core protein a single serotype HBeAg secreted protein function unknown

30. 5’- TTCACCTCTGCDR 1: direct repeat (+) DR 2: direct repeat (-) DNA POLYMERASE LEADING SEQUENCE cohesive terminus 粘性末端 Genetic structure dsDNA (-) 3200 nt (+) 50-90% of (-)

31. Genetic structure(L - ) 4 open reading frames S, C, P, X regions P region overlap with S C P

32. Genetic structure(L - ) S region: capsid protein –S gene: HBsAg –preS 1 gene: preS 1 Ag –preS 2 gene: preS 2 Ag SP1 promoter ， 2.4kb mRNA ， large protein/ 大蛋白 SP2 promoter ， 2.1kb mRNA ， middle protein/ 中蛋白 & main protein 主蛋白 HBV envelope proteins ： − 主蛋白： HBsAg ， S gene ， 226aa − 中蛋白： HBsAg+PreS2Ag ， S+PreS2 gene ， 281aa − 大蛋白： HBsAg+PreS1Ag+PreS2Ag ， S+PreS1 gene+PreS2 gene,400aa

33. Genetic structure(L - ) C region –C gene: HBcAg –preC gene + C gene : HBeAg

34. Genetic structure(L - ) P region: DNA polymerase (RDDP,DDDP, RNase H)

35. Genetic structure(L - ) X region: HBxAg 0.8 kb mRNA trans-activation factor

36. Genetic structure(L - ) 4 open reading frames S region: capsid protein –S gene: HBsAg –preS 1 gene: preS 1 Ag –preS 2 gene: preS 2 Ag C region –C gene: HBcAg –preC gene + C gene : HBeAg P region: DNA polymerase X region: HBxAg (trans-activation factor) (RDDP,DDDP, RNase H)

37. 1. absorption, uncoating Pre S1, Pre S2 2. L-DNA → dsDNA DDDP 3. dsDNA(L-)→ mRNADDRP 3.5kb, 2.4kb, 2.1kb, 0.7-0.9kb 3.5kb mRNA as template for DNA replication (pre-genome) 4. mRNA→ protein 3.5kb mRNA→ inner capsid proteins, DNA polymerase 2.4kb mRNA, 2.1kb mRNA→ outer capsid proteins 0.8kb mRNA→HBxAg 5. packaging of pre-genome and inner caspid & mRNA(pre-genome) →DNA(-) 6. DNA(-) → DNA(+) RNase H 7. virion packaging and release budding/exocytosis replication

38. Genotype Variation >=8% nt seq A-H Genotype vs. serotype Aadw Badw Cadr, adw, ayr Dayw

39. Variation HBV DNA polymerase: no proof-reading PreS/S gene Prec/C gene “a” eiptope mutation (nt in S gene encode for 145aa, 126aa ) e minus (A-G at1896nt in PreCgene ) e supression(1762/1764 ntmuationin promoter of PreCgene)

40. Isolation and culture Animal models: -Chimpanzee -Duck Cell culture: not available In vitro transfection

41. Resistance Resistant to low temperature, dry, UV, 70% ethanol, ethyl ether, chloroform, phenol Dis-infected by 100 °C 10min, pH 2.4 6h Sensitive to detergent

42. Pathogenesis

43. Transmission Routes

44. Concentration of Hepatitis B Virus in Various Body Fluids HighModerate Low/Not Detectable bloodsemenurine serumvaginal fluidfeces wound exudatessalivasweat tears breastmilk

45. Epidemiology Estimated 300 million HBV carriers worldwide High prevalence areas 10-20% – China, southeast Asia, sub- Saharan Africa Intermediate prevalence areas 2-5% – Mediterranean, Middle East, Japan, Central and S. America Low prevalence areas 0.1-2% – N. America, Europe, Australia, New Zealand

46. Natural History of HBV Infection

47. Balance between virus clearance and liver injury

48. Virions released by exocytosis, not cytolytic Ab-mediated immune responses – Type II hypersensitivity – Type III hypersensitivity Cell-mediated immune responses – Type IV hypersensitivity Immunopathogenesis

49. – Immature responses – mild symptoms, chronic infection (90%) HBV infection in infants and young children

50. HBV mutation and disease HBV pre-C mutation and chronic hepatitis HBV C mutation and fulminant 暴发性的 hepatitis

51. HBV mutation and drug resistance

52. Immune responses

53. CTL mediated anti-virus immune responses vs. liver injury

54. Antigen/Antibody Responses HBsAg & anti-HBs HBsAg –major sign of HBV infection –acute infection –chronic infection or carrier –Hepatocellular cancer patient anti-HBs –neutralization antibody HBcAg & anti-HBc HBcAg –not detectable in the serum anti-HBc, IgM – virus replication/infectious – acute infection – acute episode during chronic infection – transient response anti-HBc, IgG –do not protect individuals – chronic infections – last for a long time HBeAg & anti-HBe HBeAg –virus replication /infectious –early stage after infection anti-HBc –the sign of better prognosis –variation: ending codon in pre-C

55. Acute Hepatitis B Virus Infection with Recovery Typical Serologic Course

56. Progression to Chronic Hepatitis B Virus Infection Typical Serologic Course

57. Interpretation of Serologic Markers of HBV infection 大三阳小三阳

58. Prevention and Treatment

59. Treatment Interferon - for HBeAg +vs carriers with chronic active hepatitis. Response rate is 30 to 40%. – alpha-interferon 2b (original) – alpha-interferon 2a (newer, claims to be more efficacious and efficient) Lamivudine - a nucleoside analogue reverse transcriptase inhibitor. Well tolerated, most patients will respond favorably. However, tendency to relapse on cessation of treatment. Another problem is the rapid emergence of drug resistance. Adefovir – less likely to develop resistance than Lamivudine and may be used to treat Lamivudine resistance HBV. However more expensive and toxic Entecavir – most powerful antiviral known, similar to Adefovir Successful response to treatment will result in the disappearance of HBsAg, HBV- DNA, and seroconversion to HBeAg.

61. Prevention Vaccination - highly effective recombinant vaccines are now available. Vaccine can be given to those who are at increased risk of HBV infection such as health care workers. It is also given routinely to neonates as universal vaccination in many countries. Hepatitis B Immunoglobulin - HBIG may be used to protect persons who are exposed to hepatitis B. It is particular efficacious within 48 hours of the incident. It may also be given to neonates who are at increased risk of contracting hepatitis B i.e. whose mothers are HBsAg and HBeAg positive. Other measures - screening of blood donors, blood and body fluid precautions.

62. If you have never had hepatitis B, you can get 3 shots...... and get long lasting protection. 321 Hepatitis B can be prevented!

63. 6 months old Hepatitis B Vaccine Baby Shots for Hepatitis B if the mother has Hepatitis B 1 - 2 months old Hepatitis B Vaccine + Birth H-BIG Hepatitis B Vaccine

64. Hepatitis C virus (HCV) “Non-A Non-B Hepatitis” Identified in 1989 by molecular methods

65. Biological Properties Related to flaviviruses 黄病毒 and pestiirvuses 瘟病毒 40-60 nm particle, spherical an enveloped virion Genome: (+)ss RNA Six genotypes, regional prevalence Great heterogeneity, many “quasispecies 准种 ”

66. Biological Properties icosahedral, positive strand RNA viruses gain an envelope from host cell virus particle is about 30 to 60nm across

67. Biological Properties genome of 9,600 bases codes for ten proteins viral RNA does not have a 5’ cap or 3’ poly A tract translation of the viral RNA is mediated by the internal ribosome entry site (IRES)

68. Biological Properties HCV binds to either the CD81 antigen or low density lipoprotein (LDL) receptor on hepatocytes via its E2 glycoprotein. There is also some evidence that it may bind to glycosaminoglycans.

69. Biological Properties Highly varied genome of HCV

70. Biological Properties Highly varied genome of HCV Genotypes & subtypes Fig 1 Phylogenetic tree of HCV NS5B sequences. Nucleotide sequences for positions 7975–8196 (numbered from the polyprotein AUG initiation codon) of NS5B were analyzed using the program Phylipas described previously (76). Major branches are labeled with the type number, and minor branches with letters indicating the subtype The variant “10a” can be considered as a subtype of type 3, and the variants “7a”, “7b”, “8a,” and so forth, as subtypes of type 6 (2,3).

71. Biological Properties Highly varied genome of HCV

72. Biological Properties Highly varied genome of HCV Quasispecies& strains Quasispecies 准种：同一感染者体内同时存在同一基因亚型的不同变异株

73. Transmission Parenteral – Injected drug use – Blood transfusions (rare since screening in 1990) – Nosocomial ( 医院的 ) – Efficiency of sexual transmission is relatively low – Perinatal ( 围产期 ) risk 5%

74. Epidemiology

75. Pathogenesis Not fully understood Prolonged cell-associated state Likely low level chronic cell-mediated host immune response Progression to hepatic fibrosis and cirrhosis More severe disease progression – Alcohol – HIV co-infection

76. Acute Infection Vast majority are asymptomatic Few cases of symptomatic acute hepatitis Very rare or nonexistent fulminant cases Very high rate of chronic infection

78. Diagnosis Acute infection – HCV RNA in serum, liver biopsy – HCV Ab negative Chronic Infection – HCV Ab positive (not protective) – HCV RNA in serum, liver biopsy Virus can not be cultured

79. Prevention No vaccine available No immune globulin Behavioral interventions to reduce risk Treatment: Recombinant IFN-αalone or with ribavirin

80. Hepatitis D virus (HDV)

81. Hepatitis D “Delta Agent” Defective virus similar to plant viruses – Small single-stranded circular RNA genome – Single HDV antigen – Lipid envelope from HBV, HBsAg needed for packaging Depends on HBV for life cycle – Co-infection with acute HBV – Superinfection in chronic HBV Replicates very efficiently in hepatocytes

83. Gene structure Hepatitis delta agent. Three RNA forms. Adapted from Wagner and Hewitt.: Basic Virology. Blackwell Publishing

84. Transmission and Epidemiology Parenteral – Injected drug use – Less efficient sexual transmission than HBV Up to 5% of chronic HBV carriers may also carry HDV Varies greatly by region – Endemic in Mediterranean – Rare in the West

85. Epidemiology

86. Pathogenesis of HDV Viral replication causes hepatocyte cell death Additive to HBV-induced host inflammatory response Antibodies to HDVAg not likely to be protective

87. Clinical Consequences of Infection Increases risk of fulminant hepatitis greatly upon co-infection – Estimated 2-20% fulminant cases Increases risk of cirrhosis in chronic infection with HBV – More rapid progression – More likely progression

89. Diagnosis Detection of HDV antigen or antibody Clinical setting – Acute fulminant disease – Chronic co-infection

90. Prevention No vaccine Prevention of HBV Prevention of further exposure risks in HBV chronic infection

91. A 35-year-old man addicted to intravenous drugs has been a carrier of HBs antigen for 10 years. He suddenly develops acute fulminant hepatitis and dies within 10 days. Which one of the following laboratory tests would contribute MOST to a diagnosis: (A)Anti-HBs antibody (B)HBe antigen (C)Anti-HBc antibody (D)Anti-delta virus antibody

92. Hepatitis E virus (HEV) Used to be called “Enteric” or “Epidemic” or “Water-borne” Non-A Non B Hepatitis Identified in India in 1955

93. Non-enveloped virus Calcivirus Biological properties Mature particle Immature particle Single Strand (+) RNA 7.2-7.6 kb

94. Biological properties

95. Pathogenesis Hepatic damage by host immune response No chronic carrier state Acute infection clinical syndrome very similar to HAV, except higher rates in pregnancy Mortality 1-2%, higher than HAV –10-20% in pregnant women –Mechanism unknown Diagnosed by HEV-RNA; anti-HEV Ab, IgG and IgM

96. Transmission and Epidemiology Fecal-Oral transmission, especially from fecally contaminated water Person-to-person transmission Highest incidence in Asia, Africa, Middle East and Central America High incidence among pregnant women with 10-20% mortality

98. Sanitation No vaccine (phase III clinical trial in China) Little known about pre- or post-exposure efficacy of immune globulin No efficacy of immunoglobulin obtained from western populations Prevention

99. Viral Hepatitis - Overview A A B B C C D D E E Source of virus feces blood/ blood-derived body fluids blood/ blood-derived body fluids blood/ blood-derived body fluids feces Route of transmission fecal-oral percutaneous permucosal percutaneous permucosal percutaneous permucosal fecal-oral Chronic infection noyes no Preventionpre/post- exposure immunization pre/post- exposure immunization blood donor screening; risk behavior modification pre/post- exposure immunization; risk behavior modification ensure safe drinking water Type of Hepatitis

100. 出血热和出血热病毒 Hemorrhagic fever viruses & viral hemorrhagic fever

101. 出血热病毒 · 汉坦病毒 HANTAVIRUS 布尼亚病毒科 单负链 RNA 、有包膜 在我国引起流行性出血热（ EHF) 自然宿主啮齿类动物 黑线姬鼠、田鼠、家鼠 唾液、尿、粪便污染 呼吸道、消化道、接触传播 流行季节 10 ～ 12 月

102. 虫媒病毒 Arbovirus (arthropod-borne-viruses) 节肢动物为媒介 transmitted by means of an infected, blood-sucking, arthropod vector (arthropod borne = arboviruses) 单正链 RNA ，有包膜, 20 面体对称 我国主要流行流行性乙型脑炎病毒、登革病毒、森林脑炎病毒 有较广的宿主范围，能在脊椎动物及非脊椎动物体内繁殖，节肢动物 可长期储存和传播病毒 致病具有明显的季节性和地方性，主要引起发热、脑炎、出血热等

104. 流行性乙型脑炎病毒 （ epidemic type B encephalitis virus ） 一、生物学性状 黄病毒属，单正链 RNA 、有包膜、二十面立体对称 二、所致疾病 引起流行性乙型脑炎 动物  蚊  人 储存宿主 幼猪 传播媒介 三带喙库蚊 传播途径 蚊虫叮咬 流行季节 6 ～ 7 月（南方） 8 ～ 9 月（北方）

105. 病毒  蚊子叮咬 毛细血管内皮细胞内增殖  入血 ( 第一次病毒血症） 流感样  3 ～ 7 天   血液播散 ( 第二次病毒血症） 痊愈全身症状   血 - 脑屏障 痊愈 CNS 症状 恢复、死亡、后遗症 三、致病过程

106. 四、抗原性 稳定，只有一个血清型 五、免疫性 依赖体液和细胞免疫 免疫力稳定持久，隐性感染也可获免疫力 六、预防 灭蚊防蚊 ---- 关键 人群免疫 ---- 乙脑死疫苗 幼猪免疫 ---- 乙脑死疫苗