Sequence analysis of pre-S/surface and pre-core/core promoter genes of hepatitis B virus in chronic hepatitis C patients with occult HBV infection

Although occult hepatitis B virus (HBV) infection in individuals without detectable hepatitis B surface antigen (HBsAg) may occur and has been reported to be common in patients with chronic hepatitis C, the related molecular mechanisms remain unknown. With the polymerase chain reaction, serum HBV DNA was sought in 100 HBsAg-negative patients with chronic hepatitis C virus (HCV)-infection. In those with occult HBV infection, possible genomic variability of HBV was evaluated by amplification and direct sequencing of pre-S, surface, and pre-core/core promoter genes. In total, 10 of the 100 patients (10%) had detectable serum HBV DNA, documenting an occult HBV infection. A deletion mutant in the pre-S gene was found in one patient and mutations of the a determinant of HBsAg were observed in 2. In addition, a novel core promoter mutant (a dinucleotide substitution: T-to-C at nucleotide 1,802 and T-to-G at nucleotide 1,803, T1802C/T1803G) was found frequently in patients with occult HBV infection as compared to sex- and age-matched HBsAg-positive patients (80 vs. 10%, P < 0.001). In conclusion, the data suggest occult HBV infection is not uncommon in chronic hepatitis C patients in Taiwan, and a novel core promoter mutant may be associated with the absence of circulating HBsAg in these patients.     

Characteristics of core promoter and precore stop codon mutants of hepatitis B virus in Vietnam

In Asia, genotypes B and C are the most common genotypes of hepatitis B virus (HBV); and genotype C causes more severe liver disease. Core promoter/precore (CP/PC) mutants, known to be linked to these genotypes, could have an impact on the progression and severity of liver disease. Sera of 115 patients, including 39 acute and 76 chronic Vietnamese HBV infected patients, were tested for their liver profile, HBeAg, HBV genotypes, and HBV DNA level. Fragments of 282 nucleotides covering CP/PC were amplified, sequenced, and analysed. In the acute group, CP/PC mutants accounted for 38.4 and 25.6%, respectively. Genotype B was found to be predominant (74.3%, P < 0.05) and linked to the PC mutant (A1896) (P < 0.05). In the chronic group, CP/PC mutants accounted for 61.7 and 32.8%. CP mutants, especially the T1762/A1764 double mutant, were found to correlate with genotype C (81%, P < 0.001), liver cirrhosis, and hepatocellular carcinoma (P < 0.05). Therefore, genotype C in Vietnam, which carried high rate of C-1858 (70%), could play an important role in causing severe chronic liver disease.     

乙型肝炎病毒核心启动子及前C区突变检测基因芯片的制备及临床应用

目的 建立检测乙型肝炎病毒核心启动子及前C区突变基因芯片并探讨其临床应用的价值.方法 设计并合成针对核心启动子1762/1764、1814及前C区1896位点突变的特异性探针,制备寡核苷酸芯片.采用不对称PCR对该区域进行扩增,扩增产物与芯片杂交后分析结果,并评价该方法的特异性、灵敏度.采用该方法检测138例HBV DNA阳性血清标本.结果 该方法能够特异地检测乙型肝炎病毒核心启动子1762/1764、1814及前C区1896位点,灵敏度达1×101拷贝/μl.138例HBV DNA阳性血清标本中,T1762/A1764突变40例（28.99%）,C1814突变11例（7.97%）,A1896突变16例（11.59%）.前C区A1896突变在高拷贝组中明显高于低拷贝组（P＜0.01）.结论 本研究建立的基因芯片能够准确同时检测HBVV核心启动子区突变和前C区突变,前C区A1896突变可能与HBV复制状态有关.     

Distribution of genotype/subtype and mutational spectra of the surface gene of hepatitis B virus circulating in Hanoi, Vietnam

In order to ascertain the molecular epidemiological features and mutational spectra of hepatitis B virus (HBV) in Hanoi, Vietnam, direct sequencing of the 219-nucleotide fragment of the surface (S) gene of HBV from the sera of 40 patients mostly with chronic hepatitis were carried out. The samples were classified into genotypes by phylogenetic and genotype-specific analysis, and subtypes by the deduced amino acid sequences. The results showed that genotype B with ayw1 was predominant genotype/subtype (63%), followed by genotype C with adr (18%). The quasi-species nature of the HBV in the sera was observed in 24 of 40 samples examined. One sample (HN109) showed mixture of genotypes B and C. Among 26 amino acid substitutions, 16 were the variants and the remainders were mutations. In the "a" determinant region, three mutations with methionine to leucine (L) changes at the 133 amino acid residue were in the first loop and no mutations were in the second loop. A new mutation, threonine to methionine at 126 amino acid residue, was observed in one sample. In conclusion, the analysis of the S gene region of HBV showed that in Hanoi, genotype B with ayw1 was prevalent and the quasi-species nature of HBV was also common.     

Genotypic dominance and novel recombinations in HBV genotype B and C co-infected intravenous drug users

Pathogenic differences among hepatitis B virus (HBV) genotypes have been documented. However, the interaction between different HBV genotypes remains unclear. Herein, we chose HBV genotypes B (HBV/B) and C (HBV/C) co-infected intravenous drug users to study this issue. HBV genotype was determined in 40 HBsAg, anti-HCV, and anti-HDV co-positive intravenous drug users by using genotype-specific primers. The distribution of HBV genotype was as follows: HBV genotype B alone in 29 (72.5%); HBV genotype C alone in 4 (10.0%); and mixed HBV genotype B and HBV genotype C in 7 (17.5%). The interaction between HBV genotype B and HBV genotype C within the same individual was further studied in the seven intravenous drug users with HBV genotype B and HBV genotype C co-infection. By direct sequencing of the pre-S region, only HBV genotype B was detected. When 10-21 clones of the pre-S region were propagated from each intravenous drug user and sequenced, most of the clones were HBV genotype B. Novel recombinations between HBV genotype B and HBV genotype C occurred in four clones (M7-5, M1-10, M1-21, and M1-24) from two intravenous drug users (M7 and M1). The recombination breakpoints were estimated at nucleotide 3120-3171 for M7-5, at nucleotide 3060-3191 for M1-10, and at nucleotide 2910-2950 for M1-21 and M1-24 by SimPlot program. The recombination sites of these HBV/pre-S C-B and B-C recombinants may be within the pre-S1 region. The results in this study suggest that HBV/B is the dominant strain in HBV genotypes B and C co-infected intravenous drug users in Taiwan, and recombinations between different HBV genotype are not unusual. The impact of recombination on the evolution of HBV and their clinical significance remains to be studied.     

Genetic heterogeneity of the precore and the core promoter region of genotype C hepatitis B virus during lamivudine therapy

It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.     

乙型肝炎病毒基因型C亚型和核心启动子、前C/核心区基因变异的关系

目的 研究慢性乙型肝炎病毒（HBV）感染者中HBV基因型C亚型（HBV／C）的核心启动子、前C／核心区基因变异情况，分析HBV／C亚型的病毒学特征。方法 用酶联免疫法（ELISA）筛选出79例HBV／C，再用聚合酶链反应．限制性酶长度多态性分析方法（PCR-RFLP）进行HBV／C亚型分析；同时针对HBV核心启动子、DreC／核心区基因进行半巢式PCR及PCR产物直接测序。结果 ①79例HBV／C中，33例（41．8％）为HBV／C1亚型，46例（58．2％）为HBV／C2亚型。②HBV／C1亚型仅见于来自中国南方的患者（P〈0．0001）。③A1898位点变异仅见于HBV／C1亚型（P=0．056），V1753位点变异在HBV／C1亚型中多见（P〈0．05）；HBV／C2以T1858（90％）、A1896（40％）位点变异多见（P〈0．008）。T1762／A1764位点变异在HBV／C两种亚型中均常见。④肝细胞癌（HCC）患者中，V1753和T1762／A1764变异最常见（P〈0．05）。结论 HBV／CI和HBV／C2在中国有明显的地区差异；V1753合并T1762／A1764双变异与发展为HCC相关，尤其在HBV／C1患者。     

Prevalence of HBV core promoter/precore/core mutations in Gambian chronic carriers.

One hundred forty-two precore/core sequences were obtained from Gambian chronic hepatitis B virus (HBV) carriers and the predominant variants defined. The two point mutations, from A to T and G to A at nt positions 1762 and 1764 in the basic core promoter region, were found in only 7/99 (7%) of the samples where this region was sequenced. These mutations were found in both HBeAg-positive and -negative patients. The precore stop-codon mutation at nt position 1896 was found in 14/51 (27%) of HBeAg-negative samples, which is a lower prevalence rate in comparison with other parts of the world with high carrier rates. In HBeAg-positive patients the core amino acid sequences were conserved, but after seroconversion to anti-HBe significantly more changes were apparent. Several of the amino acid substitutions found have been described previously been in wild-type viruses of other genotypes.     

HBV前C基因突变及分型与HBV母婴传播关系的研究

目的探讨HBV(乙型肝炎病毒)前C基因突变、血清型及基因型分型与HBV母婴传播的关系。方法100份HBsAg阳性孕产妇及其新生儿的外周血,发生母婴传播血清为病例组,其他为对照组。ELISA法检测乙肝两对半,根据S基因判断HBV血清型和基因型。对HBV前C基因进行扩增及序列分析。结果99份HBV血清型全为adw型;39例发生母婴传播HBV基因C型25例;B型13例;E型1例。60例未发生母婴传播,HBV基因B型50例;C型10例。两组HBV基因分型结果差异有统计学意义(P0.05)。39例发生母婴传播中27例发生了点突变,12例无任何突变,突变发生率为69%。结论HBV母亲C基因型可能更易发生母婴传播。HBV母婴传播中大多存在HBV前C基因变异并导致氨基酸取代,其中主要涉及丝氨酸、苏氨酸、酪氨酸等的磷酸化位点,这些重要位点氨基酸的取代可能在HBV母婴传播的发病中起作用。     

HBV subtype as a marker of the clinical course of chronic HBV infection in Japanese patients

Hepatitis B virus (HBV) genotype C is predominant in Japan. However, many HBV subtypes are involved in each genotype, and the clinical manifestations in the patients associated with each subtype remain unknown. Therefore, we investigated the relationship between HBV subtype and clinical aspects of chronic HBV infection. The subtype of 237 patients with chronic HBV infection, including 74 asymptomatic carriers, was determined. The subtypes of 110 HBV carriers undergoing long-term follow-up management were determined twice to detect subtypic changes. The clinical features of the patients were also studied with regard to presence or absence of subtypic change. The subtypic distribution in the 237 HBV carriers was as follows: subtype adr, 161 (68%); subtype adw, 25 (11%); subtype adwr, 12 (5%); subtype ar, 24 (10%); subtype adyr, 4 (2%); and unclassified, 8 (3%). The proportion of asymptomatic carriers in patients with subtype adw was significantly higher than those in patients with subtype adr (56% vs. 28%, P < 0.05). In addition, the proportion of HCC in patients with subtype adwr was significantly higher than those in patients with subtype adr (25% vs. 6%, P < 0.05). The prevalence of subtype adr in 74 asymptomatic carriers tended to decrease with age (82% in carriers aged < or =35 years vs 43% in those aged > or =61 years, P < 0.05). The subtypic change and the course of chronic HBV infection had no significant correlation. These results suggest that HBV subtypes are associated with the clinical course of chronic HBV infection.     

Basal core promoter, precore region mutations of HBV and their association with e antigen, genotype, and severity of liver disease in patients with chronic hepatitis B in India

Spontaneous mutations of hepatitis B virus (HBV) could influence the severity of liver disease. Since the basal core promoter (BCP) and the precore (Pc) regions are important for viral replication, these regions were examined for naturally occurring mutations and were correlated with the genotype, e antigen status, and severity of liver disease. In 82 patients with histologically confirmed chronic hepatitis B, the BCP and Pc regions were sequenced and aligned with known wild-type sequences. Sequence based HBV genotyping was done and HBV DNA was quantified. Thirty-three (40%) patients had decompensated chronic liver disease and the remaining patients had chronic hepatitis B. Forty-six (56%) patients were HBeAg positive. HBV genotype A was found in 28%, D in 65%, and B/C in 7.3%. The Pc G1896A mutation was more common in HBeAg-negative (33% vs. 2%, P < 0.01) patients and was genotype D specific. The Pc G1862T mutation was detected more often in HBeAg-positive than HBeAg-negative (37% vs. 11%, P < 0.01) patients and was genotype A specific (P < 0.01). BCP mutations at the 1,762/64 nucleotide positions were common in HBeAg negative than positive (36% vs. 13%, P < 0.05) and were equally common in different genotypes. TA 1-3 region mutations of the BCP were significantly higher in HBeAg-negative as compared to HBeAg-positive patients (78% vs. 26%, P < 0.01). BCP mutations had significantly higher HBV DNA levels. It is concluded that Pc G1862T mutant is Genotype A-specific but is not always associated with e antigen. The TA 1-3 rich mutations of BCP region are also associated with the absence of e antigen in Indian patients.     

The prevalence of hepatitis B virus preS deletions occurring naturally in Korean patients infected chronically with genotype C

Although Korea is one of the endemic areas for hepatitis B virus infection (HBV), the prevalence of deletions in HBV preS region occurring naturally have not been determined. In the present study, the prevalence of preS deletions was determined in terms of clinical state and HBeAg serostatus in 120 patients with different clinical features [59 HBeAg positive, 61 HBeAg negative; 38 asymptomatic carriers, 21 patients with chronic hepatitis, 21 patients with liver cirrhosis, 40 patients with hepatocellular carcinoma (HCC)]. A total of 37 strains (30.8%) harbored deletions in the preS region. Overall, the frequencies of preS deletions tended to increase gradually according to the degree of the clinical severity of liver disease. The prevalence of preS1 deletions in HCC patients tended to be higher than in patients with liver cirrhosis (32.5% vs. 19%). The prevalence of preS2 deletions in HBeAg negative patients was significantly higher than in HBeAg positive patients (23% vs. 6.8%). The type of deletion encountered most frequently was one disrupting the preS1 start codon [14/37 strains (37.8%)], which showed a very high prevalence in HCC patients (9/40, 22.5%; HCC vs. asymptomatic carriers, P=0.048). These results suggest that there might be the discrepancy between preS1 and preS2 mutations in the mechanism of enhancing the progression of chronic liver disease, especially the development of HCC and to maintain tolerance during the stage of immune tolerance. Specific deletion of the type disrupting preS1 start codon may play important roles in hepatocarcinogenesis, at least in Korean patients with chronic HBV infection.     

Variations in the functional domain of basal core promoter of hepatitis B virus among Eastern Indian patients with prevalence of genotypes A, C, and D among the same ethnic population

Mutations in the basal core promoter (BCP) and precore (PC) regions are associated with persistent and intermittently high hepatitis B virus (HBV) replication in several patients. The variability in the functional domains of BCP and PC region of HBV and their association with disease progression and clinical outcome were assessed in Eastern India, an unique region where three HBV genotypes, A, D, and C are prevalent among the same ethnic group. PCR amplification and direct sequencing of BCP and PC region was done on sera obtained from 130 HBsAg positive subjects with different clinical presentations. Associations of the apparent risk factors with clinical advancement were evaluated by statistical methods including multiple logistic regression analyses (MLR). HBV genotype A was present in 33.08%, C in 25.38%, and D in 41.54% cases. Genotypes A and C were associated with higher rate of T1762/A1764 mutations than the most predominant genotype D. HBeAg negative state was associated with considerably higher rate of C1753 mutation. T1762/A1764 along with C1753 was common among cirrhosis and T1762/A1764 without C1753 was frequent among chronic liver disease cases. No significant association was found between A1896 point mutation and clinical status. Multivariate analysis revealed that T1762/A1764 double mutation, HBV/A, age ≥25 years, C1753 and A1899 were critical factors for clinical advancement while age ≥25 years and C1753 as significant predictor for cirrhosis in comparison with chronic liver disease. In conclusion, the analysis of the BCP variability may help in monitoring the progression towards advanced liver disease in Eastern Indian patients.     

HBV基因型与前C/C启动子变异及抗病毒疗效的关系

目的 了解深圳市乙型肝炎病毒（HBV）基因分型情况，探讨HBV基因型与前C／C启动子变异、乙肝的病程进展及抗病毒疗效的关系。方法 用单克隆抗体ELISA法（mAbs ELISA）对深圳市165例HBV感染者进行HBV基因分型；随机抽取24例慢性乙型肝炎（CUB）患者，用基因芯片技术检测HBV前C／C启动子变异；回顾性分析HBV基因型与干扰素、贺普丁抗HBV疗效的关系。结果 ①165例患者中，以B型106例（64.2％）和C型48例（29．1％）为主。慢性无症状乙肝病毒携带者（ASC）组B型占95．4％，肝硬化（LC）组C型占64．7％（P〈0．05）。②24例CHB患者中，16例（10例B型，6例C型）发生HBV前C／C启动子变异：前C区变异（nt1896、1862）者10例（B型9例，C型1例）。基本C区启动子变异（BCP）变异（nt1762、1764）者6例（B型1例，C型5例）。③用干扰素治疗的27例HBeAg（＋）CHB患者，达到完全应答者B型11例（62．5％）较C型1例（9．1％）多见（P〈0．05）。用贺普丁治疗的29例HBeAg（＋）CHB患者，持续应答者B型15例（78．9％）较C型3例（30．0％）多见（P〈0．05）。结论 ①深圳市HBV基因分型以B型为主，C型次之。②C型较B型易发生BCP变异，发生肝硬化机会较高，且对于扰素及贺普丁疗效较差。     

Genotype X/C recombinant (putative genotype I) of hepatitis B virus is rare in Hanoi,Vietnam—genotypes B4 and C1 predominate

There are eight known genotypes of hepatitis B virus, A–H, and several subgenotypes, with rather well‐defined geographic distributions. HBV genotypes were evaluated in 153 serum samples from Hanoi, Vietnam. Of the 87 samples that could be genotyped, genotype B was found in 67 (77%) and genotype C in 19 (22%). All genotype C strains were of subgenotype C1, and the majority of genotype B strains were B4, while a few were B2. The genotype X/C recombinant strain, identified previously in Swedish patients of indigenous Vietnamese origin, was found in one sample. This variant, proposed to be classified as genotype I, has been found recently also by others in Vietnam and Laos. The current study indicates that the genotype X/C recombinant may represent approximately 1% of the HBV strains circulating in Vietnam. J. Med. Virol. 82:1327–1333, 2010. © 2010 Wiley‐Liss, Inc.     

Prevalence of HBV genotypes in Central and Eastern Europe

The importance of hepatitis B virus (HBV) genotypes for disease progression and response to interferon-alpha-based treatment is well established. While almost all patients in the Mediterranean area are infected with HBV genotype D, HBV genotype A is dominant in Northern Europe. However, the distribution of HBV genotypes is unknown for several Central and Eastern European countries. Data are described of 1313 HBsAg-positive patients recruited at 14 referral centers in eight countries. There were only very few cases of HBV genotype B, C, E, F, and H infection while HBV genotypes A and D were found in 42% and 48% of patients, respectively. Eight percent of patients had positive bands for more than one genotype using the hybridization assay. The frequency of genotype A was higher in Poland (77%) and the Czech Republic (67%) as compared to Hungary (47%), Lithuania (41%), Croatia (8%), and Germany (32%). In contrast, HBV genotype D was most frequent in Croatian, Romanian, and Russian patients with 80%, 67%, and 93% of cases, respectively. In conclusion, HBV genotype A versus D showed significantly different distribution patterns in Central and Eastern Europe which deserves consideration for national guidelines and treatment decisions.     

乙型肝炎病毒基因B和C型与阿德福韦酯疗效关系的研究

目的探讨乙型肝炎病毒（HBV）基因B和C型与阿德福韦酯的疗效关系。方法选取应用阿德福韦酯10mg/d进行抗病毒治疗12、24、36、48周的136例慢性乙型肝炎患者作为研究对象,用聚合酶链反应（PCR）-微板核酸杂交-酶联免疫吸附试验（ELISA）法检测分析基因型,评价阿德福韦酯治疗不同基因型HBV的疗效。结果 136份样本中,HBV基因B型39份,HBV基因C型97份。在治疗12、24、36、48周时,B基因型患者丙氨酸氨基转移酶（ALT）的变化、血清HBVDNA水平下降比例≥2log10及完全抑制情况与C基因型患者相比差异均无统计学意义（P〉0.05）。结论本研究所观察的样本中,阿德福韦酯的抗病毒疗效与HBV基因B型和C型均无相关性。