Hepatitis Viruses

1. Medical Virology Hepatitis Viruses Youjun Feng Center for Infection & Immunity, Zhejiang University School of Medicine fengyj@zju.edu.cn

2. • 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

4. • Focus on the viruses – Details of hepatic disease and treatment will be demonstrated in other courses • Understand similarities and differences – Viral structure, biology – Modes of transmission – Pathogenesis – Diagnosis – Prevention • HAV • HBV • HCV • HDV • HEV Cryo-EM

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

6. Biological properties • Picornavirus classified as Enterovirus 72 in 1980s, later classified Heparnavirus • 27 nm naked (non-enveloped) icosahedral capsid(二十面体) • Extremely stable capsid Immature particle Mature particle http //www.ncbi.nlm.nih.gov/books

7. 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 3B-VPg/primer protein NCR: IRES:

8. 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 water and salt water • one serotype and 7 genotypes for 1h survive for months in fresh

9. Transmission of HAV Water/food-borne virus Ø Fecal-Oral spread Ø Contaminated water or food ( shellfish, green onions) Ø Risk factors: Poor sanitation and hygiene, overcrowding, daycare

10. Pathogenesis of HAV ØEnters bloodstreamthrough gastrointestinal epithelium ØReplicates in hepatocytes and Kupffer cells ØReleased by exocytosis, notcell lysis ØGoes into bile, intestine, excreted in feces ØShedding of virus for 10 days prior to any symptoms

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

12. Diagnosis of HAV ELISA 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

13. Prevention of HAV Sanitation Avoidance of questionable food and water in endemic regions 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 InactivatedHAV vaccine Live attenuated HAV vaccine

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

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

16. Biological properties Cryo-EM

17. 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

18. 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

19. Prevention 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

20. 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

21. Biological properties

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

23. Hepatitis B virus. Dane particle and incomplete particles that are found in patient's serum

24. Dane particle • complete 42 nm virion • nucleocapsid – partly double stranded DNA virus, the + strand not complete – DNA polymerase – HBcAg – HBeAg • is also found in the soluble forms in virus-positive sera envelope – lipid bilayer membrane – protein • HBsAg, preS1, preS2 •

25. 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

26. Geneticstructure dsDNA (-) 3200 nt (+) 50-90% of (-) LEADING SEQUENCE DNA POLYMERASE DR 1: direct repeat (+) 5’- TTCACCTCTGC DR 2: direct repeat (-)

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

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

29. Genetic structure(L-) 4 open reading frames S region: capsid protein – S gene: HBsAg – preS1 gene: preS1Ag – preS2 gene: preS2Ag C region – C gene: HBcAg – preC gene + C gene : HBeAg P region: DNA polymerase (RDDP,DDDP, RNase H) X region: HBxAg (trans-activation factor) • • • • •

30. Replication Pre S1, Pre S2 DDDP DDRP 1. absorption, uncoating 2. L-DNA → dsDNA 3. dsDNA(L-)→ mRNA 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

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

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

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

34. Pathogenesis

35. Transmission Routes

36. Concentration of Hepatitis B Virus in Various Body Fluids Low/Not High Moderate Detectable blood semen urine serum vaginal fluid feces wound exudates saliva sweat tears Breast-milk

37. 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

38. Natural History of Chronic HBV Infection

39. Acute HBV Infection • Longer incubation period prior to symptoms • Insidious onset of symptoms rather than abrupt • Only 25% of infected people will manifest the full clinical syndrome of hepatitis • Immune complex disease related to HBsAg – Seen in ~15% – Rash, arthritis, fever, necrotizing vasculitis (polyarteritis nodosum), glomerulonephritis

40. Chronic HBV Infection • Occurs in 5-10% of acute infection – 90% of perinatal – 20-50% of early childhood – 5% of adult • Usually after mild or asymptomatic infection • Source of ongoing transmission • 10% may develop cirrhosis or liver failure, mostly due to chronic active hepatitis

41. 结节性多动脉炎 肾小球性肾炎

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

43. Balance between virus clearance and liver injury

44. Balance between virus clearance and liver injury

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

46. Immune responses

47. Antigen/Antibody Responses HBsAg •surface (coat) protein •4 phenotypes adw, adr, ayw, ayr HBcAg •inner core protein •a single serotype HBeAg •secreted protein •function unknown

48. HBsAg & anti-HBs •HBsAg –major sign of HBV infection –acute infection –chronic infection or carrier –Hepatocellular cancer patient •anti-HBs –neutralization antibody

49. PreS1, PreS2 & anti-PreS1, PreS2 •PreS1, PreS2 – virus replication /infectious –newly infection •anti-PreS1, anti-PreS2 –1st appearance antibody after infection –neutralizing antibody, clearance of virus –do not routinely checked for clinical diagnosis purpose

50. 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