http://www-micro.msb.le.ac.uk/335/HBV.html

Last Updated: Friday 19th April, 2002.

Hepatitis B Virus (HBV):

'Serum hepatitis' was distinguished clinically from 'infectious hepatitis' in the 1930's indicating that at least at least 2 different infectious agents were responsible for hepatitis. Infection often results from inoculation with human serum - blood transfusions, transplants or passive immunization (common among IVDAs). However, the virus is also transmitted sexually, by ingestion and from mother to child (transplacenta and breast milk) - accounting for familial clusters. All blood/organ/tissue donations in developed countries are now tested for HBV and risk of transmission is extremely low. Incubation period 45-120 days. HBV does not grow in tissue culture and this has hindered investigations.

• Blumberg, 1963: Was searching for novel antigens useful for blood/tissue typing. Looked at U.S. haemophiliacs (multiply transfused, each transfusion pooled from 00's donors) - found Abs which recognised antigen in the serum of an Australian aborigine 'Australian antigen - Au'.

   

• 1967: It was recognised that Au was a viral antigen = HBsAg (surface antigen).

   

• Dane, 1970: Discovered 42nm 'Dane particles' in blood of hepatitis patients = HBcAg (core antigen).

   

• 1973: HBeAg discovered (endogenous antigen = a truncated version of HBcAg).

HBV is the prototype member of the family Hepadnaviridae:

Features:

Spherical, enveloped (? lipid-containing, detergent disrupted ?) particles 42-47nm diameter containing partially d/s DNA plus an RNA-dependent DNA polymerase (i.e. reverse transcriptase)
(c.f. retroviruses - package RNA + RT).

Hepadnaviruses have the among the smallest genomes of all known viruses, consisting of two uneven strands of DNA:

(-)sense strand, 3.0 - 3.3kb (size varies between different Hepadnaviruses)

(+)sense strand, 1.7 - 2.8kb (size varies between different particles)

Hepadnaviruses, together with a number of plant viruses (e.g. cauliflower mosaic virus, CaMV), have been classified as pararetroviruses. A few years ago it appeared that additional fundamental differences existed between the hepadnaviruses and the pararetroviruses, e.g. the existence of splicing in pararetroviruses (e.g. in CaMV and duck hepatitis B virus (DHBV)), but not HBV. In retroviruses, the foamy viruses have features atypical of other retroviruses, e.g. an additional internal promoters rather than a solely LTR-based promoter, and the generation of their reverse transcriptase (RT) as a separate entity rather than as a gag-pol fusion protein. Foamy virus virions even appear to carry mostly DNA rather than RNA. Thus there is some degree of overlap between the pararetroviruses and the retroviruses.

All hepadnaviruses have a restricted host range. Only humans and chimpanzees are susceptible to infection with HBV. The north american woodchuck (Marmota monax; WHV), Beechey ground squirrels (Spermophilus beecheyi; GSHV) and arctic ground squirrels all have their own hepadnaviruses and WHV has been used as a model for HBV, but has notable differences in biology from HBV. Woolly monkey (Lagothrix lagotricha) HBV (WMHBV), is a recently discovered hepadnavirus. Tree shrews (Scandentia) also have a virus similar to HBV. Pekin duck (Anas domesticus; DHBV), grey heron (HHBV) and storks all have avihepadnaviruses, but only the duck has been used as an experimental system.

HBsAg is composed of 3 polypeptides (below) and is presumably responsible for receptor binding (not known), which in turn presumably determines the tropism of the virus for hepatocytes (not known). The a component of the receptor for DHBV has been identified, a large glycoprotein termed gp180 or p170, a membrane protein of the carboxypeptidase D family. However, transfection of chicken hepatoma cells (which support replication of DHBV when transfected with DNA) does not render these cells susceptible to DHBV infection. The interpretation for this is that DHBV (and presumably, HBV) receptor has several components, of which gp180/p170 is only one.

Three major genome transcripts are produced: 3.5kb, 2.4kb, 2.1kb. All have same polarity, same 3' ends but different 5' ends (i.e. initiation sites). These transcripts are somewhat heterogeneous in size and it is not completely clear which proteins each transcript encodes, but there are 4 known genes in the virus:

• C - the core protein
• P - the polymerase
• S - the 3 polypeptides of the surface antigen (preS1, preS2 and S - produced from alternative translation start sites.
• X - a transactivator of viral transcription (and cellular genes?). HBx is conserved in all mammalian (but not avian) hepadnaviruses. Though not essential in transfected cells, it is required for infection in vivo.

How Do Animal DNA Viruses Get To The Nucleus? Ann.Rev.Microbiol. (1998) 52: 627-686

Closed circular DNA is found soon after infection in the nucleus of the cell and therefore is probably the source of these transcripts. This DNA is produced by repair of the gapped virion DNA as follows:

1. completion of the (+)sense strand
2. removal of a protein primer from the (-)sense strand and an oligoribonucleotide primer from the (+)sense strand
3. elimination of terminal redundancy at the ends of the (-)sense strand
4. ligation of the ends of the two strands

It is not known how or by which proteins (viral/cellular) these events are carried out. The 3.5kb RNA transcript, core antigen and polymerase form core particles in the cytoplasm. The polymerase converts the RNA to DNA in these particles INSIDE THE PARTICLE in the cytoplasm c.f. retroviruses, where RNA is packaged then converted to DNA as the first stage of genome replication:

The surface antigen and lipid envelope may be acquired at any stage after encapsidation of the RNA, apparently from intracellular membranes.

Assembly/release appear to be rather disordered events for Hepadnaviruses - some extracellular particles contain DNA-RNA hybrids or (-)sense DNA strands without (+)sense strands (intermediates in reverse transcription). Assembly of the particle is initiated by packaging of the RNA pregenome and the viral reverse transcriptase-DNA polymerase into a nucleocapsid. The pregenome is then reverse transcribed into single-stranded minus-polarity DNA, which is subsequently replicated to double-stranded DNA. All replicative intermediates are observable in capsids within infected liver, but only relatively mature nucleocapsids containing partially double stranded DNA are found in secreted virions. This observation suggests that maturation of the genome within the capsid is required for envelopment and secretion.

Three types of hepadnavirus particles are observed:

• complete enveloped virions, or "Dane particles"
• the nucleocapsids, or core particles
• empty envelopes called subviral, or S particles

The S particles of mammalian hepadnaviruses occur in two types, either spherical, or filamentous. The filaments, but not the spheres, contain substantial amounts of L protein, which therefore may be responsible for the different morphologies.

Release of mature particles occurs by secretory mechanisms - "reverse endocytosis", without cell lysis.

HBV infection has 3 possible outcomes:

1. Acute course with complete recovery and immunity from reinfection (>90%).
2. Fulminant hepatitis with liver failure and mortality ~90% (~1% cases).
3. Chronic infection - carrier state with virus persistence (~10% cases).

There are >350m HBV carriers worldwide (total population of world ~6bn. = 5%).

Primary Hepatocellular Carcinoma (PHC): A rare tumour in the west (<2% fatal cancers) and most cases are alcohol-related. In S.E. Asia and China PHC is the most common fatal cancer, ~5x10⁵ deaths p.a.
 

The relationship between HBV infection and PHC is not clear cut:

• Cirrhosis appears to be a prerequisite + chronic liver damage/repair results in malignant transformation.
• Co-factors such as aflatoxins and nitrosamines can induce PHC-like disease in experimental animals.

The key factor is then what determines the development of chronic vs. acute infection:

• Age (chronic infections decrease with increasing age)
   
• Sex:
   

  Syndrome:	Males : Females
  Chronic Infection:	1.5 : 1
  Cirrhosis:	3 : 1
  PHC:	6 : 1

   

• Route of infection (oral/sexual infections give rise to less chronic cases than serum infection)

72% of patients chronically infected with hepatitis B virus become negative for HBeAg (the best marker for active HBV infection) within 10 years (McMahon B.J. et al. Serologic and Clinical Outcomes of 1536 Alaska Natives Chronically Infected with Hepatitis B Virus.Ann Intern Med 2001 135: 759-768).

The HBx protein is of importance in controlling apoptosis in HBV-infected cells.

• Expression of HBx either during virus infection or alone has been found to sensitize cells to apoptosis and increase sensitivity to TNF-induced killing. This response involves activation of both c-Myc- and MEKK-dependent pathways and requires p53.
• BUT in the absence of p53, HBx is growth promoting and appears to play a role in the oncogenicity of HBV. Thus, like adenovirus E1A and polyomavirus Large T antigen, stimulation of the proliferative response activates p53-dependent apoptotic pathways.
• This implies a multi-step development for HCC, where loss of p53 is an important stage.
• Roulston, A. et al. (1999) Viruses and apoptosis. Ann. Rev. Microbiol. 53: 577-628

Prevention/Therapy:

Purified (!) HBsAg from the blood of chronic carriers has been used as a vaccine since 1981 (Hepatavax-B) and continues to be used in some areas of the world.
Recombinant HBsAg vaccines produced in yeast have been available since 1986 and are now most widely used (e.g. Engerix-B, Recombivax-HB), e.g. part of the W.H.O. expanded program on immunization. A combined hepatitis A and B vaccine (Twinrix® - GlaxoSmithKline Biologicals) is now licenced for use in persons aged 18 years. This consists of the antigenic components used in Havrix (HAV) and Engerix-B (HBV) vaccines. These are vaccines are safe and effective - one of the few recombinant vaccines to date. Effective vaccination campaigns could:

1. Save ~1m lives p.a.
2. Eradicate the virus (no animal reservoir).

a-IFN is used for therapy of chronic HBV infection. Up to 50% chronic carriers respond to this (expensive) treatment (c.f. 10-20% spontaneous loss of virus markers in untreated control groups). The response rate seems to vary depending on the genotype of the virus.

Lamivudine (3TC - 2'deoxy, 3'thiacytidine - a reverse transcriptase inhibitor) is currently being investigated for therapy of chronic HBV infection. Early results suggest this drug may be effective in patients who have previously failed to clear the virus with alpha-IFN.
A number of other nucleoside and nucleotide analogues are now known to inhibit HBV replication in vitro and in vivo, including penciclovir, lobucavir and adefovir. No specific viral kinase activity has been identified, therefore activation of these drugs is mediated by cellular kinases. Lamivudine triphosphate is thought to inhibit the elongation of minus strand DNA synthesis, whereas famciclovir may act to inhibit the priming reaction. It is of note that lamivudine is also a potent inhibitor of HIV replication, another virus with reverse transcriptase activity. Resistance to lamivudine and famciclovir in HBV has been seen in vivo, associated with specific mutations in the polymerase gene.

• Recommended Reading

   

• Books about Hepatitis

   

• Search the WWW for more information on this topic

   

• Search MEDLINE for the latest publications on this topic

   

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