1. Hepatitis Viruses Youjun Feng
Medical Virology
Lecture 10/06
Hepatitis Viruses
Youjun Feng
Center for Infection & Immunity, Zhejiang University School of Medicine

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. • HAV • HBV • HCV • HDV • HEV
• 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. • Picornavirus classified as Enterovirus 72 in 1980s, later classified
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
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7. • Picornavirus classified as Enterovirus 72 in 1980s, later classified
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:
IRES:
3B-VPg/primer protein

8. • Picornavirus classified as Enterovirus 72 in 1980s, later classified
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 bloodstream through gastrointestinal epithelium
ØReplicates in hepatocytes and Kupffer cells
ØReleased by exocytosis, not cell lysis
ØGoes into bile, intestine, excreted in feces
ØShedding of virus for 10 days prior to any symptoms

11. Ø May be mild to asymptomatic in children
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
Ø Low overall mortality from fulminant (
爆发性的
) 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
Inactivated HAV 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. Non-enveloped virus Calcivirus Single Strand (+) RNA 7.2-7.6 kb
Biological properties
Non-enveloped virus
Calcivirus
Single Strand (+) RNA kb
Mature
particle
Immature

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

20. 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

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

22. Biological properties

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

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

25. – partly double stranded DNA virus, the + strand not complete
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

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

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

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

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

30. 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) •

31. Pre S1, Pre S2 Replication DDDP DDRP 1. absorption, uncoating
2. L-DNA → dsDNA
3. dsDNA(L-)→ mRNA
3.5kb, 2.4kb, 2.1kb, kb
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

32. 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 PreC gene )
e suppression(1762/1764 nt muationin promoter of PreCgene)

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

34. Resistant to low temperature, dry, UV, 70% ethanol,
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

35. Pathogenesis

36. Transmission Routes

37. 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

38. 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

41. Natural History of Chronic HBV Infection

42. 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

43. 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

44. 结节性多动脉炎
肾小球性肾炎

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

46. Balance between virus clearance
and liver injury

47. Balance between virus clearance
and liver injury

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

49. Immune responses

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

51. 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

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

53. 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

54. 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

55. –virus replication /infectious –early stage after infection
HBeAg & anti-HBe
•HBeAg
–virus replication /infectious
–early stage after infection
•anti-HBc
–the sign of better prognosis
–variation: ending codon in pre-C

56. Laboratory Diagnosis

57. Laboratory Diagnosis • Acute infection by clinical syndrome
– Minority of infections diagnosed by syndrome
• Acute and chronic infection by patterns of
serology
• HBV DNA assay (DNA hybrid or PCR)
• No cultures performed in clinical evaluation

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

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

60. Interpretation of Serologic Markers of HBV
infection

61. Prevention and Treatment

62. – α-interferon 2b (original)
Treatment
Interferon - for HBeAg +vs carriers with chronic active hepatitis. Response rate is
30 to 40%.
– α-interferon 2b (original)
– α-interferon 2a (newer, claims to be more efficacious and efficient)
Lamivudine (拉米夫定) – 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 (阿德福韦) – less likely to develop resistance than Lamivudine and may be used to
treat Lamivudine resistance HBV. However more expensive and toxic
Entecavir (恩替卡韦) – most powerful antiviral known, similar to Adefovir
Successful response to treatment will result in the disappearance of HBsAg, HBV-
DNA, and seroconversion to HBeAg. •

63. 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 healthcare 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

64. Hepatitis B can be prevented!
If you have never had hepatitis B,
you can get 3 shots . . . 1 2 3
. . . and get long lasting protection.

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

66. Hepatitis D virus (HDV)

67. 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

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

70. 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 (地中海)
– Rare in the West

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

72. 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

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

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

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

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

78. 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.

79. 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).

80. Biological Properties Highly varied genome of HCV
Quasispecies& strains
Quasispecies

81. • Parenteral – Injected drug use
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%

82. Epidemiology

83. 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

84. Vast majority are asymptomatic
Acute Infection
Vast majority are asymptomatic
Few cases of symptomatic acute hepatitis
Very rare or non-existent fulminant cases
Very high rate of chronic infection •

86. Diagnosis • Acute infection • Chronic 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

87. Behavioral interventions to reduce risk
Prevention
No vaccine available
No immune globulin
Behavioral interventions to reduce risk
Treatment: Recombinant IFN-α alone or with
ribavirin •

88. Viral Hepatitis - Overview Type of Hepatitis
feces B
blood/ C
blood/ D
blood/ E
feces
Source of
virus
blood-derived blood-derived blood-derived
body fluids
body fluids
body fluids
Route of
transmission
fecal-oral percutaneous percutaneous percutaneous fecal-oral
permucosal permucosal permucosal
Chronic no
yes
yes
yes no
infection
Prevention
pre/post-
exposure
pre/post-
exposure
blood donor
screening;
pre/post-
exposure
ensure safe
drinking
immunization
immunization risk behavior immunization; water
modification
risk behavior
modification
经由皮肤的 >>粘膜的

89. Thank You!