西南地区快速流行基因型- - 6型HCV病毒培养体系构建及其感染与增殖特性研究

病毒体外培养体系是研究病毒感染能力、增殖速度等毒力特性，进行抗病毒药物筛选与疫苗开发的重要工具。根据基因组核酸变异而区分为多种基因型的丙型肝炎病毒，其传播特性、病程特点及药物治疗反应性也明显不同，有必要根据病毒主要流行基因型的变化，针对性建立病毒培养体系。本研究拟在前期研究已发现云南省多基因型HCV分布的基础上，选择扩增西南地区正在快速流行6型样本的5'非编码区-结构蛋白基因片段，与已建立的HCV病毒培养体系中的J6/JFH1质粒进行基因重组，构建正在快速流行HCV病毒株的嵌合体病毒培养体系，测定不同基因型嵌合体病毒间感染能力与增殖速度，通过交互回复突变及病毒毒力特性变化检测，阐明病毒核酸变异与其毒力变化的相关性。研究将建立西南地区6型HCV流行株嵌合体病毒体外培养体系，探讨6型HCV在西南地区快速传播的分子机制，为地区乃至全国丙型肝炎分子病毒学研究、抗病毒药物及疫苗研发提供技术支撑。

The virus culture system is a useful technique platform for analyzing viral infectivity and replication, for development of drugs and vaccine. Phylogenetically, HCV has been classified into at least six genotypes and more than eighty subtypes. The characters of virus transmission, disease progression and treatment response were different between various HCV genotypes. It is necessary to develop the culture system of various genotypes, particularly the emerging or fast spreading genotypes. Based on our previous finding of multiple HCV genotypes prevalence in Yunnan, in this study, we are going to amplify the partial genome of 5' un-coding region and all structure proteins coding region of genotype 6 HCV strains, which is fast spreading in southern and west-southern China. This fragment will be further inserted into the skeleton of J6/JFH-1 plasmid to construct the recombinant vector. After its linearization and subsequent transfection to Huh7 cells, the culture system of chimeric virus will be established. Then, the infectivity and replication ability of produced chimeric virus will be further determined. In comparison with JFH-1 and J6/JFH-1 culture system, which derived from genotype 2a, the natural mutation sites were revers mutated to clarify the relationship between the virulence and gene mutation of structural protein. Through the conducting of this study, the chimeric HCV culture system of genotype 6, will be established, as a technique support for the research on HCV molecular virology and development of anti-HCV drug and vaccine. Furthermore, the results of this study will shed a light on the mechanism of genotypes 6 strains' wide prevalence in Southern and South-western China.