| Abstract|| |
Inadequate and/or inappropriate host immune responses, are mainly responsible for the liver disease of chronic hepatitis B. Infection of the hepatocyte with the hepatitis B virus (HBV) results in very high levels of viral replication without actually killing the infected cell directly. The HBV uses reverse transcription to copy its DNA genome and because of a lack of proof-reading capability, mutant viral genomes or quasi species, emerge continually. All the selection pressures, both endogenous (host immune clearance) and exogenous (vaccines and antiviral drugs), readily select particular quasispecies with increased replication fitness, which then emerge as the new dominant population. The specific viral mutations or combination of mutations that play a role in the final clinical outcome of HBV infection are not fully known, but certain patterns and associations are starting to emerge. In particular, the expression of a novel protein, the hepatitis B splice protein (HBSP), has been linked to liver fibrosis and liver disease progression. Further studies are needed to identify the molecular pathological basis and subsequent clinical sequelae arising from the selection of these quasispecies in infected individuals.
|How to cite this article:|
Revill P, Locarnini S. HBV variants: Clinical significance and public health implications. Hep B Annual 2005;2:74-92
| Introduction|| |
Human HBV is the prototype member of the family Hepadnaviridae, which has a unique life-cycle, DNA genomic organization, organ trophisms, strategy of genome replication and pathogenesis. Hepatitis B virus is classified into 8 major genotypes (A to H) based on nucleotide diversity of ³ 8%,, which have a distinct global geographical distribution [Table - 1]. The clinical significance of the four major genotypes A-D is beginning to emerge. Genotype C and D are, in general, associated with more severe liver disease and less likely to respond to interferon therapy.
The HBV genome comprises a partially double-stranded 3.2 kb DNA organized into 4 overlapping open-reading frames (ORF) which are frame-shifted. The longest ORF encodes the viral polymerase (Pol), which overlaps the envelope ORF in a frame-shifted manner. Following infection of a susceptible cell, viral cores disassemble and genomic DNA is transported to the cell nucleus, where the partially double-stranded genomic viral DNA is converted by the host cell to covalently closed circular (ccc) DNA, a unique and important replicative intermediate in the HBV life cycle. Cellular repair enzymes such as the topoisomerases then chromatinise the viral ccc DNA, forming a viral minichromosome within the nucleus of the cell; this acts as the major transcriptional template for the virus. Four sets of mRNAs are then transcribed from these viral minichromosomes, again using host cell machinery (RNA polymerase II), and these molecules are transported by cellular proteins (e.g., La) to the cell cytoplasm, where they are translated to produce the viral proteins: hepatitis B core antigen (HBcAg, or nucleocapsid protein, from the 3.5 kb RNA); the soluble and secreted hepatitis B e antigen (HBeAg, from the 3.5 kb RNA); the Pol protein (from the 3.5 kb RNA); the viral envelope proteins, which express HBsAg (from the 2.4 and 2.1 kb RNAs); and hepatitis B X protein (HBx, from the 0.7 kb RNA). Expression of these four transcripts is directed by the enhancer II/basal core, large surface antigen (L), major surface antigen (S) and enhancer I/X gene promoters, respectively. In addition to serving as mRNA for the nucleocapsid and Pol proteins, the 3.5 kb pregenomic RNA also acts as the template for reverse transcription of the viral genome [Figure - 1]. The pregenomic RNA is also the template for numerous spliced RNA transcripts, one of which encodes a novel protein, the hepatitis B splice protein (HBSP), which appears to affect the natural history of liver disease.,
| Common HBV Mutations|| |
In addition to viral and host factors, exogenous selection pressures also define the predominant HBV species in an infected individual. Exogenous pressures include treatment with nucleoside or nucleotide analogues, as well as immune-based interventions such as hepatitis B immunoglobulin (HBIG), vaccination and interferon therapy. The presumably immune-based selection pressures that cause reduction in, or loss of, HBeAg and eventual elimination of HBsAg are probably responsible for the selection of particular mutants, such as those associated with HBeAg-negative chronic hepatitis B.
The frequency of HBV mutation has been estimated to be approximately 1.4 to 3.2 x 10 -5 nucleotide substitutions/site/year, around 10-fold higher than that of other DNA viruses. This is due primarily to the fact that the viral reverse transcriptase lacks proof-reading activity. The magnitude and rate of virus replication are also important in the process of mutation generation: the total viral load in serum is frequently in the range of 10 9 -10 10 virions/mL. Most theoretical estimates have placed the mean half-life of the virus in serum to be less than one day, translating to a rate of de novo HBV production exceeding 10 virions/day. The high viral loads and turnover rates coupled with poor replication fidelity, all influence mutation generation and the complexity of the HBV quasispecies pool. These mutation rates, however, are lower than those of other pararetroviruses, mainly because of the constraints imposed by the overlapping reading frames. However, most HBV mutants pre-exist within the quasispecies pool even before the introduction of the selection pressure.
(i) Mutations in the basal core promoter, precore and core genes
There are two major groups of mutations that result in reduced or blocked HBeAg expression and can be understood in terms of translational or transcriptional control mechanisms. The first group results in mutations that affect translation of a full-length precore protein. The most common form of this mutation is the introduction of a stop-codon in the precore gene by mutation at nucleotide (nt) 1896 (codon 28: TGG; tryptophan), located in the epsilon (e) structure of the precore gene. A single base substitution (G to A or G1896A) gives rise to a translational stop codon (TGG to TAG; TAG = stop codon) in the second last codon (codon 28) of the precore region. The nt G1896 forms a base pair with nt 1858 at the base of the stem loop structure of e. In HBV genotypes B, D, E and G and in some strains of genotype C, the 1858 nt is a thymidine (T) [Table - 1]. Thus, the stop-codon mutation created by G1896A (T-A) stabilizes the e structure. In contrast, the precore stop-codon mutation is rarely detected in HBV genotypes A or F or in some strains of HBV genotype C, because the nt at position 1858 is a cytidine (C), maintaining the preferred Watson-Crick (G-C) base pairing [Table - 1].
The second group of HBV mutants lacking expression of the HBeAg arise as a consequence of mutations that affect the basal core promoter (BCP) region. These mutations occur at nt 1762 and nt 1764 and result in a reduction in the transcriptional activity of the promoter region affecting precore mRNA production but not pregenomic mRNA. Mutations such as A1762T plus G1764A in the basal core promoter may be found in isolation or in conjunction with precore mutations, depending on the genotype [Table - 1]. Occurrence of the double mutation of A1762T plus G1764A results in a substantial decrease in HBeAg production and an increase in viral load. In general, this pattern of mutation is often found in genotype A infected individuals. Mutations in the basal core promoter result in reduced binding of liver-specific transcription factors, transcribing fewer precore mRNA transcripts and, consequently, less precore protein. The basal core promoter mutations however, do not affect the transcription of pregenomic RNA or the translation of the core protein or polymerase proteins, therefore HBV genomic replication proceeds normally. Thus, by removing the inhibitory effect of the precore protein on HBV replication that comes about as a consequence of precore inhibition of core dimerisation, the basal core promoter mutations actually enhance viral replication by suppressing precore mRNA relative to pregenomic RNA.
(ii) Mutations in the X gene
The exact function of HBx during HBV replication and its influence in HBV-associated hepatocellular-carcinoma are now beginning to emerge. At the cellular level, HBx functions to activate various signalling pathways, most of which are controlled by modulation of cytosolic calcium. In the nucleus, HBx can regulate transcription through a direct interaction with different transcription factors, often enhancing their binding to specific transcription elements such as NF-KB, a central molecule in cellular regulation.
Because the basal core promoter encompasses nt 1742 to 1802 and overlaps with the X gene in the concomitant reading frame, the A1762T plus G1764A core promoter mutations also cause changes in the X gene at xK130M and xV131I. Furthermore, almost all deletions or insertions in the basal core promoter result in a shift of the X gene frame and lead to the production of truncated X proteins. These shortened X proteins typically lack the domain in the C terminus (amino acids 130-140) that is required for the transactivation activity of HBxAg.
(iii) Mutations in the envelope gene
The Pre-S and S sequences exhibit the highest heterogeneity of the HBV genome, and this accounts for this region being useful in HBV genotyping. Point mutations, deletions and genetic recombinations within the Pre-S genes have been identified in HBV DNA sequences obtained from the sera of inactive carriers. HBV genomes that cannot synthesize Pre-S2 proteins occur frequently and are the dominant virus populations in these persons. These viruses are replication competent in vitro . The Pre-S2 region overlaps the spacer region of the Pol protein, which is not essential for enzyme activity.
All prophylactic hepatitis B vaccines contain the major HBsAg protein and immunization results in an immune response to the major hydrophilic region, located from residue 99 to 170, which includes the "a" determinant or neutralisation domain of HBV. The anti-HBs response produced to this region is associated with protective immunity. Mutations within this epitope have been selected during vaccination and following treatment of liver allograft recipients with HBIG. Most isolates contain a mutation from glycine to arginine at residue 145 of HBsAg (sG145R) or from aspartate to alanine at residue 144 (sD144A). Both mutations have been directly associated with vaccine failure as well as HBIG treatment breakthrough.
(iv) Polymerase mutations: Antiviral drug resistance
The advent of treatment with nucleoside and nucleotide analogues has resulted in the selection of quasispecies containing mutations in the HBV Pol gene. Antiviral resistance to lamivudine has been mapped to the YMDD locus in the C domain of HBV Pol, and the specific mutations selected are designated as rtM204I/V/S (domain C). These groups of mutations typically do not occur in isolation but are found with mutations such as rtL180M (domain B). Resistance to adefovir dipivoxil is associated with mutations in the B and D domain of the enzyme and the major mutations associated with adefovir-resistant HBV are designated as rtN236T (domain D) and rtA181T/V. Entecavir (ETV) has recently been approved for treatment of chronic hepatitis B by the FDA and from the registration studies during phase III clinical trials, ETV resistance was detected. The major mutations associated with antiviral drug resistance in the HBV Pol are shown in [Figure - 1].
a) Lamivudine resistance
Lamivudine resistance increases progressively during treatment at rates averaging 20-25% annually and approaching over 70% after 48 months of treatment. Factors that increase the risk of resistance include high pre-therapy serum HBV DNA and alanine transferase (ALT) levels (creation of replication space) and incomplete suppression of viral replication. Lamivudine resistance does not confer cross-resistance to adefovir dipivoxil.
Mutations that confer lamivudine resistance decrease in vitro sensitivity to the drug of more than 100-fold in IC 50 . The rtM204I substitution has been detected in isolation, but rtM204V and rtM204S are found only in association with other changes in the B or A motifs. Numerous other secondary changes in the rt sequence have also been found in conjunction with rtM204V/I/S, and some of these are probably acting as compensatory mutations to help restore replication efficiency (Section 5).
b) Adefovir dipivoxil resistance
HBV resistance to adefovir occurs less frequently than resistance to lamivudine. The overall prevalence is around 6% after 3 years and increasing to 18% by 4 years. The adefovir resistance genotype change of rt N236T does not affect the sensitivity of the HBV to lamivudine, but the rtA181T/V do appear to cause an increase in the IC 50 level for lamivudine.
c) Entecavir resistance
Resistance to entecavir has not been reported in any of the clinical trials where the patients are naοve to entecavir therapy, but has been observed in patients who were already resistant to lamivudine when the entecavir therapy was commenced. Mutations in the viral polymerase associated with the emergence of entecavir resistance have been mapped to domain B (rtI169I plus rtS184G), domain C (rtS202I) and domain E (rtM250V). The rtM250V does cause, by itself, an increase in IC 50 for entecavir. However, these various combinations of mutations, in association with lamivudine-resistance changes (rtL180 and rtM204), typically result in entecavir failure.
| The Hepatitis B splice protein - a possible marker of HBV pathogenesis|| |
Although there is some evidence that HBV-encoded proteins may alter cell cycle regulation and therefore influence disease progression, a marker of HBV pathogenesis is yet to be identified. Soussan and colleagues however, have recently identified a HBV-encoded gene product associated with chronic HBV infection and liver fibrosis, which may prove to be a marker of viral pathogenesis., This 10 kDa peptide, termed the hepatitis B splice protein (HBSP), was not encoded by any of the four nascent HBV mRNAs, but was instead transcribed from a 2.2 kb RNA produced as a result of pregenomic RNA splicing between nucleotides 2447 and 489. Earlier studies had shown that splicing at these sites removed approximately 1.3 kb of RNA between the 3' terminus of the core coding sequence and a region upstream of the reverse transcriptase domain in the polymerase (pol) gene.,, The resultant 2.2 kb RNA transcript was packaged and reverse transcribed by pol in trans [Figure - 2] and 2.2 kb DNA was detected in circulating extracellular virions. Rosmorduc et al subsequently showed that these defective particles were positively associated with chronic HBV infection and viral replication.
There are a number of lines of evidence that suggest the HBSP is involved in HBV pathogenesis. Soussan and colleagues, showed that HBSP was expressed in vivo , detecting anti-HBSP antibodies in sera from 46% of chronic HBV carriers. They also showed that 63% of patients with severe liver fibrosis were anti-HBSP positive. HBSP was also detected in liver tissue from chronically infected patients. The association of HBSP with liver fibrosis was also supported by in vitro assays that showed HBSP activated the profibrotic cytokine transforming growth factor (TGF)-a signalling pathway, implicating a role in liver pathogenesis. Increased levels of the cytokine tumour necrosis factor a (TNFa) were also observed in the sera of patients who were also anti-HBSP positive, suggesting HBSP was directly associated with liver damage which was perhaps mediated via the innate arm of the immune response. Soussan et al concluded that the presence of anti-HBSP antibodies in chronic carriers increased the relative risk factor of severe liver fibrosis more than 4-fold. In vitro assays also implicated a role for HBSP in cell cycle regulation, as HBSP induced apoptosis in the absence of a cell cycle block in transfected cells. The presence of HBSP was also associated with increased viral replication, as anti-HBSP antibody detection correlated with HBV DNA detection and HBeAg expression.
The association of HBSP with chronic HBV infection and liver fibrosis is of great interest, although it is still not clear whether HBSP is a pathogenesis marker. HBV is an unusual pararetrovirus in that although numerous splice-derived transcripts are detected both in vitro and in vivo , yet splicing is not an essential part of the HBV replication and infection cycle.,, This contrasts with the closely related duck hepatitis B virus (dHBV), which requires splicing to express one of its envelope genes. The identification of HBSP, a protein associated with disease progression that is encoded by a spliced transcript, suggests that RNA splicing may play a greater role in HBV pathogenesis than previously thought and further studies are clearly required in this field.
| Pathogenicity of Drug-Resistant HBV: Role of Compensatory Mutations|| |
Mutations that abolish or decrease the expression of HBeAg have now been shown to affect replication efficiency. HBeAg-negative chronic hepatitis B, which is most common in the Mediterranean region, is usually characterized by lower serum HBV DNA levels than those found in HBeAg-positive chronic hepatitis B. Hadziyannis and colleagues, studied a group of patients with HBeAg-negative chronic hepatitis B who were infected with genotype D HBV that contained both basal core promoter and precore stop-codon mutations. Development of lamivudine resistance in these patients (typically at rt M204I) was associated with relatively rapid increases in viraemia, culminating frequently in biochemical breakthrough, severe hepatitic flares and disease progression., In vitro studies by Chen and colleagues confirmed that the presence of the precore mutation (G1896A) could compensate for the replication deficiency in lamivudine-resistant HBV quasispecies. Other case reports have now established that drug-resistant HBV mutants are capable of causing severe and even even fatal disease.,,,, Such findings refute the notion that drug-resistant HBV mutants and other minority quasispecies are not virulent to the infected host.
Ogata and colleagues observed the co-occurrence of rtL80V/I (domain A) and rtL180M in conjunction with the M204V/I changes [Figure - 1] that confer lamivudine resistance in Japanese patients (genotype C) treated with lamivudine. The presence of double and triple changes has been associated with higher viral loads, increased lamivudine resistance and disease exacerbation. Longitudinal studies have shown that the mutations responsible for the sequence changes occurred almost simultaneously, just before viral breakthrough occurred and also that the mutants were displaced by wild-type genotype-C HBV after completion of therapy.
Similar observations have been recorded after liver transplantation among patients in whom life-threatening recurrence of HBV infection developed. HBV isolates from these patients contained compensatory mutations that enhanced their in vitro replication efficiency in the presence of lamivudine. Even greater enhancement and drug dependency occurred when mutations resulting in sG145R or sP120T, effecting key changes in the envelope protein were also present. None of these studies have looked at the role of the HBSP in the disease outcome.
| Pathogenesis of Fulminant Hepatitis|| |
The pathogenesis of fulminant hepatitis B still remains unclear, with the role of viral compared to host factors still unresolved. As discussed above, no single viral mutation or sequences have been identified to consistently account for all the cases. The precore stop codon (G1896A plus G1899A) has been frequently linked to fulminant hepatitis, but the association is not unequivocal., Several investigators have used bioinformatic and phylogenetic analysis to demonstrate possible lineages of HBV linked to clinical presentation. For example, mutations in precore (G1896A), mutations in the BCP region of either enhancer II or enhancer I as well as the negative regulatory element of the BCP in association with the presence of aberrant cystines and methionines in the X protein, have been found in several HBV isolates that were associated with fulminant hepatitis B (W Carman personal communication). Further molecular studies are required to confirm these possible associations.
| Public Health Aspects|| |
The genetic overlap between the polymerase, core and envelope genes as well as the transcriptional regulatory elements makes it possible that HBV mutants selected during therapy with nucleoside/nucleotide analogues can differ from wild-type virus with respect to replication competence and the nature of the envelope protein secreted. Patients receiving long-term antiviral therapy have been found to be infected with HBVs with multiple mutations in the Pol gene that have caused concomitant changes in the viral envelope. A small but significant number of these viruses have the potential to behave as vaccine or HBIG-escape mutants. These drug-resistant HBVs have also been shown to have the potential to be transmitted in particular settings, raising substantial public health concerns for the present vaccination programme. Additional compensatory mutations in other regions of the genome have also been observed in patients receiving prolonged antiviral therapy and these may restore or enhance the replication yield phenotype of the virus as the selection of these mutants has been associated with an increase in viral load and disease exacerbation.
| Conclusions|| |
Prospects for control of chronic HBV infection have never been better, given the development of more sensitive and sophisticated diagnostic tests and the renewed impetus provided by universal infant immunization programs as well as the ongoing development of newer antiviral agents by the pharmaceutical industry. However, control of HBV mutants will require new drugs, vaccines and treatment strategies and will become the next major challenge on the path to eventual elimination of HBV infection. Sufficiently potent inhibition of HBV replication should be able to prevent the development of drug resistance, mainly because mutagenesis is replication-dependent. Whatever the final outcome, an understanding of the molecular biology of HBV and a knowledge of its life cycle should provide the necessary insight to appreciate the clinical and public health significance of these recently identified variants/mutants of HBV.
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Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, 10 Wreckyn Street, North Melbourne, Victoria 3051
[Figure - 1], [Figure - 2]
[Table - 1]