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.

该研究建议的总体目标是使用鸭子 乙型肝炎病毒B病毒B病毒的乙型肝炎病毒研究步骤 肝复制的复制 抗病毒药抑制或为改善疫苗提供线索。这 提出的实验来自我们以前的工作，包括 开发针对DHBV的鼠单克隆抗体 表面抗原在体外中和病毒感染的发现 〜P基因的RNase H结构域中的几个错义突变 防止病毒RNA的封装以及对疾病的长期研究 与DHBV感染北京鸭有关。大多数实验 将使用两个细胞培养系统，即初级鸭子的病毒体感染 肝细胞和通过转染的鸡肝癌细胞系感染 克隆病毒DNA。具体目的是： 1。研究DHBV表面抗原与宿主之间的相互作用 在早期感染期间。 我们将继续表征氨基酸 在病毒中很重要的DHBV表面抗原的残基 中和，首先通过划定最小数量的残基数 在结合蛋白质印迹中的单克隆抗体和 然后按与表位相关的残基的位点特异性诱变 确定哪种氨基酸可能对附着或进入至关重要 病毒感染的阶段。我们将继续研究 与单克隆有关的DHBV中和的中和 表面抗原蛋白的5前的表位，并与 单克隆针对S部分，以确定是否有任何 单克隆可预防肝细胞受体的病毒附着。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。 这些方法会 a）针对的单克隆抗体的开发 肝细胞膜表面并将阻止感染，以用作 上面和b）抗替代型单克隆和多克隆的发展 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和使用这些抗原 白痴单克隆抗体以鉴定相互作用的细胞蛋白 每个病毒表位。如果将细胞蛋白识别为必不可少的 对于DHBV的代表或进入，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 前植物组。 RNase H域包装突变体将对野生进行测试 可以在测定中测量的P基因产物功能的类型 体外。这些包括启动和扩展减去链合成， 西北印迹中基因组RNA的结合，RNase中的反应性 H活性凝胶。目的是了解此包装活动 通过查看它是否与P基因产物的函数有关。 3。继续对肝病病毒感染的鸭子进行长期研究，以确定是否是否 DHBV持续感染与HCC的发展有关 鸭子。