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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.

Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture