Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture
Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture
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DOI：
10.1099/vir.0.80479-0
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发表时间：
2005-03-01
影响因子：
3.8
通讯作者：
Shimotohno, K
中科院分区：
文献类型：
--
作者：
Kato, N;Nakamura, T;Shimotohno, K
已知丙型肝炎病毒（HCV）基因组序列在HCV毒株之间差异很大，但迄今为止，在HCV复制的实验系统中，关于HCV的遗传变异和动力学的报道很少。在本研究中，对分别从两种HCV株1B-1和1B-2建立的两种HCV复制子细胞（50-1和1B-2 R1）的长期培养中获得的HCV复制子进行遗传分析。一个人培养50-1细胞18个月，两个人独立培养50-1细胞12个月。1B-2 R1细胞也培养了12个月。测定在几个时间点获得的三个独立复制子RNA克隆的全核苷酸序列。观察到两种复制子中的基因突变以时间依赖性方式积累，并且两种复制子的突变率约为3.0 × 10（-3）碱基置换/位点/年。两种复制子的遗传多样性也以时间依赖的方式扩大。通过将在不同时间点从两种复制子细胞分离的总RNA转染到初始HuH-7细胞中的殖民地形成测定揭示，在两种复制子中随时间积累的遗传突变明显提高了菌落形成效率。总而言之，这些结果表明HCV复制子系统对于分析HCV的进化动力学和变异是有用的。使用该复制子细胞培养系统，进一步证明了利巴韦林或其衍生物咪唑立宾均不加速HCV复制子的突变率或遗传多样性的增加。
Hepatitis C virus (HCV) genomic sequences are known to vary widely among HCV strains, but to date there have been few reports on the genetic variations and dynamics of HCV in an experimental system of HCV replication. In this study, a genetic analysis of HCV replicons obtained in long-term culture of two HCV replicon cells (50-1 and 1B-2R1), which were established from two HCV strains, 1B-1 and 1B-2, respectively, was performed. One person cultured 50-1 cells for 18 months, and two people independently cultured 50-1 cells for 12 months. 1B-2R1 cells were also cultured for 12 months. The whole nucleotide sequences of the three independent replicon RNA clones obtained at several time points were determined. It was observed that genetic mutations in both replicons accumulated in a time-dependent manner, and that the mutation rates of both replicons were approximately 3.0 x 10(-3) base substitutions/site/year. The genetic diversity of both replicons was also enlarged in a time-dependent manner. The colony formation assay by transfection of total RNAs isolated from both replicon cells at different time points into naive HuH-7 cells revealed that the genetic mutations accumulating with time in both replicons apparently improved colony formation efficiency. Taken together, these results suggest that the HCV replicon system is useful for the analysis of evolutionary dynamics and variations of HCV. Using this replicon cell culture system, it was demonstrated further that neither ribavirin nor its derivative mizoribine accelerated the mutation rate or the increase in the genetic diversity of HCV replicon.