Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT

• Published in: PloS one 2013-12, Vol.8 (12), p.e82527-e82527
• Main Authors: Arai, Masaaki, Tokunaga, Yuko, Takagi, Asako, Tobita, Yoshimi, Hirata, Yuichi, Ishida, Yuji, Tateno, Chise, Kohara, Michinori
• Format: Article
• Language: English
• Subjects: Animals; Apolipoproteins; Biology; Cell culture; Cloning; Copy number; Derivatives; Genes, Viral; Genetic aspects; Genomes; Genomics; Genotype; Genotype & phenotype; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis; Hepatitis C; Hepatitis C virus; Hepatocytes; Humans; In vivo methods and tests; Infections; Inoculation; Kinases; Laboratory animals; Lipids; Liver; Medical research; Mice; Mutation; Phosphorylation; Polymerase chain reaction; Proteins; Replication; Ribonucleic acid; RNA; RNA, Viral - genetics; Science; Transfection; Virus Replication - genetics; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0082527

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.