ANIMAL MODELS OF HBV INFECTION AND DISEASE

乙型肝炎病毒感染和疾病的动物模型

• 批准号: 2061261
• 负责人: PATRICIA L MARION
• 金额: $ 23.81万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061261
• 关键词: alternatives to animals in research; animal viral hepatitis; antigen antibody reaction; antiidiotype antibody; antiviral antibody; biological models; blocking antibody; duck hepatitis B virus; ducks; epitope mapping; hepatitis B; hepatitis B antigens; hepatocellular carcinoma; host organism interaction; laboratory mouse; laboratory rabbit; liver cells; microorganism immunology; molecular cloning; monoclonal antibody; neutralizing antibody; ribonuclease III; site directed mutagenesis; tissue /cell culture; transfection; virus infection mechanism; virus protein; virus receptors; virus replication; western blottings

The overall objectives of this research proposal are to use the duck hepatitis B virus (DHBV) model of hepatitis B virus to investigate steps of hepadnaviral replication which might eventually be susceptible to inhibition by antivirals or provide clues to improved vaccines. The proposed experiments are derived from our previous work, including the development of a panel of murine monoclonal antibodies directed to DHBV surface antigens which neutralize virus infection in vitro the finding of ~ several missense mutations in the RNase H domain of the P gene which prevent encapsidation of viral RNA, and a long-term study of the disease associated with DHBV infection of Pekin ducks. Most of the experiments will use two cell culture systems, virion infection of primary duck hepatocytes, and infection of a chicken hepatoma cell line by transfection of cloned viral DNA. The specific aims are: 1. To study the interaction between DHBV surface antigens and the host during early infection. We will continue to characterize the amino acid residues of DHBV surface antigens which are important in viral neutralization, first by delimiting the minimal number of residues that are important in binding the monoclonal antibodies in Western blots and then by site-specific mutagenesis of residues associated with the epitopes to determine which amino acids may be essential to the attachment or entry stages of viral infection. We will continue to study the mechanisms of neutralization of DHBV associated with the monoclonals directed to three epitopes on the pre-5 portion of the surface antigen proteins and with the monoclonal directed to the S portion to determine if any of the monoclonals prevent viral attachment to hepatocyte receptors. We will use several approaches to identify hepatocyte proteins or glycoproteins which interact with DHBV during virus binding and entry. These approaches will be a) development of monoclonal antibodies which are directed to hepatocyte membrane surfaces and which will block infection, to be used as above and b) development of antiidiotype monoclonal and polyclonal antibodies to existing monoclonal antibodies directed against DHBV surface antigen epitopes involved in viral neutralization and use of these anti- idiotype monoclonal antibodies to identify cellular proteins interacting with each viral epitope. If cellular proteins are identified as essential to DHBV-attachment or entry, they will be cloned and characterized. 2. Study the role of the P gene RNase H domain in packaging of the RNA pregenome. RNase H domain packaging mutants will be tested against wild type for functions of the P gene product that can be measured in assays in vitro. These include priming and extension of minus strand synthesis, binding of pregenomic RNA in Northwestern blots, and reactivity in RNase H activity gels. The goal is to understand the packaging activity of this domain by seeing if it is related to a function of the P gene product. 3. Continue long-term study of hepadnavirus-infected ducks to determine if persistent infection with DHBV is associated with development of HCC in ducks.

本研究计划的总体目标是利用鸭子 乙型肝炎病毒（DHBV）模型乙型肝炎病毒考察步骤 肝炎病毒复制最终可能容易受到影响 抗病毒药物的抑制或为改进疫苗提供线索。这 提出的实验来自我们之前的工作，包括 开发一组针对 DHBV 的鼠单克隆抗体 体外中和病毒感染的表面抗原 ~ P 基因 RNase H 结构域中的几个错义突变 防止病毒RNA衣壳化，以及对该疾病的长期研究 与北京鸭DHBV感染有关。大部分实验 将使用两种细胞培养系统，原代鸭病毒粒子感染 肝细胞以及通过转染感染鸡肝癌细胞系 克隆病毒DNA。具体目标是： 1. 研究DHBV表面抗原与宿主的相互作用 在感染早期。 我们将继续表征氨基酸 DHBV 表面抗原的残基在病毒中很重要 中和，首先通过界定残基的最小数量 在蛋白质印迹中结合单克隆抗体很重要 然后通过与表位相关的残基进行位点特异性诱变 确定哪些氨基酸对于附着或进入可能是必需的 病毒感染的阶段。我们将继续研究其机制 与单克隆抗体相关的 DHBV 中和作用针对三种 表面抗原蛋白前 5 部分上的表位和 针对 S 部分的单克隆抗体以确定是否有任何 单克隆抗体可防止病毒附着于肝细胞受体。我们将使用 鉴定肝细胞蛋白或糖蛋白的几种方法 在病毒结合和进入过程中与 DHBV 相互作用。 这些方法将 a) 开发单克隆抗体 肝细胞膜表面，可阻断感染，可用作 上述和b)抗独特型单克隆和多克隆的开发 针对 DHBV 表面的现有单克隆抗体的抗体 参与病毒中和的抗原表位以及这些抗病毒药物的使用 用于识别细胞蛋白质相互作用的独特型单克隆抗体 与每个病毒表位。如果细胞蛋白质被认为是必需的 当 DHBV 附着或进入时，它们将被克隆和表征。 2. 研究P基因RNase H结构域在RNA包装中的作用 前基因组。 RNase H 结构域包装突变体将针对野生环境进行测试 P 基因产物功能的类型，可在测定中测量 体外。这些包括负链合成的引发和延伸， Northwestern 印迹中前基因组 RNA 的结合以及 RNase 中的反应性 H活性凝胶。目标是了解此包装的活动 域，看看它是否与 P 基因产物的功能相关。 3. 继续对感染嗜肝DNA病毒的鸭进行长期研究，以确定是否 DHBV 持续感染与 HCC 的发生有关 鸭子。