Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.
The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Nao
N
Sugiyama
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Asako
A
Murayama
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Ryosuke
R
Suzuki
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Noriyuki
N
Watanabe
Department of Gastroenterology and Hepatology, Shin-Yurigaoka General Hospital, Kawasaki, Kanagawa, Japan.
Masaaki
M
Shiina
Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
T Jake
TJ
Liang
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Takaji
T
Wakita
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Takanobu
T
Kato
D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D003470:Culture Media; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D014162:Transfection; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication
D003470:Culture Media; D012367:RNA, Viral