Research for relationship between infectiosity and amino acid sequences of hepatitis C virus from human inocula and transmitted chimpanzee sera

我们分析了4种血清中丙型肝炎病毒（HCV）高变区（HVR）的核酸和氨基酸序列，为下一步用黑猩猩进行滴定实验的研究做准备。随后将稀释度为10^<-5>、10^<-3>和10^<-2>的人血清#4分别接种给编号为364,346和345的黑猩猩，随后将稀释度为10^<-4>、10^<-5>和10^<-6>的人血清#6分别接种给编号为353,215和340的黑猩猩。只有353号和215号黑猩猩血清中HCV RNA呈阳性，而接种4号和接种6号均含有高滴度的HCV RNA（通过半定量竞争测定，每个接种2 × 10^6拷贝/ml）。提取RNA，转录成cDNA，扩增，采集20个菌落，测定核酸和氨基酸序列。4号和6号克隆的遗传变异主要局限于HVR区。4号和6号分别有6个和4个克隆，并且4号克隆与6号克隆的氨基酸序列不同（表1和2）。另一方面，克隆的黑猩猩353号和215号在氨基酸序列上完全相同。来自这两只黑猩猩的所有克隆的同源性与人类接种物#6的主要类型（6a-1）相同。滴定实验结果表明，血清中HCV RNA滴度与体内传染性并不一定相关，氨基酸序列分析研究表明，稀释后接种的主要群克隆主要以传播和复制为主。

We analyzed nucleic and amino acid sequences of the hypervariable region (HVR) of the hepatitis C virus (HCV) in the 4 kinds of sera, for the study of the next step of the titration experiments using chimpanzees as follows. One ml each of 10^<-5>,10^<-3> and 10^<-2> dilutions of human serum #4 were subsequently inoculated to chimpanzees nos.364,346, and 345, respectively, and one ml of 10^<-4>,10^<-5>, and 10^<-6> dilutions of human serum #6 were subsequently inoculated to chimpanzee nos.353,215, and 340, respectively. Only chimpanzee nos.353 and 215 were positive for HCV RNA in the sera, while both of inocula #4 and #6 contained high titer of HCV RNA (2x10^6 copies/ml, each, by semi-quantitative competitive assay). RNA extraction, transcription to cDNA,and amplification were done, 20 colonies were picked up, and nucleic and amino acid sequences were determined. Genetic variations were limited mostly to the HVR region in clones from #4 and #6. There were 6 and 4 clones from #4 and #6, respectively, and clones from #4 were different from those from #6 in amino acid sequences (table 1 & 2). On the other hand, clones of chimpanzee nos.353 and 215 were completely same in amino acid sequences. Homology of all clones from both chimpanzees was identical to the major type (6a-1) of human inoculum #6. The results of titration experiment suggested that HCV RNA titer in sera was not always correlate with in vivo infectivity, and the analytical study of amino acid sequences suggested that major group clone in the diluted inocula were mainly transmitted and replicated.