Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A-F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.     

Ultra-Deep Pyrosequencing (UDPS) Data Treatment to Study Amplicon HCV Minor Variants

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%.Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.     

Analysis of Ultra-Deep Pyrosequencing and Cloning Based Sequencing of the Basic Core Promoter/Precore/Core Region of Hepatitis B Virus Using Newly Developed Bioinformatics Tools

Aims

The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS).

Methods

Four serum samples, infected with either genotype D or E, from HBeAg-positive and HBeAg-negative patients were randomly selected. UDPS and CBS were used to sequence the basic core promoter/precore region of HBV. Two online bioinformatics tools, the “Deep Threshold Tool” and the “Rosetta Tool” (http://hvdr.bioinf.wits.ac.za/tools/), were built to test and analyze the generated data.

Results

A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. In the four samples, substitutions, detected at 0.5% threshold or above, were identified at 39 unique positions, 25 of which were non-synonymous mutations. Sample #2 (HBeAg-negative, genotype D) had substitutions in 26 positions, followed by sample #1 (HBeAg-negative, genotype E) in 12 positions, sample #3 (HBeAg-positive, genotype D) in 7 positions and sample #4 (HBeAg-positive, genotype E) in only four positions. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5∶1. Compared to genotype E isolates, genotype D isolates showed greater variation in the X, basic core promoter/precore and core regions. Only 18 of the 39 positions identified by UDPS were detected by CBS, which detected 14 of the 25 non-synonymous mutations detected by UDPS.

Conclusion

UDPS data should be approached with caution. Appropriate curation of read data is required prior to analysis, in order to clean the data and eliminate artefacts. CBS detected fewer than 50% of the substitutions detected by UDPS. Furthermore it is important that the appropriate consensus (reference) sequence is used in order to identify variants correctly.     

Clinical Application of Estimating Hepatitis B Virus Quasispecies Complexity by Massive Sequencing: Correlation between Natural Evolution and On-Treatment Evolution

Aim

To evaluate HBV quasispecies (QA) complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUC) treatment.

Methods

Ultra-deep pyrosequencing of HBV preCore/Core regions in 30 sequential samples (baseline [diagnosis], treatment-free, and treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status over time: HBeAg+ (N = 4), HBeAg- (N = 2), and fluctuating HBeAg (transient seroreversion/seroconversion pattern) (N = 4). QA complexity was defined by Shannon entropy, mutation frequency, nucleotide diversity, and mutation frequency of amino acids (MfAA) in preCore and Core.

Results

The QA was less complex in HBeAg+ than in HBeAg- or fluctuating HBeAg. High complexity in preCore was associated with decreased viral replication (preCore MfAA negatively correlated with HBV-DNA, p = 0.005). QA complexity in the treatment-free period negatively correlated with values seen during treatment. Specific variants were mainly selected in the Core region in HBeAg- and fluctuating HBeAg patients, suggesting higher immune pressure than in HBeAg+.

Conclusions

The negative correlation between QA natural evolution and on-treatment evolution indicates the importance of pre-treatment QA study to predict QA changes in NUC nonresponders. Study of QA complexity could be useful for managing HBV infection.     

The state of the art of adeno-associated virus-based vectors in gene therapy

Background

Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed

Methods

A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1).

Results

Out of 295 patients, 156 (53.2%) had Acute(CAH), 71 (24.2%) were HBV Carriers, 54 (18.4%) had Chronic liver disease (CLD) Hepatitis. 14 (4.7%) were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108), Chronic (39), and Carrier (53). Cirrhosis/HCC (7) were HBV/D positive. Genotype A was the second most prevalent with 28 (13%) in acute cases, 12 (22.2%) in chronics, 14 (19.7%) in carriers and 5 (41.7) in Cirrhosis/HCC patients. Mixed genotype (A/D) was found in 20 (12.8%) of Acute patients, 3 (5.6%) of Chronic and 4 (5.6%) of carriers, none in case of severe liver conditions.

Conclusion

Mixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome.     

Hepatitis B virus infection profile in hemodialysis patients in Central Brazil: prevalence, risk factors, and genotypes

Hemodialysis patients are at high risk for hepatitis B virus (HBV) infection. A survey was conducted in the hemodialysis population of the state of Goiás, Central Brazil, aiming to assess the prevalence of HBV infection, to analyse associated risk factors, and also to investigate HBV genotypes distribution. A total of 1095 patients were interviewed in 15 dialysis units. Serum samples were screened for HBV serological markers by enzyme-linked immunosorbent assay. Hepatitis B surface antigen (HBsAg) positive samples were tested for HBV DNA by polymerase chain reaction and genotyped by restriction fragment length polymorphism. Global HBV infection prevalence was 29.8% (95% CI: 27.1-32.5). Multivariate analysis of risk factors showed that male gender, length of time on hemodialysis, and blood transfusion before 1993 were associated with HBV positivity. HBV DNA was detected in 65.4% (17/26) of the HBsAg-positive samples. Thirteen of 17 HBV DNA positive samples were genotyped. Genotype D (61.5%) was predominant, followed by A (30.8%), while genotype F was detected in only one (7.7%) sample.     

乙型肝炎病毒逆转录酶区基因序列准种与变异特点

乙型肝炎病毒(Hepatitis B Virus,HBV)P基因编码产物从功能上分为末端蛋白(1～178aa)、间隔区(179～336aa)、逆转录酶区(337～682aa)和RNA酶H区(683～816aa),各区有相应的生物学功能;逆转录酶区包含S基因主蛋白编码区.近年来的研究提出HBV感染者体内存在有准种[1,2]的假说.我们以逆转录酶区序列为研究靶区域,应用聚合酶链反应(PCR)技术扩增慢性乙型肝炎患者血清中的靶基因序列,随机选择克隆测序,比较其结果,证明了HBV准种特点的存在,并发现多种基因突变形式.     

HIV-1 Tropism Dynamics and Phylogenetic Analysis from Longitudinal Ultra-Deep Sequencing Data of CCR5- and CXCR4-Using Variants

Objective

Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships.

Methods

Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained.

Results

Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence.

Conclusions

CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.     

部分肝病患者和健康人血清中HBV基因型分析

确定了内蒙古海拉尔地区乙型肝炎病毒 (HBV)感染者HBV基因型的分布情况。采用基因型特异引物的巢式聚合酶链式反应方法对 38例无症状HBV携带者 ,3例急性肝炎 ,41例慢性肝炎 ,3例肝硬化 ,3例肝癌患者和38例健康人血清中HBV基因型进行分析。结果显示 ,12 6例中有 91例检测到HBVDNA ,其中 ,HBVB型 ,C型分别为 11例 ( 12 .1% )和 6 8例 ( 74.7% ) ,同时检测到B型和C型者有 8例 ( 8.8% ) ,有 4例 ( 4.4% )尚未确定基因型别。可见 ,在海拉尔地区HBV感染同时存在HBVB型和C型 ,而且C型是这一地区流行的主要基因型。     

Molecular epidemiology of hepatitis B viral serotypes and genotypes in taiwan

Subtypes of hepatitis B virus (HBV) have specific geographic distributions and can serve as epidemiological markers. The relationship of HBV serotypes and genotypes in Taiwan and their correlation with the domiciles of origin in 122 patients with chronic HBV infection were investigated. The serotype of HBV was determined by comparing the surface gene encoding amino acids 22-148 of the major surface protein with published sequences. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. Serotype adw accounted for 70% (85/122) of all HBVs, with the remaining belonging to serotype adr. All adr HBVs were genotype C, regardless of the patient's domicile. Of the 85 adw HBVs, 69 (81%) were genotype B, 10 (12%) were genotype C, 5 (6%) were genotype F and only 1 (1%) was genotype A. In the 31 patients originating from mainland China, the prevalence of adr/genotype C was higher than in the 91 Taiwanese patients (15/31 vs. 22/91; p < 0.05). The distribution of the HBV serotypes and genotypes was not significantly different between 17 patients born in Taiwan (6 adw/genotype B, 2 adw/genotype C, 1 adw/genotype F and 8 adr/genotype C) and 14 patients born in mainland China (5 adw/genotype B, 2 adw/genotype C and 7 adr/genotype C). Our results indicate that in Taiwan, most HBVs of serotype adw are genotype B, and all HBVs of serotype adr are genotype C. Patients with origins in mainland China have a higher proportion of serotype adr/genotype C infection.     

Clinicopathologic features and genotyping of patients with chronic HBV infection in the Upper Egypt

The aim of this study was to determine the clinicopathologic features and Hepatitis B virus genotypes in HBV-infected patients in the Upper Egypt. Eighty-three HBsAg-positive patients (28 carriers, 14 with chronic hepatitis, 32 with liver cirrhosis and 9 with hepatocellular carcinoma) were enrolled. Blood was collected and serum samples obtained were screened for Hepatitis markers genotyping was conducted for 6 HBV genotypes (A through F) using a method for genotyping HBV by primer specific polymerase chain reaction. Genotype D was the only genotype detected in different clinical forms of chronic HBV infection (carriers, chronic hepatitis, cirrhosis and hepatocellular carcinoma) and, in all patients who had elevated or normal alanine aminotransferase levels and in all ages. HBeAg was absent in 78 patients suggesting the presence of pre-core or core mutations. Positive correlation was found among serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), histological activity index and grade of hepatitis. This study provides the first indication about the clinicopathologic features of HBV-infected patients in the Upper Egypt. It also reports the predominance of genotype D in this region.     

应用系统发育树分析DDBJ基因库中HBV基因序列的基因型

为了充分利用核酸库中的HBV序列信息,探讨DDBJ核酸库中HBV基因序列的基因分型,采用Clustal X（1.8）软件比较HBV基因序列前S区序列差异并产生系统发育树。通过对下载的1471条HBV基因序列进行系统分析。获得了228条前S/S区完整的HBV基因序列,其中有66条序列的基因型已被各种方法所证实。利用软件分析绘制了基于228条HBV前S区基因序列的系统发育树。66条已知基因型HBV基因序列在系统发育树上的分型与其原有基因型完全吻合。在228条HBV基因序列中,有207条序列分别属于A、B、C、D、E、F和G等7个基因型,但另外21条序列不能归属于上述7个基因型的任何一种,而且它们又分为彼此相互独立存在的两群,暂分别称之为未分型I和未分型Ⅱ,经比较未分型I、Ⅱ和其他7个基因型前S区核苷酸序列,发现未分型I、Ⅱ和D型前S区都有33个核苷酸缺失,但三者基因缺失片段的位置和形式各不相同,但其它六型前S区无大片段基因缺失。结果说明采用基于前S区的系统发育树基因分型分析方法正确可靠,除了现已证实的7个基因型外,尚可能存在另外两个新的HBV基因型。     

Molecular Epidemiology and Genotyping of Hepatitis B Virus of HBsAg-Positive Patients in Oman

Background

Hepatitis B virus (HBV) infection is a major global health burden with distinct geographic public health significance. Oman is a country with intermediate HBV carrier prevalence; however, little is known about the incidence of HBV variants in circulation. We investigated the HBV genotype distribution, the occurrence of antiviral resistance, and HBV surface antigen (HBsAg) escape mutations in HBsAg-positive patients in Oman.

Methods

Serum samples were collected from 179 chronically HBV-infected patients enrolled in various gastroenterology clinics in Oman. HBV genotypes were determined by sequencing and phylogenetic analysis. Mutations in the HBV polymerase and the HBsAg gene were characterized by mutational analysis.

Results

HBV genotypes D (130/170; 76.47%) and A (32/170; 18.28%) are predominant in Oman. The HBV genotypes C and E were less frequent (each 1.18%), while the HBV genotypes B, G, F, and H were not detected. Four patients revealed HBV genotype mixtures (HBV-A/D and D/C). The analyses of vaccine escape mutations yield that 148/170 (87.06%) HBV sequences were wild type. 22/170 (12.94%) HBV sequences showed mutations in the “a” determinant of the HBsAg domain. Two patients showed the described HBV vaccine escape mutation sP120T. 8/146 (5.48%) HBV isolates harbored mutations in the HBV polymerase known to confer resistance against antiviral therapy. Especially the lamivudine resistance mutations rtL180M/rtM204V and rtM204I were detected.

Conclusion

This study shows the distribution of HBV genotypes, therapy resistance, and vaccine escape mutations in HBV-infected patients in Oman. Our findings will have a major impact on therapy management and diagnostics of chronic HBV infections in Oman to control HBV infection in this intermediate HBV-endemic country.     

Hepatitis B genotype G and high frequency of lamivudine-resistance mutations among human immunodeficiency virus/hepatitis B virus co-infected patients in Brazil

In this study, we evaluated the hepatitis B virus (HBV) genotype distribution and HBV genomic mutations among a group of human immunodeficiency virus-HBV co-infected patients from an AIDS outpatient clinic in S?o Paulo. HBV serological markers were detected by commercially available enzyme immunoassay kits. HBV DNA was detected using in-house nested polymerase chain reaction and quantified by Cobas Amplicor. HBV genotypes and mutations in the basal core promoter (BCP)/pre-core/core regions and surface/polymerase genes were determined by sequencing. Among the 59 patients included in this study, 55 reported prior use of lamivudine (LAM) or tenofovir. HBV DNA was detected in 16/22 patients, with a genotype distribution of A (n = 12,75%), G (n = 2,13%), D (n = 1,6%) and F (n = 1,6%). The sequence data of the two patients infected with genotype G strongly suggested co-infection with genotype A. In 10 patients with viremia, LAM-resistance mutations in the polymerase gene (rtL180M + rtM204V and rtV173L + rtL180M + rtM204V) were found, accompanied by changes in the envelope gene (sI195M, sW196L and sI195M/sE164D). Mutations in the BCP and pre-core regions were identified in four patients. In conclusion, genotype G, which is rarely seen in Brazil, was observed in the group of patients included in our study. A high prevalence of mutations associated with LAM-resistance and mutations associated with anti-HBs resistance were also found among these patients.     

A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5′UTR — the most highly conserved region of HCV — and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant™ HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant™ HCV Genotype Assay LiPA (74/78, 95%). The concordance between the two methods was 100% for genotype determination (74/74). At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively). Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant™ HCV assay. Genotype “1” subtypes (1a and 1b) were correctly identified by the Versant™ HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping.     

Occult hepatitis B virus infection among injecting drug users in the Central-West Region of Brazil

The prevalence of occult hepatitis B virus (HBV) infection was investigated in 149 hepatitis B surface antigen (HBsAg) negative injecting drug users (IDUs) in the Central-West Region of Brazil. Of these individuals, 19 were positive for HBV DNA, resulting in an occult HBV infection prevalence of 12.7% (19/149); six of these 19 individuals had anti-HBV core and/or anti-HBV surface antibodies and 13 were negative for HBV markers. All IDUs with occult hepatitis B reported sexual and/or parenteral risk behaviours. All HBV DNA-positive samples were successfully genotyped. Genotype D was the most common (17/19), followed by genotype A (2/19). These findings reveal a high prevalence of occult HBV infection and the predominance of genotype D among IDUs in Brazil''s Central-West Region.     

四份中国HCV阳性血清中包膜蛋白E1/E2准种基因的序列分析及新型插入突变的发现

为分析四份中国丙型肝炎病毒（HCV）阳性血清中包膜蛋白E1/E2基因的准种特征。本研究对从4份中国HCV阳性血清（1b亚型：274、366、383;2a亚型：283）中提取的HCV核酸,采用逆转录-聚合酶反应扩增编码全长E1/E2蛋白（191～764aa）的基因片段,随机挑取多个克隆测序。根据E1/E2基因核苷酸的序列与其他相关序列（来自于GenBank）构建亲缘性关系进化树,进行核苷酸与氨基酸同源性分析并对重要的基因位点进行分析。共获得阳性克隆序列43个（274株10个,283株12个,366株13个,383株8个）,发现高变区HVR1、HVR2的基因异质性高,而其他抗体中和表位及跨膜区I、II及N末端糖基化位点相对保守。并首次发现在HCV 2a亚型（283血清）中多个准种序列存在1279nt（E1区,313aa）处单碱基插入优势基因突变,导致HCV包膜蛋白编码突变与中断（E2区,398aa）。本研究对中国HCV代表株包膜蛋白E1/E2编码基因的准种多样性及一种新型插入突变进行了描述,可为进一步研究HCV免疫逃避与慢性化机制提供重要信息。关键词：丙型肝炎病毒;包膜蛋白;序列分析;准种;插入突变     

Genotype-specific genomic markers associated with primary hepatomas, based on complete genomic sequencing of hepatitis B virus

We aimed to identify genomic markers in hepatitis B virus (HBV) that are associated with hepatocellular carcinoma (HCC) development by comparing the complete genomic sequences of HBVs among patients with HCC and those without. One hundred patients with HBV-related HCC and 100 age-matched HBV-infected non-HCC patients (controls) were studied. HBV DNA from serum was directly sequenced to study the whole viral genome. Data mining and rule learning were employed to develop diagnostic algorithms. An independent cohort of 132 cases (43 HCC and 89 non-HCC) was used to validate the accuracy of these algorithms. Among the 100 cases of HCC, 37 had genotype B (all subgenotype Ba) and 63 had genotype C (16 subgenotype Ce and 47 subgenotype Cs) HBV infection. In the control group, 51 had genotype B and 49 had genotype C (10 subgenotype Ce and 39 subgenotype Cs) HBV infection. Genomic algorithms associated with HCC were derived based on genotype/subgenotype-specific mutations. In genotype B HBV, mutations C1165T, A1762T and G1764A, T2712C/A/G, and A/T2525C were associated with HCC. HCC-related mutations T31C, T53C, and A1499G were associated with HBV subgenotype Ce, and mutations G1613A, G1899A, T2170C/G, and T2441C were associated with HBV subgenotype Cs. Amino acid changes caused by these mutations were found in the X, envelope, and precore/core regions in association with HBV genotype B, Ce, and Cs, respectively. In conclusion, infections with different genotypes of HBV (B, Ce, and Cs) carry different genomic markers for HCC at different parts of the HBV genome. Different HBV genotypes may have different virologic mechanisms of hepatocarcinogenesis.