title: Genetic variation and dynamics of hepatitis C virus replicons in long-term cell culture.
abstract: 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.
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kato
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Dansako
authors.affiliation: First Department of Internal Medicine, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Namba
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Ken-Ichi
authors.initials: KI
authors.lastname: Abe
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Akito
authors.initials: A
authors.lastname: Nozaki
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Naka
authors.affiliation: Department of Molecular Biology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawara-cho Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan.
authors.forename: Kunitada
authors.initials: K
authors.lastname: Shimotohno
mesh_terms: D018929:Cell Culture Techniques; D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D010802:Phylogeny; D012367:RNA, Viral; D012093:Replicon / Q000235:genetics*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D012367:RNA, Viral