Global Alert and Response (GAR)

Hepatitis B


The hepatitis B virus


- Life cycle
- Morphology
- Genome and proteins
- Nomenclature of hepatitis B
- Antigenicity
- Stability

The hepatitis B virus, a hepadnavirus, is a 42 nm partially double stranded DNA virus, composed of a 27 nm nucleocapsid core (HBcAg), surrounded by an outer lipoprotein coat (also called envelope) containing the surface antigen (HBsAg).10, 11, 15, 23, 30, 31.

The family of hepadnaviruses comprises members recovered from a variety of animal species, including the woodchuck hepatitis virus (WHV), the ground squirrel hepatitis virus (GSHV), and the duck HBV. Common features of all of these viruses are enveloped virions containing 3 to 3.3 kb of relaxed circular, partially duplex DNA and virion-associated DNA-dependent polymerases that can repair the gap in the virion DNA template and have reverse transcriptase activities. Hepadnaviruses show narrow host ranges, growing only in species close to the natural host, like gibbons, African green monkeys, rhesus monkeys, and woolly monkeys.15, 30, 31

Hepatocytes infected in vivo by hepadnaviruses produce an excess of noninfectious viral lipoprotein particles composed of envelope proteins. Persistent infections display pronounced hepatotropism.15

Mammalian hepadnaviruses fail to propagate in cell culture.23, 30, 31

Intracellular HBV is non-cytopathic and causes little or no damage to the cell.6, 10, 15, 23

Click here for: Electron Microscopy (EM) picture and schematic representation of the hepatitis B virion


The hepatitis B virus life cycle
The HBV virion binds to a receptor at the surface of the hepatocyte.10

A number of candidate receptors have been identified, including the transferrin receptor, the asialoglycoprotien receptor molecule, and human liver endonexin. The mechanism of HBsAg binding to a specific receptor to enter cells has not been established yet.

Viral nucleocapsids enter the cell and reach the nucleus, where the viral genome is delivered.6, 10, 13, 23 

In the nucleus, second-strand DNA synthesis is completed and the gaps in both strands are repaired to yield a covalently closed circular (ccc) supercoiled DNA molecule that serves as a template for transcription of four viral RNAs that are 3.5, 2.4, 2.1, and 0.7 kb long.6, 10, 23, 31 

These transcripts are polyadenylated and transported to the cytoplasm, where they are translated into the viral nucleocapsid and precore antigen (C, pre-C), polymerase (P), envelope L (large), M (medium), S (small)), and transcriptional transactivating proteins (X).6, 10, 23, 31

The envelope proteins insert themselves as integral membrane proteins into the lipid membrane of the endoplasmic reticulum (ER).

The 3.5 kb species, spanning the entire genome and termed pregenomic RNA (pgRNA), is packaged together with HBV polymerase and a protein kinase into core particles where it serves as a template for reverse transcription of negative-strand DNA. The RNA to DNA conversion takes place inside the particles.10, 23

The new, mature, viral nucleocapsids can then follow two different intracellular pathways, one of which leads to the formation and secretion of new virions, whereas the other leads to amplification of the viral genome inside the cell nucleus.10, 23

In the virion assembly pathway, the nucleocapsids reach the ER, where they associate with the envelope proteins and bud into the lumen of the ER, from which they are secreted via the Golgi apparatus out of the cell.10, 23

In the genome amplification pathway, the nucleocapsids deliver their genome to amplify the intranuclear pool of covalently closed circular DNA (cccDNA).10, 23

The precore polypeptide is transported into the ER lumen, where its amino- and carboxy-termini are trimmed and the resultant protein is secreted as precore antigen (HBeAg).

The X protein contributes to the efficiency of HBV replication by interacting with different transcription factors, and is capable of stimulating both cell proliferation and cell death.10, 23

The HBV polymerase is a multifunctional enzyme. The products of the P gene are involved in multiple functions of the viral life cycle, including a priming activity to initiate minus-strand DNA synthesis, a polymerase activity, which synthesizes DNA by using either RNA or DNA templates, a nuclease activity which degrades the RNA strand of RNA-DNA hybrids, and the packaging of the RNA pregenome into nucleocapsids.6, 10, 23  Nuclear localisation signals on the polymerase mediate the transport of covalently linked viral genome through the nuclear pore.6, 10

Click here for: Scheme of genome replication

Morphology and physicochemical properties
Ultrastructural examination of sera from hepatitis B patients shows three distinct morphological forms.15

The most abundant are small, spherical, noninfectious particles, containing HBsAg, that measure 17 to 25 nm in diameter. Concentrations of 1013 particles per ml or higher have been detected in some sera. These particles have a buoyant density of 1.18 g/cm3 in CsCl, reflecting the presence of lipids, and a sedimentation coefficient that ranges from 39 to 54 S.15, 31

Tubular, filamentous forms of various lengths, but with a diameter comparable to that of the small particles, are also observed. They also contain HBsAg polypeptides.15, 31

The third morphological form, the 42 nm hepatitis B virion, is a complex, spherical, double shelled particle that consists of an outer envelope containing host-derived lipids and all S gene polypeptides, the large (L), middle (M), and small (S) surface proteins, also known as pre-S1, pre-S2 and HBsAg. Within the sphere is an electron-dense inner core or nucleocapsid with a diameter of 27 nm. The nucleocapsid contains core proteins HBcAg, a 3.2 kb, circular, partially double stranded viral DNA genome, an endogenous DNA polymerase (reverse trans-criptase) enzyme, and protein kinase activity.15, 23, 31

The sera of infected patients may contain as many as 1010 infectious virions per ml. The complete virion has a buoyant density of about 1.22 g/cm3 in CsCl and a sedimentation coefficient of 280 S in sucrose gradients.15 

Click here for: Schematic representation of viral particles found in serum of HBV-infected people

Genome and proteins
HBV virion DNA is a relaxed circular, partially duplex molecule of 3.2 kb, whose circularity is maintained by 5' cohesive ends.15, 31

The positions of the 5' ends of both strands map to the regions of short (11 nucleotides) direct repeats (DRs) in viral DNA. The 5' end of the minus strand DNA maps within the repeat termed DR1, while plus strand DNA begins with DR2. These repeats are involved in priming the synthesis of their respective DNA strands.6

The viral minus strand is unit length and has protein covalently linked to its 5' end.

The viral plus strand is less than unit length and has a capped oligoribonucleotide at its 5' end. The single-stranded region or gap is of fixed polarity but variable length.31

A virion-associated polymerase can repair this gap and generate a fully duplex genome.

Negative strand DNA is the template for the synthesis of the viral mRNA transcripts. HBV DNA has a very compact coding organization with four partially overlapping open reading frames (ORFs) that are translated into seven known proteins. Noncoding regions are not present.

Four separate viral promoters have been identified, driving expression of a) genomic, P,  and pre-C and C RNAs, b) L protein mRNA, c) M and S protein mRNAs, and d) X protein mRNA. They are referred to as the genomic, pre-S1, S, and X promoters, respectively.

Two major classes of transcripts exist: genomic and subgenomic. The subgenomic RNAs function exclusively as messenger RNAs (mRNAs) for translation of envelope and X proteins. The genomic RNAs are bifunctional, serving as both the templates for viral DNA synthesis and as messages for ORF pre-C, C, and P translation.6, 23 

ORF P encodes the viral polymerase and the terminal protein found on minus strand DNA. ORF C encodes the structural protein of the nucleocapsid and the HBeAg, and ORF S/pre-S encodes the viral surface glycoproteins. The product of ORF X is a poorly understood regulatory protein that enhances the expression of heterologous and homologous cellular genes in trans.6, 31 

Classic HBsAg, which contains the S domain only, is also called the S-protein (24 kD). Two other proteins share the C-terminal S domain, but differ by length and structure of their N-terminal (pre-S) extensions. The large L protein  (39 kD) contains  the pre-S1, the pre-S2 region and the S region, and the medium M protein (31 kD) contains the pre-S2 and the S region only. HBsAg is the most abundant of the S-related antigens.  The L and M proteins are expressed at levels of about 5-15% and 1-2% compared with S protein.31

The glycosylation of the S domain gives rise to two isoforms of each protein. In addition, the M protein contains an N-linked oligosaccharide on its pre-S2-specific domain, and the L protein carries a myristic acid group in amide linkage to its amino-terminal glycine residue. While the function of M protein is still obscure, L proteins play a role in viral assembly and infectivity.31

The three envelope glycoproteins are not distributed uniformly among the various HBV particle types. Subviral 22 nm particles are composed predominantly of S proteins, with variable amounts of M proteins and few or no L proteins. Virus particles are enriched for L proteins. L proteins carry the receptor recognition domain, which allows efficient binding to cell surface receptors.

Two in-frame AUG codons are present in ORF C. Classic HBcAg (21 kD) is the product of initiation from the more internal start codon, while initiation at the upstream AUG produces a C-related protein that is not incorporated into virions but instead is independently secreted from cells, accumulating in serum as an immunologically distinct antigen known as HBeAg (16-18 kD). The function of HBeAg is still unknown.31

HBcAg is the most conserved polypeptide among the mammalian hepadnaviruses with 68% homology between HBV and GSHV and 92% between GSHV and WHV. Core proteins spontaneously assemble into forms resembling core particles.

The polymerase protein is a DNA-dependent DNA polymerase, a reverse transcriptase, an RNAse H, and it binds to the 5' end of HBV DNA, acting thus as a primer for reverse transcription of the pregenome, an RNA intermediate, to form negative strand DNA.31 Furthermore, it plays important roles in the encapsidation of the viral pregenomic RNA. The polymerase protein is quite immunogenic during both acute and chronic infection.6

ORF X encodes the protein X (17 kD), a transactivator for the viral core and S promoters. The X protein is the least-conserved protein among hepadnaviruses with only 33% amino acid homology between GSHV and HBV, and 71% between the two rodent viruses.6

Nomenclature of hepatitis B

 

HBV

 

 

hepatitis B virus (complete infectious virion)

 

The 42 nm, double-shelled particle, originally called the Dane particle, that consists of a 7 nm thick outer shell and a 27 nm inner core. The core contains a small, circular, partially double-stranded DNA molecule and an endogenous DNA polymerase. This is the prototype agent for the family Hepadnaviridae.

 

 

HBsAg

 

hepatitis B surface antigen (also called envelope antigen)

 

The complex of antigenic determinants found on the surface of HBV and of 22 nm particles and tubular forms. It was formerly designated Australia (Au) antigen or hepatitis-associated antigen (HAA).

 

 

HBcAg

 

 

hepatitis B core antigen

 

The antigenic specificity associated with the 27 nm core of HBV.

 

 

 

HBeAg

 

 

hepatitis B e antigen

 

The antigenic determinant that is closely associated with the nucleocapsid of HBV. It also circulates as a soluble protein in serum.

 

 

Anti-HBs, anti-HBc, and anti-HBe

 

 

Antibody to HBsAg, HBcAg, and HBeAg

 

Specific antibodies that are produced in response to their respective antigenic determinants.

From: Hollinger FB, Liang TJ. Hepatitis B Virus. In: Knipe DM et al., eds. Fields Virology, 4th ed., Philadelphia, Lippincott Williams &Wilkins, 2001:2971-3036,15 with permission (http://lww.com).

Antigenicity
All three coat proteins of HBV contain HBsAg, which is highly immunogenic and induces anti-HBs (humoral immunity). Structural viral proteins induce specific T-lymphocytes, capable of eliminating HBV-infected cells (cytotoxic T-cells; cellular immunity).6, 15

HBsAg is heterogeneous antigenically, with a common antigen designated a, and two pairs of mutually exclusive antigens, d and y, and w (including several subdeterminants) and r, resulting in 4 major subtypes: adw, ayw, adr and ayr.23, 30, 31

The distribution of subtypes varies geographically.30 Because of the common determinants, protection against one subtype appears to confer protection to the other subtypes, and no difference in clinical features have been related to subtypes.

In the US, northern Europe, Asia, and Oceania, the d determinant is common, but the y determinant is found at lower frequency. The d determinant to the near exclusion of y is found in Japan. The y determinant, and rarely d, are found in Africa and in Australia aborigines. y is also frequently found in India and around the Mediterranean. In Europe, the US, Africa, India, Australia, and Oceania, the w determinant predominates. In Japan, China, and Southeast Asia, the r determinant predominates. Subtypes adw, ady, and adr are each found in extensive geographic regions of the world. Subtype ayr is rare in the world, but it is commonly found in small populations in Oceania.23, 52 

The c antigen (HBcAg) is present on the surface of core particles. HBcAg and core particles are not present in the blood in a free form, but are found only as internal components of virus particles.23, 30

The core antigen shares its sequences with the e antigen (HBeAg), identified as a soluble antigen, but no crossreactivity between the two proteins is observed.30, 31

Viral oligopeptides of 8-15 amino acids are loaded on host cell MHC-class I molecules and are transported to the surface of the cell. HBV-specific T-lymphocytes can then detect infected cells and destroy them. This cell deletion triggered by inflammation cells may result in acute hepatitis. When the infection is self-limited, immunity results. If HBV is not eliminated, a delicate balance between viral replication and immunodefence prevails which may lead to chronic hepatitis and liver cirrhosis. In chronically infected cells the HBV DNA may integrate into the host cell DNA. As a long term consequence, integration may lead to hepatocellular carcinoma.15, 23, 52

Stability
The stability of HBV does not always coincide with that of HBsAg.15

Exposure to ether, acid (pH 2.4 for at least 6 h), and heat (98°C for 1 min; 60°C for 10 h) does not destroy immunogenicity or antigenicity. However, inactivation may be incomplete under these conditions if the concentration of virus is excessively high.15

Antigenicity and probably infectivity are destroyed after exposure of HBsAg to 0.25% sodium hypochlorite for 3 min.15

Infectivity is lost after autoclaving at 121°C for 20 min or dry heat treatment at 160°C for 1 h.15, 31

HBV is inactivated by exposure to sodium hypochlorite (500 mg free chlorine per litre) for 10 min, 2% aqueous glutaraldehyde at room temperature for 5 min, heat treatment at 98°C for 2 min, Sporicidin (Ash Dentsply, York, PA) (pH 7.9), formaldehyde at 18.5 g/l (5% formalin in water), 70% isopropylalcohol, 80% ethyl alcohol at 11°C for 2 min, Wescodyne (a iodophor disinfectant, American Sterilizer Co., Erie, PA) diluted 1:213, or combined β-propriolactone and UV irradiation.15, 45

HBV retains infectivity when stored at 30°C to 32°C for at least 6 months and when frozen at –15°C for 15 years. HBV present in blood can withstand drying on a surface for at least a week.15, 31


Electron Microscopy (EM) picture and schematic representation of the hepatitis B virion

A diagrammatic representation of the hepatitis B virion and the surface antigen components

Virions are 42nm in diameter and possess an isometric nucleocapsid or "core" of 27nm in diameter, surrounded by an outer coat approximately 4nm thick. The protein of the virion coat is termed "surface antigen" or HBsAg. It is sometimes extended as a tubular tail on one side of the virus particle. The surface antigen is generally produced in vast excess, and is found in the blood of infected individuals in the form of filamentous and spherical particles. Filamentous particles are identical to the virion "tails" - they vary in length and have a mean diameter of about 22nm. They sometimes display regular, non-helical transverse striations.

A group of hepatitis B virions (right) and enlargements of the two exposed cores (indicated by arrows).

From: University of Cape Town, South Africa. http://www.uct.ac.za/depts/mmi/stannard/hepb.html


Scheme of genome replication

From: http://www.globalserve.net/~harlequin/HBV/hbvcycle.htm


Schematic representation of viral particles found in serum of HBV-infected people


Infectious HBV particle:

  • 42 to 47 nm double-shelled particle. Outer envelope containing lipid and three forms of HBsAg
  • 27 nm nucleocapsid made of 180 copies of core protein, containing the polymerase and HBV DNA

  

 

Empty noninfectious particles:

  • 22 nm spheres and filaments of variable length containing lipid and mainly one form of HBsAg usually present in 10 000 to 1 000 000-fold excess over Dane particles.

   

 

Titres of the virus in the blood can range between <104/ml and >109/ml. 

The envelope can be removed with nonionic detergents, liberating the inner core, the nucleocapsid of 27 nm. The major structural protein of the core is the C protein, a 21 kD basic phosphoprotein called hepatitis B core antigen (HBcAg).

Within the core are the viral DNA, a DNA polymerase, and a protein kinase.

The 22 nm spheres and filaments lack nucleic acid altogether and hence are noninfectious. These particles are highly immunogenic and induce a neutralizing anti-HBs antibody response.   

The number of subviral particles can exceed that of virions by a factor of 103 to 105.


HBV coding organization

From: Ganem D, Schneider RJ. Hepadnaviridae: The Viruses  and Their Replication. In: Knipe DM et al., eds. Fields Virology, 4th ed. Philadelphia Williams & Wilkins, 2001:2923-2969,10 with permission (http://lww.com).

A: Diagrammatic representation of the hepatitis B virus coding organization. Inner circle represents virion DNA, with dashes signifying the single-stranded genomic region; the locations of DR1 and DR2 sequence elements are as indicated. Boxes denote viral coding regions, with arrows indicating direction of translation. Outermost wavy lines depict the viral RNAs identified in infected cells, with arrows indicating direction of transcription. B: Fine structure of the 5’ ends of the pre-C/C transcripts (top) and pre-S2/S transcripts (bottom) relative to their respective open reading frames.10


Hepatitis B virus DNA and hepatocellular carcinoma

More than 85% of hepatocellular tumours examined harbor integrated HBV DNA, often multiple copies per cell. The viral DNA integrants are usually highly rearranged, with deletions, inversions, and sequence reiterations all commonly observed. Most of these rearrangements ablate viral gene expression, but the integrations alter the host DNA.10, 31, 52

Interestingly, tumours are clonal with respect to these integrants: every cell in the tumour contains an identical complement of HBV insertions. This implies that the integration event(s) preceded the clonal expansion of the cells. How integration is achieved is still not well understood. Since integration is not an obligatory step in the hepadnaviral replication cycle, and hepadnaviruses have no virus-encoded integration machinery, HBV DNA is probably assimilated into the nucleus by host mechanisms.10, 23

There is no similarity in the pattern of integration between different tumours, and variation is seen both in the integration site(s) and in the number of copies or partial copies of the viral genome.52

The molecular mechanisms by which hepadnaviruses predispose to malignancy are still unknown.52

Direct models

In the direct models, HBV DNA makes direct genetic contributions to the lesion by either providing cis-acting sequences deregulating host growth genes, or by providing trans-acting factors that interfere with cellular growth control.10

Indirect models

In the indirect models, HBV genes and their products make no direct genetic contribution to the transforming event. Rather, HBV-induced liver injury triggering a series of host responses that lead to liver cell regeneration increases the probability of mutation and malignant transformation.10

A better understanding of the immunologic mechanisms of liver cell injury could allow the development of therapeutic agents that would control these responses.



HBV mutants

Naturally occurring envelope, precore, core, and polymerase variants have been described.11, 15, 23 

Envelope antigenic variants may have a selective advantage over wild type under immune selection pressure, as observed in some cases after hepatitis B IG (HBIG) treatment or HBV vaccination. An epidemiological shift has not been observed yet.

A number of precore mutations preventing HBeAg synthesis have been identified in HBeAg negative carriers. The most frequent variant has a G to A point mutation at nucleotide 83 (mutant HBV83, nucleotide 1896 of the genome, amino acid 144) in the precore region, introducing a stop codon at codon 28.15, 23 The HBV83 mutant is predominantly found in Mediterranean and Asian countries but is uncommon in North America and Northern Europe. Precore mutants are found in patients with fulminant hepatitis or chronic active hepatitis, but also in asymptomatic carriers.11

HBV core gene mutations have been reported in patients from Japan, Hong Kong, United States, and Italy. Most of the mutations are concentrated in the middle-third of the core gene, but although many of these mutations are located in regions that harbor B and T cell epitopes, they have not been proven to result in loss of immune recognition.

In rare patients where the function of the polymerase gene is impaired, additional compensatory mutations were found that minimized the impact of the impaired function of the polymerase.

HBV is far more heterogeneous than is generally thought. The HBV genome seems not to be characterized by a single representative genomic molecule, but by a pool of genomes which differ both in structure and function.

The public health importance of mutant hepatitis B viruses is currently under debate. Further studies and a strict surveillance to detect the emergence of these viruses are crucial for a correct evaluation of the effectiveness of current immunization strategies.23, 52, 53  

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