Overview of hepatitis B virus mutations and theirimplications in the management of infection

Hepatitis B virus(HBV)affects approximately two billion people worldwide and more than 240 million people in the world are currently chronic carrier that could develop serious complications in the future,like liver cirrhosis and hepatocellular carcinoma.Although an extended HBV immunization program is being carried out since the early‘80s,representing effective preventive measure,leading to a dramatic reduction of HBV hepatitis incidence,globally HBV infection still represents a major public health problem.The HBV virus is a DNA virus belongs to the Hepadnaviridae family.The HBV-DNA is a circular,partial double strand genome.All coding information is on the minus DNA strand and it is organized into four open reading frames.Despite hepatitis B virus is a DNA virus,it has a high mutation rate due to its replicative strategy,that leads to the production of many nonidentical variants at each cycle of replication.In fact,it contains a polymerase without the proofreading activity,and uses an RNA intermediate(pg RNA)during its replication,so error frequencies are comparable to those seen in retroviruses and other RNA viruses rather than in more stable DNA viruses.Due to the low fidelity of the polymerase,the high replication rate and the overlapping reading frames,mutations occur throughout the genome and they have been identified both in the structural and not structural gene.The arise of mutations being to develop of a whole of viral variants called"quasi-species"and the prevalent population,which favors virus replication,was selected by viral fitness,host’s immune pressure and external pressure,i.e.,vaccination or antiviral therapy.Naturally occurring mutations were found both in acute and chronic subjects.In the present review we examine and discuss the most recent available data about HBV genetic variability and its significance.

Relationship between hepatocellular carcinoma and hepatitis B virus genotype with spontaneous YMDD mutations

AIM: To investigate the relationship between hepatitis B virus (HBV) genotype with spontaneous YMDD mu-tations and hepatocellular carcinoma (HCC) in HBV-related cirrhosis. METHODS: We investigated 264 liver cirrhosis pa-tients who were not treated with antiviral drugs, in-cluding 81 patients with HCC. YMDD mutations were detected by fluorescent hybridization bioprobe poly-merase chain reaction (PCR) and melting curve assay using the Diagnosis Kit for HBV YMDD Mutation. Serum HBV genotypes were detected by real-time PCR using genotype-specific TaqMan probes. Statistical analysis was performed according to data type using the t test, χ2 test and unconditional logistic regression analysis. RESULTS: In the HCC group, genotype C strains, spon-taneous YMDD mutations, and genotype C strains with YMDD mutations were detected in 33 (40.74%), 13 (16.05%) and 11 (13.58%) patients, respectively. In the liver cirrhosis (LC) group, HBV genotype C strains,spontaneous YMDD mutations, and genotype C strains with YMDD mutations were detected in 33 (18.03%), 7 (3.83%) and 2 (1.09%) patients, respectively. The dif-ferences in genotype C strains, spontaneous YMDD mu-tations, and genotype C strains with YMDD mutations between the two groups were statistically significant (χ2=15.441, P=0.000; χ2=11.983, P=0.001; P=0.000). In the HCC and LC groups, there were seven patients infected by genotype B strains with YMDD mutations and 13 by genotype C strains with YMDD mutations. Further research revealed that HCC oc-curred in 2 patients infected by genotype B strains with YMDD mutations and 11 infected by genotype C strains with YMDD mutations. The difference was statistically significant (P=0.000). Unconditional logistic regres-sion analysis revealed that patients infected by geno-type C strains with spontaneous YMDD mutations had a 7.775-fold higher risk for the development of HBV-related HCC than patients infected by other type HBV strains (P=0.013, 95%CI: 1.540-39.264). CONCLUSION: Genotype C strains with spontaneous YMDD mutations are an independent risk factor for HCC in LC patients and are important for early warning of HCC.

Naturally occurring hepatitis B virus reverse transcriptase mutations related to potential antiviral drug resistance and liver disease progression

The annual number of deaths caused by hepatitis B virus(HBV)-related disease, including cirrhosis and hepatocellular carcinoma(HCC), is estimated as 887000. The reported prevalence of HBV reverse transcriptase(RT) mutation prior to treatment is varied and the impact of preexisting mutations on the treatment of na?ve patients remains controversial, and primarily depends on geographic factors, HBV genotypes, HBe Ag serostatus, HBV viral loads, disease progression, intergenotypic recombination and co-infection with HIV. Different sensitivity of detection methodology used could also affect their prevalence results. Several genotype-dependent HBV RT positions that can affect the emergence of drug resistance have also been reported. Eight mutations in RT(rtL80I, rtD134N, rtN139K/T/H, rtY141F, rtM204I/V, rtF221Y, rtI224V, and rtM309K) are significantly associated with HCC progression. HBe Ag-negative status, low viral load, and genotype C infection are significantly related to a higher frequency and prevalence of preexisting RT mutations. Preexisting mutations are most frequently found in the A-B interdomain of RT which overlaps with the HBs Ag "a" determinant region, mutations of which can lead to simultaneous viral immune escape. In conclusion, the presence of baseline RT mutations can affect drug treatment outcomes and disease progression in HBVinfected populations via modulation of viral fitness and host-immune responses.

Key role of hepatitis B virus mutation in chronic hepatitis B development to hepatocellular carcinoma

Chronic hepatitis B virus(HBV) infection is a major risk factor for hepatocellular carcinoma(HCC). The HBV mutations, which include point mutation, deletion,insertion and truncation mutation of HBV gene in 4 open reading frames(S, C, P, X), are closely associated with HCC pathogenesis. Some mutations accumulated during chronic HBV infection could be regarded as a biomarker to predict the occurrence of HCC. The detection of the mutations in clinical practice could be helpful for defining better preventive and therapeutic strategies and, moreover, predicting the progression of liver disease.

Clinical utility of complex mutations in the core promoter and proximal precore regions of the hepatitis B virus genome

The core promoter and proximal precore regions are the most complex portions of the hepatitis B virus(HBV) genome. These regions cooperatively regulate viral replication and differentially regulate the synthesis of the viral proteins E,core,and X. Multiple mutations in these regions are associated with the persistency of viral infection and the development of cirrhosis and hepatocellular carcinoma(HCC). In South Korea,nearlyall HBVs are classified as HBV genotype C2; the majority of these viruses have the basal core promoter double mutation,a precore stop mutation,or both. These mutations may play a role in the alteration of viral and clinical features,and abundant and complex mutations are particularly prevalent in the core promoter and proximal precore regions. We previously demonstrated that the accumulation of ≥ 6 mutations at eight key nucleotides located in these regions(G1613A,C1653 T,T1753 V,A1762 T,G1764 A,A1846 T,G1896 A,and G1899A) is a useful marker to predict the development of HCC regardless of advanced liver disease. In addition,certain mutation combinations were predominant in cases with ≥ 4 mutations. In cases with ≤ 5 mutations,a low Hepatitis B e antigen titer(< 35 signal to noise ratio) was indicative of HCC risk. Viral mutation data of the single HBV genotype C2 suggest that the combined effect of the number and pattern of mutations in the core promoter and proximal precore regions is helpful in predicting HCC risk.

Sex disparity in viral load, inflammation and liver damage in transgenic mice carrying full hepatitis B virus genome with the W4P mutation in the preS1 region

AIM To study sex disparity in susceptibility to hepatocellular carcinoma(HCC), we created a transgenic mouse model that expressed the full hepatitis B virus(HBV) genome with the W4P mutation.METHODS Transgenic mice were generated by transferring the p HY92-1.1 x-HBV-full genome plasmid(genotype A2) into C57 Bl/6 N mice. We compared serum levels of hepatitis B surface antigen(HBs Ag), interleukin(IL)-6, and the liver enzymes alanine aminotransferase(ALT) and aspartate transaminase(AST), as well as liver histopathological features in male and female transgenic(W4PTG) mice and in nontransgenic littermates of 10 mo of age. RESULTS W4PTG males exhibited more pronounced hepatomegaly, significantly increased granule generation in liver tissue, elevated HBs Ag expression in the liver and serum, and higher serum ALT and IL-6 levels compared to W4PTG females or littermate control groups. CONCLUSION Together, our data indicate that the W4 P mutation in pre S1 may contribute to sex disparity in susceptibility to HCC by causing increased HBV virion replication and enhanced IL-6-mediated inflammation in male individuals. Additionally, our transgenic mouse model that expresses full HBV genome with the W4 P mutation in pre S1 could be effectively used for the studies of the progression of liver diseases, including HCC.

Precore/core region mutations of hepatitis B virus related to clinical severity

Despite the availability of an effective vaccine, hepatitis B virus (HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core (preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the preC/C region. Specifically, preC/C mutations in the MHC class II restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896A, are also significantly related to HBeAg-negative chronic infection. This review article mainly focuses on the HBV preC/C mutations that are related to disease severity and on the HBeAg serostatus of chronically infected patients.

Clinical implications of hepatitis B virus mutations:Recent advances

Hepatitis B virus(HBV)infection is a major cause of acute and chronic hepatitis,and of its long-term complications.It is the most variable among DNA viruses,mostly because of its unique life cycle which includes the activity of error-prone enzyme,reverse transcriptase,and the very high virion production per day.In last two decades,numerous research studies have shown that the speed of disease progression,reliability of diagnostic methods and the success of antiviral therapy and immunization are all influenced by genetic variability of this virus.It was shown that mutations in specific regions of HBV genome could be responsible for unwanted clinical outcomes or evasion of detection by diagnostic tools,thus making the monitoring for these mutations a necessity in proper evaluation of patients.The success of the vaccination programs has now been challenged by the discovery of mutant viruses showing amino acid substitutions in hepatitis B surface antigen(HBsAg),which may lead to evasion of vaccine-induced immunity.However,the emergence of these mutations has not yet raised concern since it was shown that they develop slowly.Investigations of HBV genetic variability and clinical implications of specific mutations have resulted in significant advances over the past decade,particularly in regard to management of resistance to antiviral drugs.In the era of drugs with high genetic barrier for resistance,on-going monitoring for possible resistance is still essential since prolonged therapy is often necessary.Understanding the frequencies and clinical implications of viral mutations may contribute to improvement of diagnostic procedures,more proper planning of immunization programs and creating the most efficient therapeutic protocols.

Quantitative evaluation of hepatitis B virus mutations and hepatocellular carcinoma risk:a meta-analysis of prospective studies

Background： The temporal relationship between hepatitis B virus （HBV） mutations and hepatocellular carcinoma （HCC） remains unclear. Methods： We conducted a meta-analysis including cohort and nested case-control studies to prospectively examine the HCC risk associated with common variants of HBV in the PreS, Enhancer Ⅱ, basal core promoter （BCP） and precore regions. Pertinent studies were identified by searching PubMed, Web of Science and the Chinese Biological Medicine databases through to November 2014. Study-specific risk estimates were combined using fixed or random effects models depending on whether significant heterogeneity was detected. Results： Twenty prospective studies were identified, which included 8 cohort and 12 nested case-control studies. There was an increased risk of HCC associated with any PreS mutations with a pooled relative risk （RR） of 3.82 [95% confidence interval （CI）： 2.59-5.61]. The pooled-RR for PreS deletion was 3.98 （95% CI： 2.28-6.95）, which was higher than that of PreS2 start codon mutation （pooled-RR=2.63, 95% CI： 1.30-5.34）. C1653T in Enhancer Ⅱ was significantly associated with HCC risk （pooled-RR=l.83; 95% CI： 1.21-2.76）. For mutations in BCP, statistically significant pooled-RRs of HCC were obtained for T1753V （pooled- RR=2.09; 95% CI： 1.49-2.94） and AI762T/G1764A double mutations （pooled-RR=3.11; 95% CI： 2.08- 4.64）. No statistically significant association with HCC risk was observed for G1896A in the precore region （pooled-RR=0.77; 95% CI： 0.47-1.26）. Conclusions： This study demonstrated that PreS mutations, C1653T, T1753V, and A1762T/G1764A, were associated with an increased risk of HCC. Clinical practices concerning the HCC risk prediction and diagnosis may wish to focus on patients with these mutations.

Variations and mutations in the hepatitis B virus genome and their associations with clinical characteristics

Hepatitis B virus(HBV) infection is major global issue, because chronic HBV infection is strongly associated with liver cancer. HBV spread worldwide with variousmutations and variations. This variability, called quasispecies, is derived from no proof-reading capacity of viral reverse transcriptase. So far, thousands of studies reported that the variety of genome is closely related to the geographic distribution and clinical characteristics. Recent technological advances including capillary sequencer and next generation sequencer have made in easier to analyze mutations. The variety of HBV genome is related to not only antigenicity of HBs-antigen but also resistance to antiviral therapies. Understanding of these variations is important for the development of diagnostic tools and the appropriate therapy for chronic hepatitis B. In this review, recent publications in relation to HBV mutations and variations are updated and summarized.

Hepatitis B virus genotypes and lamivudine resistance mutations in Jordan

AIM:To investigate and identify prevalent hepatitis B virus(HBV) genotypes and to explore lamivudine-resistant mutations among treated and untreated patients in Jordan.METHODS:A total of 107 cases with chronic hepatitis B were recruited from different medical centers in Jordan.Serological tests were preformed for all cases using a microparticle enzyme immunoassay.HBV Genotyping was performed for 70 cases using Line probe genotyping assay.The YMDD mutations were explored for 20 cases(4 were lamivudine naive) using the INNO-LiPA HBV DR assay.RESULTS:Genotype D was the only detected genotype.A total of 6 YMDD mutations were detected in 5 treated patients(31%) while one mutation was detected in the naive patients.Seventeen percent of cases were positive for HBeAg and had statistically significant higher levels of serum aminotransferases.CONCLUSION:HBV genotype D appears to be the only circulating type in Jordanian patients.The YMDD mutations were detected in 31% of lamivudine-treated cases with similar patterns to those found in the literature.We also found a relatively low prevalence of HBeAg expression among examined cases(17%).Awareness of these serologic,genotypic and resistance patterns might help in the formulation of management plans and for predicting clinical outcomes.Further larger scale studies are needed to confirm our results and to examine possible associations among clinical,serologic,and genetic patterns of HBV infections in Jordan.

X region mutations of hepatitis B virus related to clinical severity

Chronic hepatitis B virus (HBV) infection remains a major health problem, with more than 240 million people chronically infected worldwide and potentially 650000 deaths per year due to advanced liver diseases including liver cirrhosis and hepatocellular carcinoma (HCC). HBV-X protein (HBx) contributes to the biology and pathogenesis of HBV via stimulating virus replication or altering host gene expression related to HCC. The HBV X region contains only 465 bp encoding the 16.5 kDa HBx protein, which also contains several critical cis-elements such as enhancer II, the core promoter and the microRNA-binding region. Thus, mutations in this region may affect not only the HBx open reading frame but also the overlapped cis-elements. Recently, several types of HBx mutations significantly associated with clinical severity have been described, although the functional mechanism in most of these cases remains unsolved. This review article will mainly focus on the HBx mutations proven to be significantly related to clinical severity via epidemiological studies.

In vitro resistance to interferon of hepatitis B virus with precore mutation

AIM: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-α), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. METHODS: HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. RESULTS: We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind lll and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result: showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. CONCLUSION: These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Hepatitis B virus mutations related to liver disease progression of Korean patients

Hepatitis B virus(HBV)infection is a global health problem and more than 350 million people worldwide are chronic carriers of the virus.Despite the recent dramatic decline in HBV chronic patients through successful programs of hepatitis B surface antigen vaccination,South Korea is still recognized as an endemic area of HBV infection.HBV infections in South Korea exhibit several distinct features in epidemiologic and clinical aspects.In this review paper,we summarize the distinct HBV mutation patterns related to clinical severity and the molecular epidemiologic traits in Korean chronic patients based on previous reports.Generally,several lines of evidence,including our previous results,have led to the conclusion that a combination of the exclusive predominance of genotype C2,which is prone to mutations,the high prevalence of basal core promoter double mutations,and the presence of distinct immune responses against HBV proteins in the Korean population may generate the distinct HBV variants rarely or not encountered in other areas,which results in distinct clinical manifestations in Korean chronic patients.This may provide a novel insight into the relationships between clinical severity,HBV genotype distribution,and HBV naturally occurring variants.

Hepatitis B virus genetic mutations and evolution in liver diseases

Hepatitis B virus(HBV)belongs to the genus Orthohepadnavirus of the Hepadnaviridae family and is approximately 3.2 kb in length.Owing to a lack of proofreading capacity during reverse transcription and a high replication rate,HBV exhibits as quasispecies.To detect the genetic mutations of HBV,many methods with different sensitivities and throughputs were developed.According to documentary records,HBV mutation and evolution were important vial parameters in predicting disease progression and therapeutic outcome.In this review,we separately discussed the correlation between HBV genomic mutations in four open reading frames and liver disease progression.Since some of the results were controversial from different laboratories,it remains to be seen whether functional analyses will confirm their role in modifying the course of infection.

Biological impacts of "hot-spot" mutations of hepatitis B virus X proteins are genotype B and C differentiated

AIM: To investigate the biological impacts of 'hot-spot'mutations on genotype B and C HBV X proteins (HBx).METHODS: Five types of'hot-spot' mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I,I127T+K130M+V131I, or K130M+V131I+F132Y, respectively,were generated by means of site-directed mutagenesis.To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity,and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were lysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test.RESULTS: As compared to standard genotype B HBx,mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity,compared to standard genotype C HBx, F132Y and K130M+V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity,while the mutants of I127T and I127T+K130M+V131I showed no differences.CONCLUSION: 'Hot-spot' mutations affect the antiproliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most 'hot-spot' mutations on HBx are genotype B and C differentiated.

A mutation of the start codon in the X region of hepatitis B virus DNA in a patient with non-B,non-C chronic hepatitis

There are cases of hepatitis involving occult hepatitis B virus(HBV)infection in which,even though the HB surface antigen(HBsAg)is negative,HBV-DNA is detected by a polymerase chain reaction(PCR).We con-ducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-yearold female.A diagnosis of non-B non-C chronic hepatitis was made.Although HBV markers,such as HBs antibody(anti-HBs),anti-HBc,HBeAg and anti-HBe,were negative,HBV-DNA was positive.Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR.Sequence analysis of the two obtained bands was conducted by direct sequencing.Compared with the control strains,the ATG(Methionine)start codon in the X region had mut ated to GTG(Valine).It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.

Hepatitis B virus mutations potentially conferring adefovir/ tenofovir resistance in treatment-naive patients

Anti-hepatitis B virus(HBV)therapy leads to the emer- gence of mutant viral strains during the treatment of chronic hepatitis B with nucleos(t)ides analogues. The existence of HBV variants with primary antiviral resistance may be important for treatment choice. We studied two patients with chronic HBV infection by sequencing the HBV polymerase gene.They had adefovir-and tenofovir-related mutations in the viral polymerase,although they had never been treated. These mutations were rtV214A/rtN238T in one patient and rtA194T in the other.Thus,mutations in untreated patients deserve cautious surveillance.These data indicate that mutations that can theoretically confer adefovir or tenofovir resistance may emerge in treatmentnaive patients.

Mutations in hepatitis B virus core regions correlate with hepatocellular injury in Chinese patients with chronic hepatitis B

AIM: To elucidate the relationship between the frequency of core mutations and the clinical activity of hepatitis B virus (HBV)-related liver disease and to characterize the amino acid changes in the core region of HBV.METHODS: We studied 17 Chinese patients with chronic hepatitis B according to their clinical courses and patterns of the entire core region of HBV.RESULTS: Amino acid changes often appeared in the HBV core region of the HBV gene in patients with high values of alanine aminotransferase (ALT) or with the seroconversion from HbeAg to anti-HBe. The HBV core region with amino acid changes had high frequency sites that corresponded to HLA Ⅰ/Ⅱ restricted recognition epitopes reported by some investigators.CONCLUSION: The core amino acid changes of this study occur due to influence of host immune system. The presence of mutations in the HBV core region seems to be important for predicting the clinical activity of hepatitis B in Chinese patients.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.