T_(1762)A_(1762)变异对乙型肝炎病毒C启动子活性影响

国的乙型肝炎病毒（ＨＢＶ）Ｃ基因调节序列存在较多变异,为探讨在诸多变异中,Ｔ１７６２Ａ１７６４变异对Ｃ区启动子（ＣＰ）活性究竟起何作用。方法用克隆及ＰＣＲ产物直接测序,检测Ｔ１７６２Ａ１７６４变异;并将含调节序列的ＰＣＲ产物插入到氯霉素乙酰转移酶（ＣＡＴ）表达质粒中,转染ＨｅｐＧ2细胞并瞬时表达。结果通过比较野生株及变异株在ＣＰ区序列及ＣＡＴ表达水平显示,Ｔ１７６２Ａ１７６４变异在使ＣＡＴ表达下调中起主要作用。结论Ｔ１７６２Ａ１７６４变异在使ＣＰ活性下调中起主要作用。     

T1762A1764变异对乙型肝炎病毒C启动子活性影响

目的 乙型肝炎病毒Ｃ基因调节序列存在较多变异,为探讨在诸多变异中,Ｔ１７６２Ａ１７６４变异对Ｃ区启动子活性空间起何作用。方法 用克隆及ＰＣＲ产物直接测序,检测Ｔ１７６２Ａ１７６４变异;并将含调节序列的ＰＣＲ产物插入到氯霉素乙酰转移酶表达质粒中,转染ＨｅｐＧ２细胞并瞬时表达。结果 通过比较野生株及变异株在ＣＰ区序列及ＣＡＴ表达水平显示,Ｔ１７６２Ａ１７６４变异在使ＣＡＴ表达下调中起主要作用。结论Ｔ１     

快速简便筛检乙型肝炎病毒C基因启动子变异的方法及应用

目的明确中国乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子（ＢＣＰ）区段变异特点及其可能的临床意义。方法应用错配聚合酶链反应限制性片段长度多态性（ＰＣＲ－ＲＦＬＰ）方法，结合核苷酸序列分析，检测１４３例ＨＢｓＡｇ阳性的ＨＢＶ慢性感染者中Ｘ基因／ＢＣＰ区的核苷酸（ｎｔ）１７６２碱基由Ａ→Ｔ和１７６４碱基由Ｇ→Ａ变异。结果在１１４份ＨＢＶＤＮＡ阳性血清中，３７例感染ＢＣＰ变异株，其中３１例为慢性肝炎病例，６例为慢性ＨＢＶ无症状感染者（ＡｓＩ）；其中４１例ＨＢｅＡｇ阳性ＡｓＩ中仅２例检出，１８例ＨＢｅＡｇ阴性ＡｓＩ中也有４例检出。ＨＢｅＡｇ阳性的６５例中，１３例有ＢＣＰ区变异，而ＨＢｅＡｇ阴性的４９例中，２４例有ＢＣＰ区变异（Ｐ＜０．０１）。结论错配ＰＣＲ－ＲＦＬＰ检测这一对点突变，具有快速、简便、适用的优点；ＨＢＶ毒株ＢＣＰ变异可能与肝病变活动有一定关系。     

HBV X基因／C基因启动子变异在重型肝炎病人的发生率

目的：了解重型乙型肝为病人乙型肝炎病毒（ＨＢＶ）Ｘ基因／Ｃ基因启动子（ＢＣＰ）变异性发生情况，方法；用错配聚合酶链反应（ＰＣＲ）限制性片长度多态性分析（ＲＦＬＰ）检测中国不同地区５８例重型乙型肝炎病人ＨＢＶ毒株ＢＣＰ区变异，并与前Ｃ区变异情况比较，部分病例经测序证实，结果：５８列中可见ＨＢＶ毒株ＢＣＰ区和／或前Ｃ区变异者４３例（７４％），ＢＣＰ区第２个ＡＴ丰富区有一对点突变核苷酸（ｎｔ）１７６２碱     

乙型肝炎病毒基因组变异对干扰素治疗的影响

目的 研究乙型肝炎病毒基因组Ｘ区和前Ｃ／Ｃ区多部位变异对干扰素（ＩＦＮ）治疗的影响。方法 利用套式聚合酶链反应产物直接测序技术，测定１７例慢性乙型肝炎患者ＩＦＮ治疗前病毒核苷酸序列。结果 ５例Ｔ＾１７６２－Ａ＾１７６４变异株感染者均对ＩＦＮ治疗有应答，而在１２例无Ｔ＾１７６２－Ａ＾１７６４突变患者中仅３例有应答。在８例Ａ＾１８９６变异株感染者中，５例同时伴Ｃ区Ｂ细胞表位ａａ１０７￣１１８突变，其中     

乙型肝炎病毒前C区基因变异和外周血单个核细胞增殖反应

一、资料与方法１．对象：选取１９９６－１９９７年宁波市传染病医院８４例住院乙型肝炎患者,按１９９５年（北京）全国肝炎诊断标准,急性肝炎６例,慢性肝炎轻度２２例,中度ＺＩ例,重度２２例,慢性重型肝炎１３例。ＨＢｓＡｇ和ＨＢＶＤＮＡ均阳性,其中６９例ＨＢｅＡｇ阳性,１１例抗一ＨＢｅ阳性,对照组１１例为健康献血员,ＨＢＶ血清学标志物均阴性。２．ＨＢＶ前Ｃ区基因１８９６位点突变的检测：用错配ＰＣＲ限制片段长度多态性分析［１］。ｐＣＲ产物按酶切后ＤＮＡ条带不同,分为野生株,野生株优势变异,变异株优势变异和…     

乙型肝炎病毒X蛋白对NFкB启动子和增强子反式激活作用

应用杆状病毒穿梭质粒载体与ＨＢＶＸ表达质粒共转染ＨｅＰＧ２细胞,研究ＨＢＶＸ蛋白对杆状病毒载体中的ＮＦкＢ启动子和增强子反式激活作用,作为进一步杆状病毒载体应用研究的基础。 １．材料与方法：（１）为构建ＮＦкＢ启动子和增强子驱动的氯霉素乙酰转移酶（ＣＡＴ）报告基因质粒,合成寡核苷酸引物 Ｆ１含ＮＦкＢ启动子、增强子序列（３０ ｂｐ）及 ＣＡＴ启动于 ５’末端序列（２０ｂｐ）,引物 Ｆ２含 ＣＡＴ基因 ３’端序列,以ｐｃＤＮＡ ３．１／ＣＡＴ（Ｉｎｖｉｔｒｏｇｅｎ）质粒为模板, ＰＣＲ扩增获得ＮＦкＢ启动子…     

抗—HBe阳性慢性乙型肝炎病毒感染：病毒前C变异与病变活动

抗－ＨＢｅ（＋）的慢性活动性肝炎５１例，以聚合酶链反应检出ＨＢＶ ＤＮＡ４８例（９４．１％）。对６例以反应产物直接序列分析，发现前Ｃｎｔ８３发生Ｇ→Ａ（Ａ８３）点突变，使密码２８由于ＴＧＧ变异为终止密码ＴＡＧ而不能编码ＨＢｅＡｇ。以抗－ＨＢｅ（＋）的慢性无症状ＨＢＶ携带者３０例为对照，仅１２例（４０％）检出弱ＨＢＶ，ＤＮＡ带。５例序列分析未发现Ａ８３变异。结果提示：变异病毒逃避免疫清除而继续活跃复     

抗－HBe阳性慢性乙型肝炎病毒感染：病毒前C变异与病变活动

抗－ＨＢｅ（＋）的慢性活动性肝炎５１例，以聚合酶链反应检出ＨＢＶＤＮＡ４８例（９４．１％）。对６例以反应产物直接序列分析，发现前Ｃｎｔ８３发生Ｇ→Ａ（Ａ８３）点突变，使密码２８由ＴＧＧ变异为终止密码ＴＡＧ而不能编码ＨＢｅＡｇ。以抗－ＨＢｅ（＋）的慢性无症状ＨＢＶ携带者３０例为对照，仅１２例（４０％）检出弱ＨＢＶＤＮＡ带。５例序列分析未发现Ａ８３变异。结果提示：变异病毒逃避免疫清除而继续活跃复制，与病变持续活动有关。     

乙型肝炎病毒e抗原阴性重型肝炎病人前C基因信号酶位点变异

为进一步了解乙型肝炎病毒变异所致肝细胞内过度表达乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）前体对肝细胞可能带来的影响，用聚合酶链反应（ＰＣＲ）产物直接和克隆后序列分析，在中国ＨＢｅＡｇ阴性重型肝炎病人中发现一种新ＨＢＶ变异株：前Ｃ区第１７位密码子由缬氨酸变为苯丙氨酸，变异影响信号酶切功能，导致ＨＢｅＡｇ加工和分泌障碍，ＨＢｅＡｇ前体在肝细胞内过度积聚。认为细胞毒Ｔ淋巴细胞攻击此靶抗原可能是肝细胞严重坏死的原因，但因例数太少尚难肯定。     

Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan

It was recently reported that mutations in the precore and core promoter region of hepatitis B virus (HBV) are associated with fulminant hepatitis. The aim of this study was to investigate the association of mutations in the precore and core promoter region of HBV with fulminant and severe acute hepatitis. We studied Japanese patients with acute HBV infection, including seven patients with fulminant hepatitis, 12 with severe acute hepatitis and 41 with acute self-limited hepatitis. The presence of HBV mutants was examined by using a point mutation assay to detect a G to A transition at position 1896 in the precore region and an A to T transition at position 1762 and a G to A transition at position 1764 in the core promoter region. Significant differences in the proportion of mutations in the precore or core promoter region were present between patients with fulminant hepatitis and self-limited acute hepatitis (7/7 (100%) vs 4/41 (9.8%), P < 0.01) and between severe acute hepatitis and self-limited acute hepatitis (6/12 (50.0%) vs 4/41 (9.8%), P < 0.01). The frequency of mutation increased proportionately with the severity of disease in patients with acute HBV infection. Fulminant hepatitis B in Japan is closely associated with mutations in the core promoter and precore gene of HBV. Point mutation assays for HBV precore and core promoter analysis may be useful to predict the outcome of liver disease in patients with acute HBV infection.     

重型乙型肝炎e抗原阴性患者前C变异株及其体外翻译

目的：探讨HBeAg阴性的慢性重型乙型肝炎患者HBV前C区变异及前C区T1862突变体对e抗原合成和分泌影响,方法：选取9例重型乙型重病毒性肝炎患者,用PCR方法扩增血清中HBV前C／C基因区,并克隆后测序和序列分析,构建HBV前C区T1862位点突变体表达质粒pGEMT,经体外翻译比较野毒株和变异株的表达产硪,结果：HBV前C区有3个位点异使氨其酸序列改变,A1896,A1899和T1862,A1899并不单独出现,前C区T1862突变并不阻止HBVe前体外合成,同时,有2例并未检测到前C区的变异,结论：部分重型肝炎HBeAg阳性原因降了T1896变异外,还存在T1862变异,前C1862突变对HBVe抗原前体蛋白合成并无影响,可能e抗原前在分泌过程中受阻。     

Reversion from precore/core promoter mutants to wild-type hepatitis B virus during the course of lamivudine therapy

The effect of lamivudine administration on the evolution of precore/core promoter mutation is unknown. The aim of this study was to determine the changes of precore/core promoter sequences in chronic type B hepatitis patients treated with lamivudine. Serial sera were obtained from 11 patients before, at the beginning of, and during therapy. Serum samples were polymerase chain reaction-amplified, and nucleotide sequences of hepatitis B virus (HBV) were analyzed. At baseline, precore and core promoter mutations were found in 6 and 4 of 11 patients, respectively. A precore stop codon mutant was replaced by a wild-type virus in all 6 patients infected with precore mutant at a median treatment of 12 months (vs. before therapy; P =.011). Mutations in the core promoter appeared in only 1 of 10 patients (vs. before therapy; P =.021). However, precore and core promoter mutations appeared in 5 and 7 of 10 patients at a median treatment of 21 months, respectively. Acute exacerbation occurred after lamivudine withdrawal in 2 patients who had hepatitis B e antigen (HBeAg) loss or seroconversion. The serum remained HBeAg-negative throughout the study period, and each of 2 patients had precore wild-type virus during acute exacerbation. HBV mutants with core gene deletions are not eliminated completely during prolonged therapy in 2 patients in whom the HBV genomes had core gene deletions at baseline. In conclusion, lamivudine therapy resulted in reversion from precore/core promoter mutants to wild-type. However, mutations in the precore and core promoter region reappeared during prolonged therapy. HBeAg-negative wild-type precore hepatitis B virus could be selected after lamivudine withdrawal in patients who had HBeAg loss or seroconversion.     

Distribution of hepatitis B viral genotypes and mutations in the core promoter and precore regions in acute forms of liver disease in patients from Chiba, Japan

BACKGROUND: Although it has been reported that different hepatitis B virus (HBV) genotypes induce different clinical characteristics in patients with chronic liver diseases (CLD), there have been few reports that have detailed the distribution of HBV genotypes in acute forms of liver disease. METHODS: HBV genotypes were determined in 61 patients who had acute forms of liver disease (45 had acute self limited hepatitis (AH) and 16 had fulminant hepatitis (FH)) and in 531 patients with CLD, including 19 patients with severe acute exacerbation of CLD. We also analysed the enhancer II, core promoter, and precore region sequences for the presence of mutations. RESULTS: Expression of genotype B in patients with acute forms of liver disease was significantly greater than in those with CLD (39.3% v 11.7%, respectively; p<0.001). Furthermore, expression of genotype B was significantly greater in patients with FH than in those with AH (62.5% v 31.1%, respectively; p=0.027). The precore mutation A1896 and the core promoter mutation at nt 1753 and 1754 were found more frequently in FH than in AH, and genotype B was predominant in FH regardless of the presence of these mutations. CONCLUSIONS: HBV genotype B was found more frequently in patients with acute forms of liver disease than in patients with CLD, and more frequently in patients with FH than in those with AH. These results suggest that this HBV genotype may induce more severe liver damage than other viral genotypes, at least in patients from Chiba, Japan.     

An outbreak of fulminant hepatitis B in immunocompromised hemodialysis patients

Background We aimed to clarify the pathogenesis of an outbreak of fulminant hepatitis B in hemodialysis (HD) patients whose compromised cell-mediated immunity in turn contributed to chronic hepatitis B virus (HBV) carriage.Methods Five consecutive adult HD patients with acute hepatitis B were evaluated. Viral genotype, mutations, and HBV-DNA levels were studied in relation to viral clearance, liver disease severity, and liver histology by immunostaining.Results All five patients had hepatitis B surface antigen (HBsAg) genotype C, a G-to-A stop codon mutation at nucleotide (nt) 1896 in the precore region, an A-to-T mutation at nt 1762 and an G-to-A mutation at nt 1764 in the basal core promoter. The possible index patient, who suffered from liver cirrhosis, had HBsAg genotype C, anti-hepatitis B envelope (HBe), and these mutations. The level of HBV-DNA declined by about 10 percent per week and no difference in viral kinetics between the patients who died and the survivor was found, irrespective of therapies. The amount of liver cell apoptosis, as assessed by single-stranded DNA, was scarce. The risk of fulminant hepatic failure did not correlate with the preexistent liver histopathological changes. Acute HBV superinfection was associated with hepatitis C virus (HCV) elimination and increased mortality.Conclusions This outbreak of fulminant hepatitis B suggests that HD patients can foster highly virulent HBV strains (possibly owing to their compromised immune responses), which may place others at risk of severe, life-threatening acute liver damage and at increased risk of mortality if chronic carriers of HCV should be infected.     

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5 terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.     

Hepatitis B virus precore mutants are identical in carriers from various ethnic origins and are associated with a range of liver disease severity.

Hepatitis B virus carriers in Israel are mostly HBeAg negative, of whom 5% to 10% have circulating hepatitis B virus. Recently, a hepatitis B virus variant with a stop codon in the precore region was identified, and it was suggested that specific mutations are associated with fulminant or severe chronic active hepatitis. We have analyzed serum samples from HBeAg-positive and HBeAg-negative patients by polymerase chain reaction, using primers spanning the precore/core region. Nucleotide sequence analysis (by direct sequencing) from amplified hepatitis B virus DNA demonstrated that viral genomes from all HBeAg-negative patients contain G to A mutation (nucleotide 1896), leading to the formation of a stop codon. An additional G to A mutation was identified three nucleotides downstream (nucleotide 1899). These patients are of various ethnic origins, with no unique clinical characteristics and with normal liver histology, chronic hepatitis or cirrhosis. No mutation at the precore/core region was observed in the HBeAg-positive patients. In conclusion, the precore mutations identified in hepatitis B virus carriers in Israel are identical regardless of the carrier's ethnic origin and are associated with mild-to-severe liver disease.     

Possible association of vigorous hepatitis B virus replication with the development of fulminant hepatitis

A 49 year-old man was referred to our hospital for fear of developing fulminant hepatic failure. There had been an outbreak of fulminant hepatitis B in a dialysis clinic in the western part of Honshu, Japan, that resulted in four deaths among six patients. After the sixth patient contracted severe hepatitis, all patients in the unit were screened biweekly for hepatitis B surface antigen (HBsAg) to detect newly infected patients as soon as possible. Our patient was the seventh victim, and on the day he gave a positive result for HBsAg, his hepatitis B virus (HBV) DNA level had reached 1.1 × 10¹¹ copies/ml as assessed by real time polymerase chain reaction. Sequence analysis of the causal HBV revealed the presence of a mutation in the precore region (nt 1896), two mutations in the core promoter (nt 1762 and nt 1764), and some minor mutations in the P gene that were restricted to the upstream region. These mutations are indicative of a virus with a high replicative rate that cannot secrete HBeAg. Taken together, these findings indicate that it is very likely that the replicative ability of the causal virus was as vigorous as that of HBV in hepatitis B e antigen-positive asymptomatic carriers with markedly high viral titers. The present case report provides clinical evidence of a possible association between the rapid spread of highly replicative HBV before host immunological recognition and the development of fulminant hepatitis.