重型乙型肝炎病人中乙型肝炎病毒C基因启动子变异的研究

目的　了解重型乙型肝炎（ 乙肝） 病人血清乙肝病毒（ＨＢＶ）ＤＮＡＣ基因启动子（ＣＰ） 的变异。方法　对用聚合酶链反应（ＰＣＲ） 法扩增的血清ＨＢＶＤＮＡ 直接测序。结果　７ 例亚急性重型肝炎病人的ＨＢＶ 分离株ＣＰ区分别有２～１２ 个替代变异，１ 例病人有１１ｂｐ 的碱基插入。ＣＰ变异主要发生于ＣＰ的第１ 和第２ 个ＡＴ丰富区，ｎｔ１ ７６２ 和ｎｔ１ ７６４ 的替代变异见于７ 例亚急性重型肝炎病人的４ 例中，是ＣＰ变异的热点，其中３ 例ＨＢｅＡｇ 阴性，说明和ＨＢｅＡｇ 阴性表型相关。ＣＰ的第３ 个ＡＴ丰富区、ＨＢＶ逆转录起始位点（ＤＲ１） 和前Ｃ基因、前基因组转录起始位点未见变异。结论　重型肝炎病人的ＨＢＶＣＰ区存在较多的变异，ＣＰ变异主要发生于和前Ｃ基因相关的第１ 和第２ 个ＡＴ丰富区，可能和ＨＢｅＡｇ 阴性表型相关     

乙型肝炎前C区核苷酸1896G→A变异与1858碱基的关系

乙型肝炎前Ｃ区核苷酸１８９６Ｇ→Ａ变异与１８５８碱基的关系邱望龙李福元骆抗先侯金林乙型肝炎病毒（ＨＢＶ）前Ｃ区核苷酸（ｎｔ）１８９６Ｇ→Ａ终止变异是近来研究的热点。在ＨＢＶ前基因组ＲＮＡ的茎襻结构（ｓｔｅｍｌｏｏｐ）中ｎｔ１８９６与ｎｔ１８５８碱基配...     

乙型肝炎病毒C基因调节序列变异分析

ＨＢＶＣ基因上游约 2 0 0ｂｐ序列(ｎｔ 16 0 0～ｎｔ 185 0 )是重要ＨＢＶ基因调控区。Ｔ 176 2Ａ 176 4(ｎｔ 176 4Ａ→Ｔ ,ｎｔ 176 4Ｇ→Ａ ,)变异在慢性ＨＢＶ感染者是变异的热点。为了解我国ＨＢＶ流行株Ｃ基因调节序列及变异情况 ,分别对 7例慢性无症状ＨＢＶ携带者和 7例亚急性重症乙型肝炎患者ＨＢＶＣＰ区序列进行了分析。1　研究对象　 7例慢性无症状ＨＢＶ携带者 (ＡｓＣ) (3例来自广东地区 ,4例自东北地区 )、7例亚急性重症乙型肝炎患者 (ＦＨ) (为我科 1993～ 1996年间收治的住院患者 )。酶联免疫法检测ＨＢＶ血清标志物。2…     

粒细胞—巨噬细胞集落刺激因子与HBsAg融合蛋白基因的 …

目的 研究粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）的免疫活性。方法 用聚合酶链反应（ＰＣＲ）技术扩增出粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）３８４ｂｐ的基因片段及ＨＢｓＡｇ８４６ｂｐ基因片段，扩增产物经柱纯化后，由Ｔ４ＤＮＡ酶连结因子１２３０ｂｐ的片段，将此片段命名为ＬＧＨ，克隆到ｐＵＣ１９质粒中，利用菌落ＰＣＲ法快速筛选阳性克隆及限制酶切鉴定片段的大小。结果 该基因全长１２３０ｂｐ，经     

重型乙型肝炎病人中乙型肝炎病毒C基因启动子变？…

目的 了解重型乙型肝炎（乙肝）病人血清乙肝病毒（ＨＢＶ）ＤＮＡ Ｃ基因启动子（ＣＰ）的变异。方法 对用聚合酶链反应（ＰＣＲ）法扩增的血清ＨＢＶ ＤＮＡ直接测序。结果 ７例亚急性重型肝炎病人的ＨＢＶ分离株ＣＰ区分别有２￣１２个替代变异,１例病人有１１ｂｐ的碱基插入。ＣＰ变异主要发生于ＣＰ的第１和第２个ＡＴ丰富,ｎｔｌ７６２和ｎｔｌ７６４的替代变异见于７例亚急性重型肝炎病人的４例中,是ＣＰ变异的热点,     

20例中国人HCVⅡ/1b型高变区1序列变异的动态观察

目的 动态研究中国人群ＨＣＶⅡ／１ｂ型包膜蛋白Ｅ２／ＮＳ１高变区１（ＨＶＲ１）序列变异规律、意义及影响因素。方法 应用逆转录巢式ＰＣＲ技术从２０例ＨＣＶⅡ／１ｂ型感染的中国病人血清中扩增了ＨＣＶ部分包膜区基因片段（ｎｔ１４４９～１５８６，ＨＣＶ－Ｊ），纯化后直接采用双脱氧链末端终止法进行序列分析。结果 中国人群ＨＣＶ ＨＶＲ１位于氨基酸（ＡＡ）３８４～４１０位，有６个较保守的ＡＡ位点；３８５位Ｔｈ     

外周血单核细胞中HCV RNA与急慢性丙型肝炎关系的研究(简报)

丙型肝炎易发生慢性化，目前致病机制尚不清楚。我们采用逆转录－巢式ＰＣＲ技术对急慢性丙型肝炎患者血清及外周血单核细胞（ＰＢＭＣ）中ＨＣＶＲＮＡ进行检测，同时对ＰＢＭＣ中的ＨＣＶＲＮＡ进行限制性酶切片段长度多态性分析（ＲＦＬＰ）。结果１０例急性丙型肝炎患...     

乙/丙型肝炎病毒双重感染患者前C区终止变异低频率

目的了解乙型肝炎病毒（ＨＢＶ）与丙型肝炎病毒（ＨＣＶ）双重感染患者前Ｃ区基因变异，及其可能的临床意义。方法用聚合酶链反应（ＰＣＲ）与限制片段长度多态性（ＲＦＬＰ）来分析２５例ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性（Ａ组）和３１例ＨＢｓＡｇ和ＨＢＶＤＮＡ阳性但抗－ＨＣＶ和ＨＣＶＲＮＡ均阴性（Ｂ组）的慢性肝病患者前Ｃ区密码２８终止变异（终２８）。结果ＨＢＶ和ＨＣＶ双重感染患者（Ａ组）血清ＨＢＶＤＮＡ第１次ＰＣＲ阳性率（１６％）明显低于单独ＨＢＶ感染组（６５％）（Ｐ＜０．００１）；前Ｃ终２８检出率（２８％）亦明显低于单独ＨＢＶ感染（６８％）（Ｐ＜０．００１）。结论提示双重感染患者ＨＢＶ前Ｃ终止变异低频率可能与ＨＢＶ低水平复制有关     

乙型肝炎病毒C基因启动子及前C变异的动态研究

目的明确乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子（ＢＣＰ）与前Ｃ变异株在慢性乙型肝炎患者中的动态消长,及与ＨＢｅＡｇ血清学状态的关系。方法对３２例慢性乙型肝炎患者的１０５份系列血清进行错配聚合酶链反应（ＰＣＲ）－限制性片段长度多态性分析,并对其中１５例患者系列血清标本的ＰＣＲ产物进行直接测序。结果ＢＣＰ和Ａ８３变异株感染率均明显增高（６２．５％＞４６．９％;３１．３％＞１２．５％）;ＢＰＣ变异株更多地（１４／１６）表现为优势积累,且先于ＨＢｅＡｇ血清阴转;ＢＣＰ野株／变异株比例变化影响ＨＢｅＡｇ血清学的稳定性。结论两种类型的变异株在炎症活动中均具有选择优势,最终造成ＨＢｅＡｇ（－）的ＨＢＶ感染;可能与ＨＢＶ感染之持续有关。     

湖北汉族人群C4限制性片段长度多态性的研究

用限制性内切酶ＴａｑＩ和Ｃ４基因５￣＇末端探针ｐＡＴ－Ａ检测５８份湖北地区健康献血员基因组ＤＮＡ的Ｃ４基因限制性片段长度多态性（ＲＦＬＰ），观察到７．０ｋｂ、６．０ｋｂ和５．４ｋｂ三种Ｃ４基因片段，它们共组成三种常见的Ｃ４基因ＲＦＬＰ表型，即Ａ：７．０５－４ｋｂ（频率为２４．１４％））；Ｂ：７．０－６．０ｋｂ（频率为２０．６９％）和Ｃ：７．０－６．０－５．４ｋｂ（频率为５５．１７％，在所检测的样本中未观察到６．４ｋｂ的Ｃ４基因片段。     

Prevalence of basal core promoter and precore mutations in Chinese chronic hepatitis B patients and correlation with serum HBeAG titers

The A1762T and G1764A mutations in the basal core promoter (BCP) region and the G1896A mutation in the precore (PC) region of hepatitis B virus (HBV) genome are found commonly in HBeAg‐negative patients. Experiments in vitro suggest that BCP and PC mutation reduce and abolish HBeAg expression, respectively. In the present study, the prevalence of the BCP and PC mutations were determined in 207 patients with HBeAg positive chronic hepatitis B from China and correlated with the titers of serum HBeAg. None of the patients received antiviral therapy. The HBV genotype was determined by direct sequencing of the HBsAg gene. The BCP and PC mutations were detected by the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) and confirmed by DNA sequencing. The HBeAg titer was measured by the microparticle enzyme immunoassay. Fifty‐one of the 207 patients (24.6%) were infected with genotype B and the remainder with genotype C. The BCP mutations were detected in 103 patients (50%) while the PC mutation was present in 43 (20.8%). Thirteen patients (6.3%) harbored both BCP and PC mutations. No significant difference in the titers of HBeAg was found between patients infected with the two HBV genotypes, but the presence of either the BCP or PC mutation was associated with reduced HBeAg titer (P < 0.05). The presence of both the BCP and PC mutations was accompanied by even lower HBeAg titer (P < 0.05). These findings confirm that in patients with HBeAg, the BCP and PC mutations reduced the expression of HBeAg. J. Med. Virol. 81:807–814, 2009. © 2009 Wiley‐Liss, Inc.     

Hepatitis B e antigen‐negative mutations in the precore and core promoter regions in Korean patients

Most patients with hepatitis B e antigen (HBeAg)‐negative chronic hepatitis B have variants of the hepatitis B virus (HBV) that include mutations in the precore or core promoter regions of the HBV genome. The aim of this study was to investigate the patterns of precore and core promoter mutations and their relationship to HBeAg expression in Korean patients. Four hundred seventy‐five Korean patients with chronic HBV infection between February 1995 and December 2003 were enrolled in this study. There were 236 HBeAg‐positive and 239 HBeAg‐negative patients. Blood samples were tested for HBsAg, anti‐HBs, HBeAg, hepatitis B e antibody (anti‐HBe), liver function tests, and serum HBV DNA. Mutations in the precore and core promoter regions were determined by direct sequencing. In the core promoter region, the C1740, C1753, T1762/A1764, and T1766 mutations were associated with HBeAg escape (all; P < 0.05). In the precore region, a higher frequency of the C1802, A1828, T1846, A1850, C1858, T1862, and A1896 mutations was found in HBeAg‐negative patients (all; P < 0.05). In particular, the A1896 mutation was associated with high serum levels of ALT and HBV DNA in HBeAg‐negative patients (P = 0.014 and 0.026, respectively). Mutations around the Kozak sequence (nucleotides 1809–1812) were found in 6.7% of patients and were not associated with undetectable HBeAg (P = 0.13). In Korean patients, various mutations in the precore and core promoter regions were associated with HBeAg escape and amelioration of hepatic inflammation in HBeAg‐ negative patients. Only the A1896 mutation contributed to HBeAg‐negative chronic hepatitis B. J. Med. Virol. 81:594–601, 2009 © 2009 Wiley‐Liss, Inc.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

Epidemiology of precore mutants of hepatitis B in the United Kingdom

A point mutation assay was used to study the codon 28 and codon 1 precore mutant status of 310 chronic hepatitis B carriers (82 HBeAg positive and 228 HBeAg negative). Fourteen of 228 (6%) of HBeAg negative carriers had high levels of serum HBV DNA. Nine of these were explained by precore variants, three by core promoter variants, and two were not explained by recognised precore changes. Nested PCR detected serum HBV DNA in 36% (82/228) of HBeAg negative carriers and 63% (52/82) of these had precore variants. Four of 82 (4%) of the HBeAg positive carriers had precore variants, all as mixed mutant/wild type populations and evidence indicated that these carriers were seroconverting. Overall 23% (52/228) of HBeAg negative carriers had both serum HBV DNA and codon 1 or 28 precore mutations. A sexual transmission event from an HBeAg negative carrier with a relatively low serum HBV DNA level (10(4)-10(6) genome copies/ml) and only core promoter mutations was observed. Despite high rates of variant carriage in the antenatal sub-group perinatal transmission was not observed. The results of direct sequencing on 45 carriers validated the point mutation assay and also showed that codon 28 mutations were only seen in carriers with the genotype CCT at codon 15. For the Caucasian population a higher prevalence of codon 28 mutations (13/25 or 52%) than expected was seen. Liver biopsy data indicated that there was no link between the presence or absence of precore mutants and the severity of liver disease.     

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.     

Comparison of genotypes C and D of the hepatitis B virus in Japan: a clinical and molecular biological study

The genotype-related differences between genotype C and genotype D of the hepatitis B virus (HBV) remain unknown. The relationship was studied between the HBV genotypes and their clinical features, paying special attention to genotypes C and D. Serum samples from 413 HBV carriers were genotyped using an enzyme immunoassay (EIA) and by restriction fragment length polymorphism. The nucleotide sequences at the basic core promoter (BCP) and precore (PreC) regions were analysed by direct sequencing. The full genome sequences of three HBV genotype D cases were also examined. Almost all carriers with HBV genotype D were asymptomatic carriers (84.2%). Genotype D was not found in patients with liver cirrhosis and hepatocellular carcinoma. In contrast, carriers with genotype C had mainly chronic liver disease (63.2%; P<0.001). The ratio of hepatitis B e antigen (HBeAg)/anti-HBe was significantly higher in genotype C than in genotype D in the young age-matched group (P<0.01). The mutation at BCP (T1762, A1764) was significantly lower in genotype D than in genotype C among HBeAg-negative patients (P<0.05). The HBV full-genome sequences are very similar to certain HBV genotype D sequences from Europe. In conclusion, genotype C was associated with chronic liver disease, whereas genotype D was related to asymptomatic carriers with earlier HBeAg seroconversion. Thus, the outcome of chronic HBV infection may be different in persons infected with HBV genotypes C and D.     

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.