Genetic variability of hepatitis B virus and response to antiviral treatments: Searching for a bigger picture
Authors: Lilly K.W. Yuen, Stephen A. Locarnini
Journal: Journal of Hepatology (2008)

The profile of mutational clusters associated with lamivudine resistance can be constrained by HBV genotypesJournal of HepatologyVol. 50Issue 3PreviewTo investigate the different clusters of mutations associated with lamivudine resistance in HBV genotypes D and A. Full-Text PDF Chronic infection with hepatitis B virus (HBV) remains a global public health concern. There are approximately 350 million people in the world who are persistently infected with this virus [[1]WHO. Hepatitis B fact sheet. http://www.who.int/mediacentre/factsheets/fs204/en/. Accessed October 2008.Google Scholar], and they have higher risks of developing complications of the disease that can range from mild to chronic hepatitis to the more advanced disease manifestations which include cirrhosis with hepatic decompensation and hepatocellular carcinoma (HCC). The only means currently to prevent such complications in individuals who are already chronically infected with the virus is to control viral replication and reduce liver damage with the use of interferon-alpha (IFN-α) and or nucleos(t)ide analogs (NA). Nonetheless, efficacies of these treatment methods are limited, and there is increasing evidence to suggest that these limitations are associated with the genetic variability of HBV [2Wiegand J. Hasenclever D. Tillmann H.L. Should treatment of hepatitis B depend on hepatitis B virus genotypes? A hypothesis generated from an explorative analysis of published evidence.Antivir Ther. 2008; 13: 211-220PubMed Google Scholar, 3Kramvis A. Kew M.C. Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy.J Viral Hepat. 2005; 12: 456-464Crossref PubMed Scopus (159) Google Scholar, 4Liu C.J. Kao J.H. Genetic variability of hepatitis B virus and response to antiviral therapy.Antivir Ther. 2008; 13: 613-624PubMed Google Scholar]. The HBV genome is complex and has at least four open reading frames (ORF) that overlap one another so that the total coding capacity is around one and a half times the genomic length. This viral family is classified into eight geographically defined genotypes (A to H) based on ⩾8% diversity of their entire genome nucleotide sequences. HBV genotypes have significant associations with severity of liver disease, clinical outcome, and response to antiviral therapies [[3]Kramvis A. Kew M.C. Relationship of genotypes of hepatitis B virus to mutations, disease progression and response to antiviral therapy.J Viral Hepat. 2005; 12: 456-464Crossref PubMed Scopus (159) Google Scholar]. C-genotype infections tend to have higher disease progression rates [5Chu C.M. Liaw Y.F. Genotype C hepatitis B virus infection is associated with a higher risk of reactivation of hepatitis B and progression to cirrhosis than genotype B: a longitudinal study of hepatitis B e antigen-positive patients with normal aminotransferase levels at baseline.J Hepatol. 2005; 43: 411-417Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 6Orito E. Mizokami M. Sakugawa H. Michitaka K. Ishikawa K. Ichida T. et al.A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group.Hepatology. 2001; 33: 218-223Crossref PubMed Scopus (384) Google Scholar] and lower spontaneous HBeAg seroconversion rates [7Kao J.H. Chen P.J. Lai M.Y. Chen D.S. Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers.J Med Virol. 2004; 72: 363-369Crossref PubMed Scopus (149) Google Scholar, 8Nakayoshi T. Maeshiro T. Nakayoshi T. Nakasone H. Sakugawa H. Kinjo F. et al.Difference in prognosis between patients infected with hepatitis B virus with genotype B and those with genotype C in the Okinawa Islands: a prospective study.J Med Virol. 2003; 70: 350-354Crossref PubMed Scopus (44) Google Scholar] than B-genotype infections. C-genotype infections also require longer for HBeAg seroconversion to take place [[9]Miyakawa Y. Okamoto H. Mayumi M. The molecular basis of hepatitis B e antigen (HBeAg)-negative infections.J Viral Hepat. 1997; 4: 1-8Crossref PubMed Scopus (47) Google Scholar], thus active infections can persist longer than B-genotype infections, and in turn may enhance the development of severe liver diseases such as cirrhosis and HCC. Similarly, chronic infections appear to be more common with European A-genotype viruses [[10]Mayerat C. Mantegani A. Frei P.C. Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection?.J Viral Hepat. 1999; 6: 299-304Crossref PubMed Scopus (246) Google Scholar], and A-genotype infections seem less likely to persist after hepatitis B e antigen (HBeAg) seroconversion [[11]Rodriguez-Frias F. Buti M. Jardi R. Cotrina M. Viladomiu L. Esteban R. et al.Hepatitis B virus infection: precore mutants and its relation to viral genotypes and core mutations.Hepatology. 1995; 22: 1641-1647Crossref PubMed Google Scholar] when compared with D-genotype infections. The latter observation is probably due to the inability for the European A-genotype viruses to select the G1896A mutation. This mutation introduces a stop codon in the precore (pc) gene to inhibit the production of HBeAg, and in turn allows the virus to evade the immune system of the host. However, G1896 is in the epsilon region of the pregenomic RNA that is involved with RNA encapsidation and pairs with the nucleotide at genome position 1858. HBV A-genotype has a cytosine at genome position 1858, thus without a corresponding C1858T mutation, the G–C bond would prevent a G1896A selection. However, relationships between clinical outcomes and HBV genotypes are not always unambiguous. Individuals infected with the same HBV genotype can exhibit dissimilar clinical and virological characteristics due to infections by different HBV subgenotypes [[12]Miyakawa Y. Mizokami M. Classifying hepatitis B virus genotypes.Intervirology. 2003; 46: 329-338Crossref PubMed Scopus (255) Google Scholar]. Importantly, some of the subgenotypes were derived by recombination with other HBV genotypes. For example, Sugauchi et al. [[13]Sugauchi F. Kumada H. Sakugawa H. Komatsu M. Niitsuma H. Watanabe H. et al.Two subtypes of genotype B (Ba and Bj) of hepatitis B virus in Japan.Clin Infect Dis. 2004; 38: 1222-1228Crossref PubMed Scopus (66) Google Scholar] classified HBV B-genotype into Ba and Bj subgenotypes, and demonstrated Ba-genotype was in fact a B-genotype that had a recombination event(s) with the pc and core regions of C-genotype. This finding provided an insight into why individuals with B-genotype infections can develop HCC at an earlier age than those with C-genotype infections in Taiwan [[14]Kao J.H. Chen P.J. Lai M.Y. Chen D.S. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B.Gastroenterology. 2000; 118: 554-559Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar]. IFNs are naturally occurring cytokines primarily produced by the innate immune system that have antiviral, antiproliferative and immunomodulatory effects [[15]Farrell P.J. Sen G.C. Dubois M.F. Ratner L. Slattery E. Lengyel P. Interferon action: two distinct pathways for inhibition of protein synthesis by double-stranded RNA.Proc Natl Acad Sci USA. 1978; 75: 5893-5897Crossref PubMed Scopus (157) Google Scholar]. As an antiviral agent, IFN-α functions to promote viral control by immune stimulation and direct cytokine-mediated suppression of HBV replication [[16]Gunther S. Paulij W. Meisel H. Will H. Analysis of hepatitis B virus populations in an interferon-alpha-treated patient reveals predominant mutations in the C-gene and changing e-antigenicity.Virology. 1998; 244: 146-160Crossref PubMed Scopus (36) Google Scholar]. However, this immune modulator is limited in its use in clinical practice due to a number of factors: expense, parenteral administration, significant side effects and poor tolerance. Thus, knowledge gained by studying the natural clinical outcomes of infections caused by different HBV genotypes/subgenotypes could be used to predict potential benefits of IFN-α for individuals infected with HBV and to improve the overall response rate of this drug. Influences that HBV genotypes have on the response rates of IFN-based treatments (IFN-α and PEG-IFN-α) have been summarised in a recent review [[4]Liu C.J. Kao J.H. Genetic variability of hepatitis B virus and response to antiviral therapy.Antivir Ther. 2008; 13: 613-624PubMed Google Scholar]. The majority of these studies showed consistent findings in that European A-genotype infections had higher response rates to IFN-based therapies compared with D-genotype infections, and similarly B-genotype infections had higher response rates than C-genotype infections. However, HBV genotypes/subgenotypes cannot be used as the sole predictor for IFN responsiveness since efficacy of IFN-based treatments is also influenced by a number of other factors such as pre-treatment HBV DNA and alanine aminotransferase (ALT) levels, age, and the presence of basal core promoter (bcp) and pc mutations in the viruses [[17]Zampino R. Marrone A. Cirillo G. del Giudice E.M. Utili R. Karayiannis P. et al.Sequential analysis of hepatitis B virus core promoter and precore regions in cancer survivor patients with chronic hepatitis B before, during and after interferon treatment.J Viral Hepat. 2002; 9: 183-188Crossref PubMed Scopus (20) Google Scholar], as well as human leukocyte antigen-DRB1 (HLA-DRB1) types in the host [[18]Chu R.H. Ma L.X. Wang G. Shao L.H. Influence of HLA-DRB1 alleles and HBV genotypes on interferon-alpha therapy for chronic hepatitis B.World J Gastroenterol. 2005; 11: 4753-4757PubMed Google Scholar]. Further explorations of the relationships between these host as well as viral factors and IFN-α response are required. NAs are designed to inhibit the function of HBV reverse transcriptase (rt), and are widely used for treating patients with chronic hepatitis B. To date, five NAs have been licensed for treatment (lamivudine/LMV, telbivudine/Ld-T, entecavir/ETV adefovir/ADV, and tenofovir/TDF). Although these drugs are highly effective in viral replication control, efficacy is often hindered by the selection of drug-resistant viruses. The primary resistance mutations have been identified for LMV [[19]Allen M.I. Deslauriers M. Andrews C.W. Tipples G.A. Walters K.A. Tyrrell D.L. et al.Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group.Hepatology. 1998; 27: 1670-1677Crossref PubMed Scopus (785) Google Scholar], Ld-T [[20]Yang H. Qi X. Sabogal A. Miller M. Xiong S. Delaney WEt Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV.Antivir Ther. 2005; 10: 625-633PubMed Google Scholar], ETV [[21]Colonno R.J. Rose R.E. Pokornowski K. Baldick C.J. Klesczewski K. Tenney D.J. Assessment at three years shows high barrier to resistance is maintained in entecavir-treated nucleoside naive patients while resistance emergence increases over time lamivudine refractory patients.Hepatology. 2006; 44: 229AGoogle Scholar] and ADV [[22]Angus P. Vaughan R. Xiong S. Yang H. Delaney W. Gibbs C. et al.Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase.Gastroenterology. 2003; 125: 292-297Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar], and they are all confined to eight HBV-rt codons (169, 180, 181, 184, 202, 204, 236 and 250) [[23]Locarnini S. Primary resistance, multidrug resistance, and cross-resistance pathways in HBV as a consequence of treatment failure. Hepatol Int 2008: doi:10.1007/s12072-12008-19048-12073.Google Scholar]. However, some of these primary resistance mutations can compromise viral replication fitness, and to compensate, these particular HBV mutants would often co-select other rt mutations to partially restore replication efficiency. Most compensatory mutations reported to date are associated with LMV resistance (LMVr); and rtL180M is the most widely reported. While rtL180M alone does not affect LMV susceptibility, the level of LMVr does increase substantially when co-selected by rtM204V mutants (153- to ⩾10,000-fold increase in EC50 compared to wild-type virus) [[19]Allen M.I. Deslauriers M. Andrews C.W. Tipples G.A. Walters K.A. Tyrrell D.L. et al.Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group.Hepatology. 1998; 27: 1670-1677Crossref PubMed Scopus (785) Google Scholar]. Interestingly, rtL180M is nearly always co-selected with rtM204V mutants while prevalence for rtL180M+rtM204I mutants is comparatively lower. In contrast, rtL80I/V is commonly co-selected by rtM204I mutants. The association between infection with rtL80I/V HBV and severe liver disease in C-genotype infections was first noted by Ogata et al. [[24]Ogata N. Fujii K. Takigawa S. Nomoto M. Ichida T. Asakura H. Novel patterns of amino acid mutations in the hepatitis B virus polymerase in association with resistance to lamivudine therapy in japanese patients with chronic hepatitis B.J Med Virol. 1999; 59: 270-276Crossref PubMed Scopus (81) Google Scholar], but the ability of the rtL80I/V change to enhance the replication efficiency of rtM204I/V mutants has only been confirmed recently [[25]Warner N. Locarnini S. Kuiper M. Bartholomeusz A. Ayres A. Yuen L. et al.The L80I Substitution in the Reverse Transcriptase Domain of the Hepatitis B Virus Polymerase Is Associated with Lamivudine Resistance and Enhanced Viral Replication In Vitro.Antimicrob Agents Chemother. 2007; 51: 2285-2292Crossref PubMed Scopus (90) Google Scholar]. This latter study also showed rtL80I/V occurs almost exclusively in association with LMVr, and co-selection with rtM204I or rtM204V occurred in approximately 46% and 9% of the HBV sequences respectively using a large database [[26]Yuen L.K. Ayres A. Littlejohn M. Colledge D. Edgely A. Maskill W.J. et al.SEQHEPB: A sequence analysis program and relational database system for chronic hepatitis B.Antiviral Res. 2007; 75: 64-74Crossref PubMed Scopus (56) Google Scholar]. Another important compensatory mutation associated with LMVr is rtV173L, and this change is nearly always detected in association with rtL180M and rtM204V mutations. Prevalence for the "triple" rtV173L+rtL180M+rtM204V mutant is overall low, but its incidence rate varied significantly between immuno-competent (mono-infection – 9%) and immuno-suppressed (liver transplantation/ HIV co-infection – 20%) individuals with LMVr chronic HBV infections [[27]Delaney WEt Yang H. Westland C.E. Das K. Arnold E. Gibbs C.S. et al.The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro.J Virol. 2003; 77: 11833-11841Crossref PubMed Scopus (236) Google Scholar]. These viral mutants can replicate at approximately 90% efficiency of the wild-type viruses, while the rtL180M+rtM204V mutants could only replicate at 40% efficiency. More importantly, the rtV173L and rtM204V changes in the "triple mutant" result in two corresponding hepatitis B surface antigen (HBsAg) changes (sE164D and sI195M) due to the overlapping nature of HBV genomes. Such mutants with both HBsAg changes have markedly reduced anti-HBs binding ability to the HBsAg [[28]Torresi J. Earnest-Silveira L. Deliyannis G. Edgtton K. Zhuang H. Locarnini S.A. et al.Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy.Virology. 2002; 293: 305-313Crossref PubMed Scopus (191) Google Scholar], which suggests potential vaccine-escape characteristics. This is a serious public health concern since there is increased likelihood that these drug-associated mutants will propagate among the vaccinated individuals, particularly in the setting of HIV infection. Cross- and multi-drug resistance further complicate the issue of HBV drug resistance. Some rt mutations can exhibit high levels of cross-resistance between NAs [[20]Yang H. Qi X. Sabogal A. Miller M. Xiong S. Delaney WEt Cross-resistance testing of next-generation nucleoside and nucleotide analogues against lamivudine-resistant HBV.Antivir Ther. 2005; 10: 625-633PubMed Google Scholar], and can accumulate over time to produce complex mutation patterns leading to multi-drug resistance. Nonetheless, the ability to discover novel drug resistance profiles for HBV is limited due to the large variations of rt mutation patterns that can occur (naturally occurring and drug-associated mutations) in HBV isolates. In this issue of the Journal, Svicher et al. [[29]Svicher V. Gori C. Trignetti M. Visca M. Micheli V. Bernassola M. et al.The profile of mutational clusters associated with lamivudine resistance can be constrained by HBV genotypes.J Hepatol. 2009; 50: 461-470Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar] addressed this problem by using a combination of statistics and advanced data mining techniques to identify two distinctive HBV mutation clusters that are associated with LMVr in European A- and D-genotype isolates. In a cohort of 89 LMV-failing patients (61 HBV mono-infections and 28 HIV/HBV co-infections), trend analysis showed HIV/HBV co-infected patients tended to be younger, had a lower proportion of negative serum HBeAg, and required less time to develop LMVr. These findings correlated with previous European studies [10Mayerat C. Mantegani A. Frei P.C. Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection?.J Viral Hepat. 1999; 6: 299-304Crossref PubMed Scopus (246) Google Scholar, 11Rodriguez-Frias F. Buti M. Jardi R. Cotrina M. Viladomiu L. Esteban R. et al.Hepatitis B virus infection: precore mutants and its relation to viral genotypes and core mutations.Hepatology. 1995; 22: 1641-1647Crossref PubMed Google Scholar] when the data were stratified in the context of HBV genotypes – higher prevalence for A-genotype infections was noted among the HIV/HBV co-infected patients (57%), and D-genotype infections for the HBV mono-infected patients (90.1%). Importantly, clustal analysis of LMVr associated mutations revealed rtM204V mutants always co-select rtL180M, and have a tendency to occur with other mutations in the HBV-rt B domain such as rtV173L and rtT184A/S, which are also associated with ETV resistance [[21]Colonno R.J. Rose R.E. Pokornowski K. Baldick C.J. Klesczewski K. Tenney D.J. Assessment at three years shows high barrier to resistance is maintained in entecavir-treated nucleoside naive patients while resistance emergence increases over time lamivudine refractory patients.Hepatology. 2006; 44: 229AGoogle Scholar]. In contrast, rtM204I alone is sufficient to result in LMVr, but viral mutants that carry this mutation can occur with other mutations such as those in the HBV-rt A domain, including rtS53N, rtT54Y and rtL80I/V. Interestingly, preferences for these two distinct LMVr mutation clusters differ between the two HBV genotypes studied – A-genotype preferentially select rtM204V (68.2%) while D-genotype showed similar prevalence for rtM204V(39.1%) and rtM204I (45.3%). Another important contribution made by this study is the demonstration that these data mining techniques used can be applied to analyse the interactions between HBV polymerase and envelope during antiviral treatments. Data exploratory techniques have been utilised to study drug resistance mutation profiles in HIV for a number of years, but only recently for HBV [[26]Yuen L.K. Ayres A. Littlejohn M. Colledge D. Edgely A. Maskill W.J. et al.SEQHEPB: A sequence analysis program and relational database system for chronic hepatitis B.Antiviral Res. 2007; 75: 64-74Crossref PubMed Scopus (56) Google Scholar]. One of the earliest studies conducted in the HIV area was by Gonzales et al. [[30]Gonzales M.J. Wu T.D. Taylor J. Belitskaya I. Kantor R. Israelski D. et al.Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors.Aids. 2003; 17: 791-799Crossref PubMed Scopus (107) Google Scholar] who used two advanced data mining methods, binomial (phi) correlation coefficient calculations and principal component analysis, to aid in the identification of novel mutations that may be associated with cross- and multi-drug resistance in antiretroviral therapy. Using experiences gained in similar HIV drug resistance studies, Svicher et al. [[29]Svicher V. Gori C. Trignetti M. Visca M. Micheli V. Bernassola M. et al.The profile of mutational clusters associated with lamivudine resistance can be constrained by HBV genotypes.J Hepatol. 2009; 50: 461-470Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar] demonstrated that these data exploratory techniques can also be applied to study HBV drug resistance profiles. Another tool available for antiretroviral research that can also benefit HBV drug resistance studies is the public database and datamining system hosted by Stanford University [[31]Rhee S.Y. Gonzales M.J. Kantor R. Betts B.J. Ravela J. Shafer R.W. Human immunodeficiency virus reverse transcriptase and protease sequence database.Nucleic Acids Res. 2003; 31: 298-303Crossref PubMed Scopus (677) Google Scholar], a central location where data can be pooled and utilised by all researchers. Combining such public database systems and advanced data mining methods [[26]Yuen L.K. Ayres A. Littlejohn M. Colledge D. Edgely A. Maskill W.J. et al.SEQHEPB: A sequence analysis program and relational database system for chronic hepatitis B.Antiviral Res. 2007; 75: 64-74Crossref PubMed Scopus (56) Google Scholar] will enable HBV researchers to further study and explore relationships between disease progression, drug-associated rt mutations as well as corresponding HBsAg mutations, and can contribute significantly to the development of treatment algorithms which can be used for better management of patients with chronic hepatitis B.