乙型肝炎病毒-Alu-聚合酶链反应检测肝细胞癌中乙型肝炎病毒DNA的整合

目的 了解肝细胞癌(HCC)中HBV DNA的整合情况.方法 收集24例HBsAg阳性患者的HCC组织,提取其DNA,应用套式PCR原理,在设计的引物中引入U碱基,根据已知基因序列和人Alu重复序列分别设计引物,建立克隆整合的HBV DNA及其相邻的细胞基因序列的PCR技术.PCR产物测序所获结果经美国国家生物技术信息中心(NCBI)BLAST及Map Viewer检索确定HBV整合在染色体上的精确位置.结果 24份HCC组织中,14份存在HBV整合现象,整合的标本中11份正向插入宿主基因,8份反向插入宿主基因,其中5份标本既有正向插入也有反向插入,从病毒基因分析,整合可发生于X基因的任何长度,且均以截短形式插入宿主细胞DNA.另有8份标本未测到整合.结论 HBV在HCC细胞染色体上的整合呈不均衡分布.     

Complex HBV populations with mutations in core promoter, C gene, and pre-S region are associated with development of cirrhosis in long-term renal transplant recipients

Long-term immunosuppressed renal transplant recipients with chronic hepatitis B virus (HBV) infection often develop liver cirrhosis (LC) and end-stage liver disease (ESLD). This study investigated accumulation and persistence of specific HBV mutants in relation to the clinical course in these patients (n = 38; mean follow-up, 3.5 years). HBV was analyzed longitudinally via length polymorphism of polymerase chain reaction (PCR) fragments (median, 6.5 serum samples per patient) as well as by cloning and partial sequencing of 346 full-length HBV genomes. Fourteen patients (group 1) developed LC or died from ESLD, whereas 24 patients (group 2) showed no evidence of LC during follow-up. Development of LC and ESLD was associated with persistence of HBV mutant populations characterized by deletions/insertions in core promoter plus deletions in the C gene and/or deletions in the pre-S region (86% of group 1 vs. 17% of group 2; P <.0001). HBV without these mutations or with core promoter mutations alone were predominantly found in group 2 (14% of group 1 vs. 75% of group 2). In patients infected with core promoter mutants, the additional appearance and persistence of deletions in the C gene and/or the pre-S region were accompanied or followed by development of LC and ESLD. The mutations were distributed on individual genomes in various combinations, leading to a high complexity of the virus population. In conclusion, these data suggest that accumulation and persistence of specific HBV populations characterized by mutations in 3 subgenomic regions play a role in pathogenesis of LC and ESLD in long-term renal transplant recipients.     

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

Background/Aim: During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of anti-hepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon.Methods: Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced.Results: In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitutions in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found.Conclusion: HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.     

Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes

Human cytomegalovirus (HCMV) has a large 240 kb genome that may encode more than 700 gene products with many of them remaining uncharacterized. Mutagenesis of bacterial artificial chromosome (BAC)-cloned CMV genomes has greatly facilitated the analysis of viral gene functions. However, the roles of essential proteins often remain particularly elusive because their investigation requires the cumbersome establishment of suitable complementation systems. Here, we show that HCMV genomes can be introduced into cells with unprecedented efficiency by applying a transfection protocol based on replication-defective, inactivated adenovirus particles (adenofection). Upon adenofection of several permissive cell types with HCMV genomes carrying mutations in essential genes, transfection rates of up to 60% were observed and viral proteins of all kinetic classes were found expressed. This enabled further analyses of the transfected cells by standard biochemical techniques. Remarkably, HCMV genomes lacking elements essential for viral DNA replication, such as the lytic origin of replication, still expressed several late proteins. In conclusion, adenofection allows the study of essential HCMV genes directly in BAC-transfected cells without the need for sophisticated complementation strategies.     

HBsAg-negative hepatitis B virus infections in hepatitis C virus-associated hepatocellular carcinoma

This study was conducted to evaluate reports that hepatitis B virus (HBV) DNA sequences can be found in the serum and/or tumour tissue from some hepatocellular carcinoma (HCC) patients who have no detectable hepatitis B surface antigen (HBsAg) in their sera. Such HBV infections would be highly atypical, because prospective studies have shown a clear succession of specific serologic markers during and after most HBV infections. As most HBsAg-negative HCC patients in Japan have hepatitis C virus (HCV) infections, the present study was conducted to determine whether some of these patients actually have unrecognized HBV infections. Thirty newly diagnosed HCC patients from Kurume, Japan, with antibody to the hepatitis C virus (anti-HCV) were studied. None of the 30 had HBsAg detectable in their serum. Of 22 for whom test results for antibodies to the hepatitis B core antigen (anti-HBc) and antibodies to HBsAg (anti-HBs) were available, 14 (64%) had anti-HBc and anti-HBs, four (18%) had anti-HBc alone, and four (18%) had no HBV markers. Nested polymerase chain reaction was used to detect the HBV surface (S), core (C), polymerase (P) and core promoter gene sequences in the HCC tissues and in the adjacent nontumorous liver tissues. HBV DNA was detected in HCC and/or adjacent nontumorous liver in 22 of 30 (73%) patients [detected in both HCC and nontumorous liver in 19/30 patients (63%)]. Among the 22 patients with detectable HBV DNA, more than one HBV gene was detected in 10 (46%). Among the four patients whose sera were negative for all HBV markers, three had HBV DNA in either HCC and nontumorous liver (two cases) or only in the nontumorous liver (one case); HBV DNA could not be detected in tissues from the fourth patient. In 18 of 21 (86%) patients with detectable HBV core promoter sequences, mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found. No deletions were detected in the core promoter gene region of the type reported to be associated with some cases of HBsAg-negative HBV infection. Thus, HBV DNA was detectable in 22 (73%) HBsAg-negative, anti-HCV-positive HCCs, including three (10%) who were also negative for anti-HBc and anti-HBs. HBV mutations at both nucleotides 1762 (A-GT) and 1764 (G-A) in the core promoter region were found in the majority of cases, mutations that have previously been reported in HBV that is integrated in HCC DNA. In serologic surveys to determine etiologic associations of HCC, patients such as those in this study would have been incorrectly designated as having 'HCV-associated HCC,' whereas the data in this study suggest that HBV could have played a role in the development of their HCCs.     

Rapid and high throughput detection of HBV YMDD mutants with fluorescence polarization

AIM: To develop a simple and rapid detection of HBV gene variants and prediction of lamivudine-resistance in patients.METHODS: Initially, plasmids harboring the wild-type or mutant HBV DNA fragments were used in a model system.The technique was then applied to clinical samples for an analysis of YMDD mutations. The sera were extracted from chronic hepatitis patients who had received lamivudine treatment for more than one year. P region gene of HBV was amplified by polymerase chain reaction. The excess primers and dNTPs in PCR products were removed by cleaning-up reagents. Template-directed dye-terminator incorporation reaction was performed and Rl10 or TAMRA labeled acyclo-terminator was added on the 3‘ end of TDI-primer specifically. Fluorescence polarization value was measured with Victor 2 multilabel counter and the genotypes of HBV were analyzed. RESULTS: The YMDD genotypes in recombined positive plasmid and 56 serum samples of HBV infected patients were analyzed by using our TDI-FP method and the specificity and sensitivity were confirmed by DNA sequencing. Five of 56 serum samples showed YVDD phenotype (9 %), including 1 YMDD and YVDD mixed infection. Four of 56 showed YIDDphenotype (7.1%). CONCLUSION: This is a simple, rapid, low cost and high throughput assay to detect HBV polymerase gene variants and suitable for large-scale screening and prediction of the lamivudine-resistance in clinical samples.     

乙型肝炎病毒表达载体pHY106在筛选抗病毒药物中的意义

目的应用新的HBV表达载体,建立体外筛选临床抗HBV药物的方法。方法克隆对拉米夫定耐药的CHB患者体内HBV全基因组,然后将其亚克隆到HBV真核表达载体pHY106,体外转染Huh7细胞,检测转染后不同时间HBsAg、HBeAg、HBV DNA及HBV复制中间体水平。分析拉米夫定和阿德福韦对HBV基因表达和复制的抑制作用,指导临床用药。结果成功构建全部8个HBV临床分离株全基因组真核表达质粒,HBV聚合酶基因YMDD有5个产生YVDD突变,3个产生YIDD突变。该质粒上HBV基因能在Huh7细胞中进行复制和表达,拉米夫定不能体外抑制HBV复制,阿德福韦抑制了HBV在Huh7细胞中的复制,抑制程度与药物浓度相关。阿德福韦在患者体内也抑制了HBV的复制。结论新建立的体外筛选抗HBV药物的方法能用于临床快速筛选抗HBV药物,对CHB的治疗用药具有指导意义。     

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.     

Identification of a novel hepatitis B virus precore/core deletion mutant in HIV/hepatitis B virus co-infected individuals

OBJECTIVES: Although HAART has resulted in improved health outcomes for most HIV-infected individuals, liver failure has emerged as a major cause of morbidity and mortality in people co-infected with hepatitis B virus (HBV). In HBV mono-infected individuals, core deletion mutants are associated with more aggressive liver disease. As HIV accelerates HBV liver disease progression, we hypothesized that HIV-HBV co-infected individuals have increased frequency of core mutations including deletions. To test this hypothesis, we have analysed genome-length sequences of HBV DNA from patients both prior to and during antiviral therapy. SETTING: Prospective HIV/HBV co-infected cohort study. METHODS: Genomic length HBV DNA was amplified by PCR from the serum samples of ten HIV/HBV co-infected individuals and five HBV mono-infected individuals prior to the commencement of lamivudine therapy and again after nine to 74 months of treatment. The complete genomes were sequenced and in order to further analyse some mutations, their frequency was determined in additional HIV/HBV co-infected and HBV mono-infected individuals. RESULTS: A novel -1G mutation was identified in the HBV precore and overlapping core genes that truncated the deduced precore/core proteins. The mutant genome was the dominant species in some HIV/HBV co-infected individuals and was more prevalent in HIV/HBV co-infected individuals than HBV mono-infected individuals. The mutation was also associated with high HBV DNA concentrations in HIV/HBV co-infected individuals. Additional mutations were identified in the core/precore and polymerase genes and regulatory regions. CONCLUSION: Mutations in the HBV core and precore genes may be contributing to disease pathogenesis in HIV/HBV co-infected individuals.     

小分子乙型肝炎病毒基因组缺失突变体结构分析

目的 了解小分子HBV基因组缺失突变体的基因结构及特点.方法 采用一步法PCR从慢性乙型肝炎患者血清中扩增全基因组HBV DNA,回收并克隆＜1 kh的小分子HBV DNA,测序并以BioEdit及VectorNTI 6.0软件分析基因结构特点.结果 获得基因长度介于174～986 bp之间的64种、共124个小分子HBV缺失突变体DNA,按基因结构特点分为3类,即3种GT-AG剪接变异体、29种"常规"缺失突变体及32种基因组内部含polyA的缺失突变体.所有小分子基因组缺失突变体在HBV各编码区及基因调控区均存在不同程度缺失,其中66%(42/64种)保留与HBV复制(包装)相关的所有顺式调控序列,48%(31/64种)保留X编码区.结论 乙型肝炎患者血清中普遍存在小分子HBV基因组缺失突变体,深入了解这些变异体的结构和功能,有助于进一步探索HBV的致病机制.     

拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测

目的建立拉米夫定治疗过程中乙型肝炎病毒多聚酶变异的检测方法,对该部位变异类型、发生率及其与病毒滴度的关系进行初步分析。方法拉米夫定治疗的慢性乙型肝炎患者164例,在其中治疗64周时HBVDNA阳性的101份血清中,选择9份血清进行分析。设计台成一对引物SI、A1,采用聚台酶链反应法扩增得到包括YMDD基序（motif）的部分P区基因,片断长926bp,对扩增片断的部分校苷酸（约500bp）进行序列分析。HBVDNA定量用分枝DNA信号扩增祛。结果在9例患者中,发现YMDD（酪氨酸－蛋氨酸－天门冬氨酸－天门冬氨酸）基序变异的5例,占56％,其中M（蛋氨酸）－I（异亮氨酸）3例,M（蛋氨酸）－V（缬氨酸）2例,后者合并D（天门冬氨酸）→G（甘氨酸）变异1例。在2例血清HBVDNA低滴度（＜100MEq／ml）患者中未发现YMDD基序变异,7例中、高滴度（＞300ME／ml）患者中5例发现YMDD基序变异,占71％。结论拉米夫定是一种新的治疗慢性乙肝较有潜力的抗病毒药物,若能在治疗过程中对HBVDNA滴度变化及病毒变异发生进行监测,可指导临床的合理用药及联台用药。     

拉米夫定耐药患者乙型肝炎病毒聚合酶基因G743C和G743A点突变的检测及分析

目的 探讨拉米夫定耐药患者乙型肝炎病毒聚合酶(HBVP)基因点突变。方法 聚合酶链反应(PCR)扩增拉米夫定耐药患者的HBVP基因，产物经直接测序检测其YMDD变异；同时，用PCR-限制性片段长度多态性(PCR—RFLP)分析测序后的17例YIDD变异样本，先用3对引物扩增HBVP基因，再分别用3个限制性内切酶FokⅠ、SspⅠ、Alw441酶切扩增产物，酶切产物用8．0％聚丙烯酰胺凝胶电泳分析。结果 拉米夫定耐药患者HBVP基因PCR产物序列分析结果与GenBank的HBV标准株相比，发现16例存在HBVP基因G743C点突变和1例存在G743A点突变。其核苷酸序列由ATG变为ATC和ATA，YMDD基因序列发生变异，即由YMDD变异为YIDD；但是，PCRRFLP不能确定这17例样本存在YIDD变异。结论拉米夫定耐药患者存在HBVP基因G743C和G743A的点突变，同样引起YIDD变异；而PCR—RFLP法不能准确地检测出G743C和G743A的点突变；该发现对HBVP基因YMDD变异检测和临床均有指导意义。     

Uncommon mutation pattern of a hepatitis B virus isolate from genotype F infecting a patient with AIDS

OBJECTIVE: To study the genomic variations of a hepatitis B virus (HBV) isolate in a patient coinfected with human immunodeficiency virus type 1 (HIV-1) who developed severe hepatitis and died of AIDS. METHODS: Two blood samples were collected, the first one during the asymptomatic phase of HIV-1 infection, and the other, 3 years later, few months before the death of the patient. Both samples were HBsAg and anti-HBe positive. Pre-S/S and precore-core genome regions were PCR amplified and analyzed. RESULTS: The HBV isolate belonged to genotype F, cluster IV. A number of unique amino acid substitutions were found in the surface antigen gene and the overlapping polymerase coding region of HBV genomes derived from both samples. However, these substitutions reflected natural variations rather than mutations of clinical significance. The precore stop codon mutation A(1896) was present in both genomes. Furthermore, the HBV genome derived from the second, but not first sample, showed two out-of-frame core interval deletions, one and 103 nucleotides in length, respectively. CONCLUSIONS: This is the first report of an HBV isolate from genotype F with core internal deletions. Our results suggest an association between specific core mutations and the severe hepatitis developed by the patient.     

Effect of virological response on post-treatment durability of lamivudine-induced HBeAg seroconversion

Lamivudine-induced HBeAg seroconversion may not be durable in Korean patients with hepatitis B virus (HBV) infection. It is unknown whether virological response during lamivudine treatment affects the post-treatment outcome. We retrospectively analysed 124 consecutive HBeAg-positive chronic hepatitis B (CHB) patients treated with lamivudine. Lamivudine was given at a dose of 100 mg per day. HBV DNA levels in sera obtained before and during therapy were measured by the Digene Hybrid Capture II assay and Digene Ultrasensitive Hybrid Capture II assay, respectively. HBeAg seroconversion was achieved in 42 of the 124 patients (33.8%) treated with lamivudine. Mean duration of treatment in HBeAg seroconverters was 12.86 +/- 4.44 months. During the follow-up period, the cumulative relapse rates at 3 months and 6 months post-treatment in 42 patients with HBeAg seroconversion were 40.5% and 57.4%, respectively. Among 31 seroconverted patients whose sera were available at the second month of treatment, HBV DNA remained at > 4.7 x 103 genomes/mL in 15 patients and decreased to < 4.7 x 103 genomes/mL in the remaining 16 patients. HBV DNA levels at the second month of treatment was not related with relapse after discontinuation of treatment (66.7% vs. 43.8%, P= 0.2). At the time of HBeAg seroconversion, HBV DNA remained at > 4.7 x 103 genomes/mL in five patients and decreased to < 4.7 x 103 genomes/mL in the remaining 37 patients. Relapse rates were increased in patients who remained at > 4.7 x 103 genomes/mL compared with patients with HBV DNA levels < 4.7 x 103 genomes/mL (100% vs. 51.4%, P < 0.001). Thus, monitoring of serum HBV DNA at the time of HBeAg seroconversion may be helpful for predicting relapse in patients with lamivudine-induced HBeAg seroconversion.     

Genomic mutations with amino acid substitutions of circulating hepatitis B virus found in non-B,non-C patients with hepatocellular carcinoma

OBJECTIVE: Most hepatocellular carcinoma (HCC) in Japan is caused by chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). HBV DNA has been detected in the serum and liver tissue of some proportion of those patients who are HBs antigen-negative and HCV antibody-negative; i.e., non-B, non-C (NBNC) patients with HCC. We sought to detect HBV DNA in the serum from NBNC HCC cases and to investigate genomic mutations of HBV in seronegative cases. PATIENTS AND METHODS: The sera from 26 NBNC HCC patients were examined by polymerase chain reaction (PCR) followed by southern blotting for existence of HBV DNA. The precore/core and polymerase regions of the HBV genome in the sera from five seronegative cases were analyzed by direct sequence. RESULTS: HBV DNA was detected in 17 of 26 patients (65.4%). Demographic factors such as age, gender, anti-HBs positivity, anti-HBc positivity, complication with cirrhosis, and excessive alcohol intake did not affect circulating HBV positivity. Genomic mutations with amino acid substitutions were detected in the polymerase and the precore regions from one of the five cases, and in the core region from four of the five cases. CONCLUSIONS: PCR-based HBV screening is necessary in patients suffering from liver diseases of unknown etiology, although its etiological importance and benefit of viral elimination have not been established. Genomic mutations in the precore/core and the polymerase region detected in this study might be involved in the lack of HBsAg in NBNC HCC cases.