MOLECULAR BIOLOGY OF HEPATITIS VIRUS IN VIVO

这项建议的目的是应用分子技术来研究 土拨鼠肝炎病毒感染的生物学与进展的 与人类乙肝病毒(乙肝病毒)相关的三种动物病毒， WHV是已知的唯一一种引起肝细胞癌的病毒，与乙肝病毒一样 确实如此。这项研究将定义病毒感染的环境 肝癌的发生，它将提供基线数据 需要开发WHV作为治疗乙肝病毒的实验模型 感染。 使WHV的血清学检测类似于用于研究乙肝病毒的方法 人类WHV的表面、核心和e抗原将从 受感染动物的适当组织，用于免疫兔子。这个 这样获得的抗原和抗体将被用来建立酶 免疫分析。 将详细绘制感染肝脏中存在的WHV特异性RNA物种的图谱 通过基于引物延伸的方法和编码该基因的转录本 表面和核心抗原将通过体外翻译来鉴定。这个 病毒DNA链的末端将相对于 已知的基因组核苷酸序列。 将通过以下方式研究急性和慢性WHV感染的进展 血清学采样和对重复试验获得的核酸的分析 感染动物的肝脏活检。RNA抄本将通过以下方式进行分析 “Northern”杂交，DNA复制的中间产物 Southern杂交，同时共价闭合环状和整合的病毒DNA 在BND-纤维素分离后也将进行分析 层析法。希望这种研究方法能够揭示出 在慢性感染过程中导致转变的分子变化 感染性病毒的产生本身就能表达表面抗原， 以及提供有关解决慢性感染的信息。

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.