MOLECULAR BIOLOGY OF HEPATITIS VIRUS IN VIVO

这项建议的目的是应用分子技术来研究 土拨鼠肝炎病毒（WHV）感染的生物学和进展。 的 与人B肝炎病毒（HBV）相关的三种动物病毒， WHV是唯一一种已知引起肝细胞癌的HBV 可以这 这项研究将确定病毒感染的环境， 它将提供基线数据， 需要开发WHV作为治疗HBV的实验模型 感染 为了使WHV的血清学检测类似于用于研究HBV的检测， WHV的表面抗原、核心抗原和e抗原将从 感染动物的适当组织，并用于免疫兔子。 的 如此获得的抗原和抗体将用于建立酶 免疫测定。 将详细绘制感染肝脏中存在的WHV特异性RNA种类 通过基于引物延伸的方法，以及编码 通过体外翻译鉴定表面和核心抗原。 的 病毒体DNA链的末端将相对于 已知的基因组核苷酸序列。 急性和慢性WHV感染的进展将通过 血清学采样和通过重复获得的核酸分析 受感染动物的肝活检。 RNA转录本将通过以下方法进行分析： “北方”印迹杂交和DNA复制中间体 Southern印迹，而共价闭合的环状和整合的病毒DNA 将在通过BND-纤维素分离后同样进行分析 层析 希望这种研究方法能揭示 在慢性感染过程中， 产生感染性病毒以表达其自身的表面抗原， 以及提供关于慢性感染的解决方案的信息。

The object of this proposal is to apply molecular techniques to study the biology and progression of Woodchuck Hepatitis Virus (WHV) infection. Of the three animal viruses that are related to human Hepatitis B Virus (HBV), WHV is the only one that is known to cause hepatocellular carcinoma as HBV does. This study will define the environment in which viral hepatocarcinogenesis takes place, and it will provide baseline data required to develop WHV as an experimental model for treatment of HBV infection. To make serological assays for WHV analogous to those used to study HBV in Man the surface, core and e antigens of WHV will be purified from appropriate tissues of infected animals and used to immunise rabbits. The antigens and antibodies so obtained will be used to set up enzyme immunoassays. WHV-specific RNA species present in infected liver will be mapped in detail by methods based on primer extension, and the transcripts coding for the surface and core antigens will be identified by in vitro translation. The ends of the virion DNA strands will be precisely located relative to the known nucleotide sequence of the genome. The progression of acute and chronic WHV infections will be studied by serological sampling and by analysis of nucleic acids obtained by repeated liver biopsy of infected animals. RNA transcripts will be analysed by "Northern" blot hybridisation, and DNA intermediates of replication by Southern blot, while covalently closed circular and integrated viral DNA will be analysed likewise after separation by BND-cellulose chromatography. It is hoped that this method of study will reveal the molecular changes that underlie the shift during chronic infection from production of infectious virus to expression of surface antigen on its own, as well as providing information about the resolution of chronic infection.