HBV VARIANTS AND HEPATOCELLULAR CARCINOMA

B肝炎病毒（HBV）感染是慢性肝病最常见的原因 疾病在世界范围内的基础上，并密切相关，随后 肝细胞癌（HCC）的发展。 我们和其他人发现 通过单克隆抗体和分子技术， 慢性肝病和肝细胞癌患者 此前认为与乙型肝炎病毒感染无关。 我们希望描述 在分子和抗原水平上更详细地描述这些病毒因子， 研究其变异生物学行为的生化基础， 探讨其在肝癌发病中的意义。 我们计划做 以下内容：1）评价临床和流行病学意义 感染低水平HBV和/或变异体，并评估 丙型肝炎病毒（HCV）感染的贡献。 我们将采用 聚合酶链反应检测、扩增和表征低水平 血清和肝脏中的病毒序列的HBsAg阴性患者， 肝细胞癌 在这方面，我们的目标如下：a. 通过快速、灵敏和特异的技术， 单克隆抗-HBs抗体捕获血清中的灭活病毒粒子 随后PCR扩增HBV基因组的不同区域。 B. MAb捕获/PCR后使用限制性内切酶片段分析 以快速表征HBV相关因子。 这种方法可以让我们 来快速评估基因组异质性。 C. 证明存在 低水平的HBV DNA在这些患者的肝脏，并比较我们的结果， 血清中的发现。 D. 试图确定HCV的患病率 通过测量HCV抗体和HCV基因组的存在， 抗-HCV诊断试剂盒和PCR技术。 2)从HBsAg阴性患者中克隆和测序HBV DNA， 确定核苷酸和氨基酸的变异性，这可能是 生物学行为的分子基础 我们还计划探索 病毒因子在肝病发病机制中的作用。 3)审查 乙型肝炎病毒变异株基因组突变的体外生理学意义 为了了解它们在肝细胞中的生物学特性， carcinoma. 我们将对变异的HBV基因组进行重组和测序， 人肝癌细胞系和原代肝细胞培养物和病毒分析 抗原产生和组成，转录调节，和 这些变体的复制能力。 根据我们的初步数据 获得，我们乐观地认为，新的信息将获得的 乙型肝炎病毒基因型的分子特征及临床意义 变体。 我们认为这些代理人可能发挥了以前未被认识到的作用， 在慢性肝病发病机制中的作用导致或 为HCC做贡献 我们还认为，通过研究这些变体， 体外和体内，进一步了解的生物学意义， 将获得HBV的遗传突变体。

Hepatitis B virus (HBV) infection is the most common cause of chronic liver disease on a worldwide basis and closely associated with the subsequent development of hepatocellular carcinoma (HCC). We and others have detected by monoclonal antibodies and molecular techniques HBV and/or variants in individuals with chronic liver disease and hepatocellular carcinoma previous thought to be unrelated to HBV infection. We wish to characterize these viral agents in more detail at the molecular and antigenic level, to investigate the biochemical basis of their variant biological behavior, and to evaluate their significance in the pathogenesis of HCC. We plan to do the following: 1) Evaluate the clinical and epidemiological significance of infection with low level HBV and/or variants and to assess the contribution of hepatitis C viral (HCV) infection. We will employ the polymerase chain reaction to detect, amplify and characterize low level viral sequences in serum and liver of HBsAg-negative patients with hepatocellular carcinoma. In this regard, our aims are as follows: a. Identify such agents by a rapid, sensitive and specific technique using monoclonal anti-HBs antibody to capture encapsidated virions in serum following by PCR amplification of different regions of the HBV genome. b. Use a restriction endonuclease fragment analysis following MAb capture/PCR to rapidly characterize the HBV related agents. This method will allow us to rapidly assess genomic heterogeneity. c. Demonstrate the presence of low level HBV DNA in liver of these patients and compare our results to findings in serum. d. Attempt to determine the prevalence of HCV infection by measuring the presence of antibody to HCV and HCV genome using recently available anti-HCV diagnostic kit and PCR technique, respectively. 2) Clone and sequence HBV DNA from HBsAg-negative patients in order to determine the nucleotide and amino acid variability which may be the molecular basis for their biological behavior. We also plan to explore the role of viral factor(s) in the pathogenesis of liver disease. 3) Examine the physiologic significance of genomic mutations in HBV variants in vitro in order to understand their biological properties in hepatocellular carcinoma. We will reconstruct and transfect the variant HBV genomes into human hepatoma cell lines and primary hepatocyte cultures and analyze viral antigen production and composition, transcriptional regulation, and replication competence of these variants. Based on our preliminary data obtained, we are optimistic that new information will be obtained on the clinical significance and molecular characteristics of HBV genetic variants. We believe that these agents may play a previously unrecognized role in the pathogenesis of chronic liver disease leading to or contributing to HCC. We also believe that by studying these variants in vitro and in vivo, further insights into the biological significance of genetic mutants of HBV will be obtained.