title: Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems.
abstract: BACKGROUND
  The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells.
  
  
  RESULTS
  Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5'-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10(-3) and 2.7-4.0×10(-3) base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents.
  
  
  CONCLUSIONS
  Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kato
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Hiroe
authors.initials: H
authors.lastname: Sejima
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Youki
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Mori
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Satoh
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Dansako
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Okayama, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Ikeda
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000821:virology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003062:Codon; D019143:Evolution, Molecular; D017353:Gene Deletion; D005814:Genes, Viral; D014644:Genetic Variation; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000821:virology; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D010802:Phylogeny; D012367:RNA, Viral / Q000096:biosynthesis*
chemical_list: D000998:Antiviral Agents; D003062:Codon; D012367:RNA, Viral