反向斑点杂交法检测HBV基本核心启动子区A1762T／G1764A突变的实验研究

目的建立一种基于尼龙膜的反向斑点杂交法,用于检测乙型肝炎病毒（HBV）基本核心启动子区（BCP）A1762T／G1764A突变。方法根据我国HBV主要流行的基因型为B和C,从GenBank上查出4种HBVBCP序列。利用在线工具ClustalW进行比对,针对该突变位点设计引物和检测探针。探针经合成和修饰后点在带正电的尼龙膜上。将反向斑点杂交法结合地高辛检测试剂盒用于检测A1762T／G1764A突变,以测序法确定该区域序列的标本为检测对象。结果反向斑点杂交法分别检测5例A1762／G1764病毒株、2例T1762／G1764病毒株、5例A1762／A1764病毒株和4例T1762／A1764病毒株,结果与测序完全相同。结论应用本方法可以快速、准确地HBV相关的热点突变。     

Genome replication,virion secretion,and e antigen expression of naturally occurring hepatitis B virus core promoter mutants

The core promoter mutants of hepatitis B virus (HBV) emerge as the dominant viral population at the late HBeAg and the anti-HBe stages of HBV infection, with the A1762T/G1764A substitutions as the hotspot mutations. The double core promoter mutations were found by many investigators to moderately enhance viral genome replication and reduce hepatitis B e antigen (HBeAg) expression. A much higher replication capacity was reported for a naturally occurring core promoter mutant implicated in the outbreak of fulminant hepatitis, which was caused by the neighboring C1766T/T1768A mutations instead. To systemically study the biological properties of naturally occurring core promoter mutants, we amplified full-length HBV genomes by PCR from sera of HBeAg(+) individuals infected with genotype A. All 12 HBV genomes derived from highly viremic sera (5 x 10(9) to 5.7 x 10(9) copies of viral genome/ml) harbored wild-type core promoter sequence, whereas 37 of 43 clones from low-viremia samples (0.2 x 10(7) to 4.6 x 10(7) copies/ml) were core promoter mutants. Of the 11 wild-type genomes and 14 core promoter mutants analyzed by transfection experiments in human hepatoma cell lines, 6 core promoter mutants but none of the wild-type genomes replicated at high levels. All had 1762/1764 mutations and an additional substitution at position 1753 (T to C), at position 1766 (C to T), or both. Moreover, these HBV clones varied greatly in their ability to secrete enveloped viral particles irrespective of the presence of core promoter mutations. High-replication clones with 1762/1764/1766 or 1753/1762/1764/1766 mutations expressed very low levels of HBeAg, whereas high-replication clones with 1753/1762/1764 triple mutations expressed high levels of HBeAg. Experiments with site-directed mutants revealed that both 1762/1764/1766 and 1753/1762/1764/1766 mutations conferred significantly higher viral replication and lower HBeAg expression than 1762/1764 mutations alone, whereas the 1753/1762/1764 triple mutant displayed only mild reduction in HBeAg expression similar to the 1762/1764 mutant. Thus, core promoter mutations other than those at positions 1762 and 1764 can have major impact on viral DNA replication and HBeAg expression.     

乙型肝炎病毒变异与肝细胞癌发生关系

乙型肝炎病毒（hepatitisBvirus,HBV）基因组复制时,以病毒前基因组RNA作为模板合成子代病毒DNA,催化该过程的逆转录酶缺乏校对功能,所以HBV易出现变异。近年来,各国学者通过比较肝细胞癌（hepatocellular carcinoma,HCC）患者和非HCC患者的HBV基因序列,发现HBV基本核心启动子区的A1762T／G1764A变异或T1753V变异、增强子Ⅰ区的G1053A或G1229A变异、前S蛋白的F141L变异、前s2区基因缺失变异和x基因的截短变异,分别是HCC的易患因素,而前c区常见的G1896A变异,与HCC的发生无关。增强子Ⅱ区的C1653T变异在c基因HBV感染中可能与发生HCC有关,而在A基因型可能无关。     

Low frequency of mutations in the core promoter and precore regions of hepatitis B virus in anti-HBe positive Brazilian carriers

Background  

Mutations in the core promoter and precore regions of the hepatitis B virus (HBV) genome, notably the double substitution (AGG to TGA) at nt positions 1762-1764 in the core promoter, and the precore stop codon mutation G to A at nt 1896, can often explain the anti-HBe phenotype in chronic carriers. However, the A1896 mutation is restricted to HBV isolates that have T at nt 1858. The double substitution at positions 1762-1764 has been described to occur preferentially in patients infected with strains showing C instead of T at nt 1858.     

Pre-S Deletion and Complex Mutations of Hepatitis B Virus Related to Young Age Hepatocellular Carcinoma in Qidong,China

Background/Aim

To investigate the roles of biomedical factors, hepatitis B virus (HBV) DNA levels, genotypes, and specific viral mutation patterns on the progression of hepatocellular carcinoma (HCC) patients below 40 years of age in Qidong, China.

Methods

We conducted a case-control study within a cohort of 2387 male HBV carriers who were recruited from August, 1996. The HBV DNA sequence was determined in 49 HCC and 90 chronic hepatitis (CH) patients below 40 years of age. Mutation exchanges during follow-up in 32 cases were compared with 65 controls with paired serum samples. In addition, a consecutive series of samples from 14 HCC cases were employed to compare the sequences before and after the occurrence of HCC.

Results

After adjustment for age, history of cigarette smoking and alcohol consumption, HBeAg positive, HBV DNA levels ≥4.00 log₁₀ copies/mL, pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations were associated with risk of young age HCC. Moreover, the presence of an increasing number of HCC-related mutations (pre-S deletion, T1762/A1764, and T1766 and/or A1768 mutations) was associated with an increased risk of young age HCC. Paired samples analysis indicated that the increased HCC risk for at-risk sequence mutations were attributable to the persistence of these mutations, but not a single time point mutation. The longitudinal observation demonstrated a gradual combination of pre-S deletion, T1762/A1764 double mutations, and T1766 and/or A1768 mutations during the development of HCC.

Conclusion

High HBV DNA levels and pre-S deletion were independent risk factors of young age HCC. Combination of pre-S deletion and core promoter mutations increased the risk and persistence of at-risk sequence mutations is critical for HCC development.     

Novel Point and Combo-Mutations in the Genome of Hepatitis B Virus-Genotype D: Characterization and Impact on Liver Disease Progression to Hepatocellular Carcinoma

Background

The contribution of chronic hepatitis B virus (HBV) infection in the pathogenesis of hepatocellular carcinoma (HCC) through progressive stages of liver fibrosis is exacerbated by the acquisition of naturally occurring mutations in its genome. This study has investigated the prevalence of single and combo mutations in the genome of HBV-genotype D from treatment naïve Indian patients of progressive liver disease stages and assessed their impact on the disease progression to HCC.

Methods

The mutation profile was determined from the sequence analysis of the full-length HBV genome and compared with the reference HBV sequences. SPSS 16.0 and R software were used to delineate their statistical significance in predicting HCC occurrence.

Results

Age was identified as associated risk factor for HCC development in chronic hepatitis B (CHB) patients (p≤0.01). Beyond the classical mutations in basal core promoter (BCP) (A1762T/G1764A) and precore (G1862T), persistence of progressively accumulated mutations in enhancer-I, surface, HBx and core were showed significant association to liver disease progression. BCP_T1753C, core_T147C, surface_L213I had contributed significantly in the disease progression to HCC (p<0.05) in HBeAg positive patients whereas precore_T1858C, core_I116L, core_P130Q and preS1_S98T in HBeAg negative patients. Furthermore, the effect of individual mutation was magnified by the combination with A1762T/G1764A in HCC pathogenesis. Multivariate risk analysis had confirmed that core_P130Q [OR 20.71, 95% CI (1.64–261.77), p = 0.019] in B cell epitope and core_T147C [OR 14.58, 95% CI (1.17–181.76), p = 0.037] in CTL epitope were two independent predictors of HCC in HBeAg positive and negative patients respectively.

Conclusions

Thus distinct pattern of mutations distributed across the entire HBV genome may be useful in predicting HCC in high-risk CHB patients and pattern of mutational combinations may exert greater impact on HCC risk prediction more accurately than point mutations and hence these predictors may support the existing surveillance strategies in proper management of the patients.     

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.     

Variability in the precore and core promoter regions of HBV strains in morocco: characterization and impact on liver disease progression

Background

Hepatitis B virus (HBV) is one of the most common human pathogens that cause aggressive hepatitis and advanced liver disease (AdLD), including liver cirrhosis and Hepatocellular Carcinoma. The persistence of active HBV replication and liver damage after the loss of hepatitis B e antigen (HBeAg) has been frequently associated with mutations in the pre-core (pre-C) and core promoter (CP) regions of HBV genome that abolish or reduce HBeAg expression. The purpose of this study was to assess the prevalence of pre-C and CP mutations and their impact on the subsequent course of liver disease in Morocco.

Methods/Principal Findings

A cohort of 186 patients with HBeAg-negative chronic HBV infection was studied (81 inactive carriers, 69 with active chronic hepatitis, 36 with AdLD). Pre-C and CP mutations were analyzed by PCR-direct sequencing method. The pre-C stop codon G1896A mutation was the most frequent (83.9%) and was associated with a lower risk of AdLD development (OR, 0.4; 95% CI, 0.15–1.04; p = 0.04). HBV-DNA levels in patients with G1896A were not significantly different from the other patients carrying wild-type strains (p = 0.84). CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were associated with higher HBV-DNA level and increased liver disease severity. Multiple logistic regression analysis showed that older age (≥40 years), male sex, high viral load (>4.3 log₁₀ IU/mL) and CP mutations C1653T, T1753V, A1762T/G1764A, and C1766T/T1768A were independent risk factors for AdLD development. Combination of these mutations was significantly associated with AdLD (OR, 7.52; 95% CI, 4.8–8; p<0.0001).

Conclusions

This study shows for the first time the association of HBV viral load and CP mutations with the severity of liver disease in Moroccan HBV chronic carriers. The examination of CP mutations alone or in combination could be helpful for prediction of the clinical outcome.     

野生型及突变型乙型肝炎病毒X基因在pGEX-6P-2系统中的克隆、表达、纯化及免疫鉴定

构建野生型和A1762T/G1764A双突变型乙型肝炎病毒(HBV)X基因原核表达系统,为深入研究HBV X基因及其双突变后对HBV慢性感染者病情发展和肿瘤发生的作用奠定基础。从慢性乙型肝炎患者血清中抽提HBV基因组DNA,经PCR扩增和基因测序,证实并获得野生型及A1762T∕G1764A双突变型HBV X基因。应用TA克隆及亚克隆技术将2基因分别插入pGEX-6P-2载体,构建pGEX-6P-2-hbvxw(野生型)及pGEX-6P-2-hb-vxm(A1762T∕G1764A突变型)重组表达载体;转化入宿主菌进行诱导表达;包涵体复性后,GSTrap FF蛋白纯化柱纯化带有GST标签的野生型和A1762T∕G1764A突变型HBV x抗原(HBxAg);应用SDS-PAGE、Westernblot和ELISA方法对表达的野生型和突变型HBxAg进行鉴定。结果SDS-PAGE证实本研究构建的2个表达系统均能高效表达目的蛋白;复性、纯化后野生型和突变型GST-HBxAg分别达到4.88mg/mL和5.07mg/mL;Western blot鉴定证实所纯化的野生型和突变型HBxAg均能被特异性的单克隆抗体所识别;ELISA方法检测显示2种抗原都能成功包被于微量滴定板上并与乙肝患者血清反应。证实本研究成功地构建了野生型和A1762T∕G1764A突变型HBxAg表达系统,为进一步研究A1762T∕G1764A基因突变对肿瘤发生学和慢性HBV感染者疾病进展的作用和分子机制奠定了基础。     

乙型肝炎病毒全基因序列的测定

测定７ 例慢性 Ｈ Ｂ Ｖ 携带者 Ｈ Ｂ Ｖ 基因组全序列,经同源性比较,确定基因型。２ 例基因型为 Ｂ 型,余均为 Ｃ 型;血清型ａｄｒ ４ 例,ａｄｗ ３ 例。各序列间 Ｘ 基因差异最大。未见 Ａ１８９６ 、 Ｔ１７６２ Ａ１７６４等重要位点的变异。结合已有的２ 株 Ｈ Ｂ Ｖ 中国流行株全基因序列,初步建立以中国流行株序列为基础的 Ｈ Ｂ Ｖ 标准序列,该标准序列与国外标准序列仅有２２ 个位点的差异     

Specific mutations of basal core promoter are associated with chronic liver disease in hepatitis B virus subgenotype D1 prevalent in Turkey

The role of hepatitis B virus (HBV) genetics in the clinical manifestations of infection is being increasingly recognized. Genotype D is one of eight currently recognized major HBV genotypes. The virus is ubiquitous worldwide, but shows different features in different regions. One hundred and ninety‐eight patients with chronic HBV infection were enrolled in this study, 38 of whom had been diagnosed with cirrhosis of the liver and/or hepatocellular carcinoma. HBV DNA was isolated from the patients' blood samples and the entire genome and/or the basal core promoter/core promoter region sequenced. Phylogenetic analysis of the complete genomes revealed that subgenotype D1 is the most prevalent subgenotype in Turkey, but there was no definite phylogenetic grouping according to geography for isolates from different regions within Turkey, or for isolates in Turkey relative to other parts of the world. Turkish isolates tended to be genetically similar to European and central Asian isolates. Overall, HBV‐infection in Turkey appears to be characterized by early HBeAg seroconversion, a high incidence of the A1896 core promoter mutation and a small viral load. Genotype D characteristic mutations A1757 and T1764/G1766 were found in the BCP region. T1773 was associated with T1764/G1766 and a larger viral load. In conclusion, infection with HBV genotype D in Turkey has a similar clinical outcome to that of Europe and central Asia. Genotypic mutations in genotype D may be linked with disease prognosis in Turkey, but further studies with higher sample numbers and balanced clinical groups are needed to confirm this.     

乙型肝炎病毒全基因序列的测定

Seven cases of chronic hepatitis B virus carriers were included for sequencing of whole gene sequences of hepatitis B virus (HBV). The serotype of 4 strains of HBV were adr, and 3 strains were adw. Two strains were classified to genotype B, and the other 5 strains to genotype C. No significant mutations, such as A¹⁸⁹⁶, T¹⁷⁶²A¹⁷⁶⁴ were present. With other reported 2 complete sequences of HBV strains prevailing in mainland China, 7 strains of HBV whole sequences of genotype C from mainland China were analyzed to produce the consensus sequence of HBV of China. Comparing of the consensus sequence with that from Genbank, there were only 22 sites with different nucleotides.     

The G1613A mutation in the HBV genome affects HBeAg expression and viral replication through altered core promoter activity

Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg) and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC.     

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.     

我国常见乙型肝炎病毒基因型与基本核心启动子及前C区突变关系的初步研究

收集81份HBV DNA阳性血清标本,经PCR扩增和序列测定确定其中有50份属于基因型C,31份属于基因型B;C基因型的基本核心启动子BCP T1762/A1764的突变率(38%)明显高于B基因型(12.9%,P<0.05);前C区A1896的突变在B、C两基因型间无显著性差异,B基因型为9.7%,C基因型为12%,P>0.05;HBeAg的表达与否与BCP双突变或前C区A1896突变均无明显相关性。经定量PCR检测证明,HBeAg阳性组中的HBV DNA含量明显高于抗-HBe阳性组,P<0.05。组内BCP双突变株和野生株及前C1896突变株和野生株的HBV DNA含量无显著性差异。     

High Endemicity and Low Molecular Diversity of Hepatitis B Virus Infections in Pregnant Women in a Rural District of North Cameroon

Background

A program, supported by the GEMHEP (Groupe d''étude Moléculaire des Hépatites), was established in 2007 in the sanitary district of Tokombéré, to prevent perinatal transmission of hepatitis B virus (HBV). It comprises screening for HBV surface antigen (HBsAg) in all pregnant women and vaccinating the newborn if tests are positive.

Methods/Principal Findings

1276 women were enrolled in the study after providing informed consent. Demographic data and blood samples were available for 1267 of the enrolled patients. HBsAg was determined locally using a rapid test (Vikia HBsAg, Biomerieux). Tests for HBV and HDV virological markers (HBeAg, anti-HDV antibodies (Ab), HBV-DNA, HDV-RNA, HBV and HDV genotypes) were performed on the confirmed HBsAg-positive samples in the virology unit of the Angers University Hospital (France). HBsAg was found in 259 of the 1267 pregnant women (20.4%) between January 2009 and April 2010, of whom 59 were HBeAg-positive (22.7%) with high levels of HBV-DNA. Anti-HDV Ab were found in 19 (7.3%) of the HBsAg-positive women. The prevalence rates of HBsAg and HDV were not age-dependent whereas HBeAg carriers were statistically younger than non carriers. Basal core promoter (BCP) and precore (PC) mutations and genotypes were determined by sequencing. Of 120 amplified sequences, 119 belonged to HBV genotype E (HBV/E) and the 9 HDV strains belonged to HDV clade 1. In the PC region, 83/228 patients (36.4%) harbored a G1896A mutant or mixed phenotype virus. In the BCP region, the double mutation A1762T/G1764A and the G1757A substitution were detected respectively in 26/228 patients (11.4%) and 189/228 patients (82.8%).

Conclusions

Our results confirm the high prevalence and low molecular diversity of HBV in Far Northern Cameroon; more than 20% of the infected women were highly viremic, suggesting a high rate of HBV perinatal transmission and supporting the WHO recommendation to vaccinate at birth against hepatitis B.     

HLA-DP Polymorphisms Affect the Outcomes of Chronic Hepatitis B Virus Infections,Possibly through Interacting with Viral Mutations

Genetic polymorphisms of HLA-DP have been associated with hepatitis B virus (HBV) persistence. We aimed to determine the effect of HLA-DP polymorphisms on the generation of HBV mutations and their interactions on the outcomes of HBV infection. rs3077, rs3135021, rs9277535, and rs2281388 were genotyped in 1,342 healthy controls, 327 HBV clearance subjects, and 2,736 HBV-positive subjects, including 1,108 hepatocellular carcinoma (HCC) patients, using quantitative PCR. HBV mutations were determined by sequencing. Multiplicative interactions of HLA-DP polymorphisms and viral mutations were assessed by multivariate logistic regression. rs3077 (from subjects with genotype CT combined with those from subjects with genotype TT [CT+TT] versus CC), rs3135021 (GA+AA versus GG), rs9277535 (GA+AA versus GG), and rs2281388 (CC versus CT+TT) significantly decreased HBV persistence. This effect was found only in genotype B HBV-infected subjects compared to HBV clearance subjects. HLA-DP polymorphisms promoting HBV clearance were associated with a lower prevalence of mutations increasing HCC risk (C1653T, T1674C/G, A1846T, G1896A and pre-S2 mutations and pre-S deletion in genotype C) and a higher prevalence of mutations decreasing HCC risk (G1652A, T1673C, T1674C, G1719T, G1730C, and G1799C in genotype B and A1727T in genotype C). Significant effects of viral mutations on cirrhosis and HCC were selectively evident in those with HLA-DP polymorphisms promoting HBV persistence. The interactions of C1653T, T1674C/G, and G1896A mutations with HLA-DP polymorphisms promoting HBV clearance significantly decreased cirrhosis risk. The interaction of rs9277535 AA with the T1674C/G or G1719T mutation in genotype C significantly decreased HCC risk. In conclusion, HLA-DP polymorphisms affect genotype B HBV clearance, regulate immune selection of viral mutations, and influence cirrhosis and HCC risks contributed by HBV mutations.