Hepatitis Viruses

Medical VirologyLecture 05/06

Youjun Feng

Center for Infection & Immunity, Zhejiang University School of Medicine

fengyj@zju.edu.cn

• 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

• 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

Hepatitis A Virus (HAV)

• “Epidemic jaundice”

• “Infectious hepatitis” (1912)

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

Mature particle

Immature particle

http //www.ncbi.nlm.nih.gov/books

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

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,for 1h survive for months in freshacid (pH 1.0 for 2h), 60

water and salt water

• one serotype and 7 genotypes

℃

Fecal-Oral spread

Contaminated water or food ( shellfish, green onions)

Risk factors:

Transmission of HAV

Poor sanitation and hygiene, overcrowding, daycare

Water/food-borne virus

Enters bloodstream through gastrointestinal epithelium

Replicates in hepatocytes and Kupffer cells

Released by exocytosis, not cell lysis

Pathogenesis of HAV

Goes into bile, intestine, excreted in feces

Shedding of virus for 10 days prior to any symptoms

Acute Hepatitis A Infection

May be mild to asymptomatic in children

Abrupt onset of disease in adults

“Self-limited”-controlled by immune system

Low overall mortality from fulminant ( ) hepaptitis

Higher risk with simultaneous liver disease such ascirrhosis due to alcohol or chronic Hepatitis B or C

爆发性的

Clinical syndrome

Detection of anti-HAV specificantibodies

IgM titer in acute infection, positivefor 4-6 months

Diagnosis of HAV

IgG titer present for decades

Research testing

-Virus feces by electron microscopy(no cell culture available)

RNA PCR

EILSA

Sanitation

Avoidance of questionable food and water in endemic regions

Prevention of HAV

Passive immunizationPolyclonal anti-HAV antibodies that persist for 6 monthsOnly effective for 2 weeks prior to exposureExpensive, painful, IM injection site reactions

Active immunization Inactivated HAV vaccine

Live attenuated HAV vaccine

Hepatitis E virus (HEV)

Used to be called “Enteric” or “Epidemic”or “Water-borne”

Non-A Non B Hepatitis

Identified in India in 1955

Matureparticle

Immatureparticle

Biological properties

Non-enveloped virus

Calcivirus

Single Strand (+) RNA

7.2-7.6 kb

Biological properties

Cryo-EM

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, IgGand IgM

Transmission and Epidemiology

•Fecal-Oral transmission, especially from fecally-contaminated water

•Person-to-person transmission

•Highest incidence in Asia, Africa, Middle East andCentral America

•High incidence among pregnant women with 10-20%mortality

Sanitation

No vaccine (Phase III clinical trial in China)

Little known about pre- or post-exposure efficacy of immuneglobulin

Prevention

No efficacy of immunoglobulin obtained from westernpopulations

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

Biological properties

tubular particle

Electron microscopy of hepatitis B virus-positive serum reveals 3morphologically distinct forms of particles

Dane particle

(complete virion )

spherical particle

(HBsAg)

Hepatitis B virus. Dane particle and incomplete particles that arefound in patient's serum

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

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

Genetic structure

dsDNA(-) 3200 nt(+) 50-90% of (-)

DR 1: direct repeat (+) 5’- TTCACCTCTGCDR 2: direct repeat (-)

DNA POLYMERASE

LEADING SEQUENCE

Genetic structure(L-)

• P region: DNA polymerase

(RDDP,DDDP, RNase H)

Genetic structure(L-)

• X region: HBxAg

0.8 kb mRNA

trans-activation factor

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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)

1. absorption, uncoating

2. L-DNA → dsDNA

3. dsDNA(L-)→ mRNA

Replication

Pre S1, Pre S2

DDDP

DDRP

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

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 PreC gene )

e supression(1762/1764 nt muationin promoter of PreCgene)

Isolation and culture

•Animal models:

-Chimpanzee

-Duck

•Cell culture: not available

•In vitro transfection

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

℃

Pathogenesis

Transmission Routes

High Moderate

Concentration of Hepatitis B Virusin Various Body Fluids

Low/NotDetectable

blood semen urineserum vaginal fluid feces

wound exudates saliva sweat

tearsBreast-milk

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

Natural History of Chronic HBV Infection

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 (polyarteritisnodosum), glomerulonephritis

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

肾小球性肾炎结节性多动脉炎

– Immature responses

– mild symptoms, chronic infection (90%)

HBV infection in infants and young children

Balance between virus clearanceand liver injury

Balance between virus clearance and liver injury

• Virions released by exocytosis, not cytolysis

• Ab-mediated immune responses– Type II hypersensitivity

– Type III hypersensitivity

Immunopathogenesis

• Cell-mediated immune responses– Type IV hypersensitivity

Immune responses

CTL mediated anti-virus immune responses vs. liver injury

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

HBsAg & anti-HBs

•HBsAg

–major sign of HBV infection

–acute infection

–chronic infection or carrier

–Hepatocellular cancer patient

•anti-HBs

–neutralization antibody

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 clinicaldiagnosis purpose

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

Laboratory Diagnosis

Laboratory Diagnosis

• Acute infection by clinical syndrome– Minority of infections diagnosed by syndrome

• Acute and chronic infection by patterns ofserology

• HBV DNA assay (DNA hybrid or PCR)

• No cultures performed in clinical evaluation

Acute Hepatitis B Virus Infection with RecoveryTypical Serologic Course

Progression to Chronic Hepatitis B Virus InfectionTypical Serologic Course

Interpretation of Serologic Markers of HBVinfection

Prevention and Treatment

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TreatmentInterferon - for HBeAg +vs carriers with chronic active hepatitis. Response rate is30 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 oncessation 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 totreat 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.

Prevention• Vaccination – highly effective recombinant vaccines are now

available. Vaccine can be given to those who are at increasedrisk of HBV infection such as health care workers. It is also givenroutinely to neonates as universal vaccination in many countries.

• Hepatitis B Immunoglobulin – HBIG may be used to protectpersons who are exposed to hepatitis B. It is particularefficacious within 48 hours of the incident. It may also be givento neonates who are at increased risk of contracting hepatitis Bi.e. whose mothers are HBsAg and HBeAg positive.

• Other measures - screening of blood donors, blood and body fluid precautions

If you have never had hepatitis B,you can get 3 shots . . .

321

Hepatitis B can be prevented!

. . . and get long lasting protection.

Baby Shotsfor Hepatitis B

if the mother has Hepatitis B

Birth

+ H-BIGHepatitis B

Vaccine

6 months old

Hepatitis BVaccine

1 - 2 months old

Hepatitis BVaccine

Hepatitis D virus (HDV)

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

Gene structure

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

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

Pathogenesis of HDV

• Viral replication causes hepatocyte cell death

• Additive to HBV-induced host inflammatoryresponse

• Antibodies to HDVAg not likely to beprotective

Clinical Consequences of Infection

• Increases risk of fulminant hepatitis greatlyupon co-infection– Estimated 2-20% fulminant cases

• Increases risk of cirrhosis in chronic infectionwith HBV– More rapid progression– More likely progression

Diagnosis

• Detection of HDV antigen or antibody

• Clinical setting– Acute fulminant disease– Chronic co-infection

Hepatitis C virus (HCV)

•“Non-A Non-B Hepatitis”

•Identified in 1989 by molecular methods

Biological Properties

• Related to flaviviruses ( 黄病毒） and pestivirus ( 瘟病毒 )

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40-60 nm particle, spherical

an enveloped virion

Genome: (+)ss RNA

Six genotypes, regional prevalence

Great heterogeneity, many “quasispecies”

Prevention

• No vaccine

• Prevention of HBV

• Prevention of further exposure risks in HBVchronic infection

Biological PropertiesHCV 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.

Biological PropertiesHighly varied genome of HCV

Genotypes & subtypes

Fig 1 Phylogenetic tree of HCV NS5B sequences. Nucleotide sequences for positions 7975–8196 (numberedfrom the polyprotein AUG initiation codon) of NS5B were analyzed using the program Phylipas describedpreviously (76). Major branches are labeled with the type number, and minor branches with letters indicating thesubtype 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).

Biological PropertiesHighly varied genome of HCV

Quasispecies& strains

Quasispecies

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%

Epidemiology

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

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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

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

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Prevention

No vaccine available

No immune globulin

Behavioral interventions to reduce risk

Treatment: Recombinant IFN-α alone or withribavirin

Source ofvirus

Afeces

Bblood/

body fluids

Cblood/

body fluids

Dblood/

blood-derived blood-derived blood-derivedbody fluids

Efeces

Viral Hepatitis - Overview

Type of Hepatitis

Route oftransmission

fecal-oral percutaneous percutaneous percutaneous fecal-oralpermucosal permucosal permucosal

Chronicinfection

no yes yes yes no

Prevention pre/post-exposure

immunization

pre/post-exposure

blood donorscreening;

modification

pre/post-exposure

risk behaviormodification

ensure safedrinking

immunization risk behavior immunization; water

经由皮肤的 >> 粘膜的

1. Each of the following statements concerning hepatitis C virus (HCV) and hepatitis Dvirus (HDV) is correct EXCEPT:

(A) HCV is an important cause of post-transfusion hepatitis(B) Delta virus is a defective virus with an RNA genome and a capsid composed of

hepatitis B surface antigen(C) HDV is transmitted primary by the fecal-oral route(D) People infected with HCV commonly become chronic carriers of HCV and are

predisposed to hepatocellular carcinoma

Self control questions-part I

2. A 35-year-old man addicted to intravenous drugs has been a carrier of HBs antigen for10 years. He suddenly develops acute fulminant hepatitis and dies within 10 days. Whichone 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

3. The routine screening of transfused blood for HBs antigen has not eliminated the problemof post-transfusion hepatitis. For which one of the following viruses screening has eliminated a large number of cases of post-transfusion hepatitis?

(A) Hepatitis A virus(B) Hepatitis C virus(C) Cytomegalovirus(D) Epstein-Barr virus

•Please summarize the similarities and differences of hepatitis viruses (Viral structure;Modes of spread; Pathogenesis; Diagnosis; Prevention)

•What is the structure of a complete HBV particle (Dane particle)?

•What is the antigenic composition of a Dane particle?

•How many ORFs (open reading frame) does a HBV gene (long form) have? What are thegene products from each ORF?

•What kinds of antigen and antibody can be detected in the sera of HBV infected patients?What is the diagnostic value of each item?

•Term explanation: Dane particle

Thank You!