Establishment of novel HCV cell-culture system using HCV strains other than JFH-1

• Project/Area Number: 23790513
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Virology
• Research Institution: National Institute of Infectious Diseases
• Principal Investigator: MURAYAMA Asako 国立感染症研究所, ウイルス第二部, 協力研究員 (40415534)
• Project Period (FY): 2011 – 2012
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2012: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2011: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: ウイルス / 細胞培養 / ゲノム複製 / C型肝炎ウイルス

Research Abstract

We established a novel HCV genotype 2a cell-culture system using J6CF strain harboring five mutations in NS4A, NS5B and 3'UTR. We also established an HCV genotype 2b cell-culture system using a chimeric genome of MA strain harboring minimal regions of JFH-1. These cell-culture systems will be useful for characterizing genotype 2a and 2b viruses and developing antiviral strategies. Our data provides information that will prove essential for the establishment of replication-competent variants of HCV strains that are currently replication incompetent in cultured cells.

Research Products

• [Journal Article] Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection (2014)
  • Author(s): Sugiyama N, Murayama A, Suzuki R, Watanabe N, Shiina M, Liang TJ, Wakita T, Kato T
  • Journal Title: PLoS One Volume: 9(5)
• [Journal Article] Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I-α in Infectious Virus Production (2014)
  • Author(s): Masaki T, Matsunaga S, Takahashi H, Nakashima K, Kimura Y, Ito M, Matsuda M, Murayama A,Kato T, Hirano H, Endo Y, Lemon SM, Wakita T, Sawasaki T, Suzuki T
  • Journal Title: Journal of Virology Volume: in press
  • Peer Reviewed
• [Journal Article] NS5B-RNAポリメラーゼの構造と機能 (2013)
  • Author(s): 村山麻子、加藤孝宣
  • Journal Title: 肝胆膵 Volume: 67(6) Pages: 918-923
• [Journal Article] Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays (2012)
  • Author(s): Murayama A, Sugiyama N, Watashi K, Masaki T, Suzuki R, Aizaki H, Mizuochi T, Wakita T, Kato T
  • Journal Title: J Clin Microbiol Volume: 50(6) Pages: 1943-1949
• [Journal Article] Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone (2012)
  • Author(s): Date T, Kato T, Kato J, Takahashi H, Morikawa K, Akazawa D, Murayama A, Tanaka-Kaneko K, Sata T, Tanaka Y, Mizokami M, Wakita T
  • Journal Title: J Virol Volume: 86(19) Pages: 10805-10820
• [Journal Article] A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest (2012)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara-Sugano M, Masaki T, Kim S, Wakita T, Mishiro S, Kato T
  • Journal Title: PLoS One Volume: 7(12)
• [Journal Article] Production of Infectious Chimeric Hepatitis C Virus Genotype 2b Harboring Minimal Regions of JFH-1 (2012)
  • Author(s): Murayama A, Kato T, Akazawa D, Sugiyama N, Date T, Masaki T, Nakamoto S, Tanaka Y, Mizokami M, Yokosuka O, Nomoto A, Wakita T
  • Journal Title: J Virol Volume: 86(4) Pages: 2143-2152
• [Journal Article] A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest. (2012)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara-Sugano M, Masaki T, Kim S, Wakita T, Mishiro S, Kato T
  • Journal Title: PLoS One Volume: 7(12) Issue: 12 Pages: e52697-e52697
  • DOI: 10.1371/journal.pone.0052697
  • Peer Reviewed
• [Journal Article] Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone (2012)
  • Author(s): Date T,Tanaka Y.
  • Journal Title: J Virol. Volume: 86(19) Issue: 19 Pages: 10805-10820
  • DOI: 10.1128/jvi.07235-11
  • Peer Reviewed
• [Journal Article] Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays. (2012)
  • Author(s): Maruyama, A.
  • Journal Title: J. Clin. Microbiol. Volume: 50(6) Issue: 6 Pages: 1943-1949
  • DOI: 10.1128/jcm.00487-12
  • Peer Reviewed
• [Journal Article] 25-Hydroxyvitamin D3 suppresses hepatitis C virus production (2012)
  • Author(s): Matsumura T, Kato T, Sugiyama N, Tasaka-Fujita M, Murayama A, Masaki T, Wakita T, Imawari M
  • Journal Title: Hepatology Volume: 56(4) Issue: 4 Pages: 1231-9
  • DOI: 10.1002/hep.25763
  • Peer Reviewed
• [Journal Article] Production of Infectious Chimeric Hepatitis C Virus Genotype 2b Harboring Minimal Regions of JFH-1 (2011)
  • Author(s): Murayama A., et al
  • Journal Title: J Virol Volume: 86 Issue: 4 Pages: 2143-2152
  • DOI: 10.1128/jvi.05386-11
  • Peer Reviewed
• [Journal Article] In vivo adaptation of hepatitis C virus in chimpanzees for efficient virus production and evasion of apoptosis (2011)
  • Author(s): Saeed M, Shiina M, Date T, Akazawa D, Watanabe N, Murayama A, Suzuki T, Watanabe H, Hiraga N, Imamura M, Chayama K, Choi Y, Krawczynski K, Liang TJ, Wakita T, Kato T
  • Journal Title: Hepatology Volume: 54 Issue: 2 Pages: 425-433
  • DOI: 10.1002/hep.24399
  • Peer Reviewed
• [Journal Article] Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2 (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Munakata T, Nomoto A, Nakamoto S, Yokosuka O, Watanabe H, Wakita T, Kato T
  • Journal Title: Biochem Biophys Res Commun Volume: 410(3) Issue: 3 Pages: 404-9
  • DOI: 10.1016/j.bbrc.2011.05.144
  • Peer Reviewed
• [Presentation] An adaptive mutation in NS4A region enhances Hepatitis C Virus replication and virus production in cell culture (2013)
  • Author(s): 村山麻子、杉山奈央、脇田隆字、加藤孝宣
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸
• [Presentation] HCV NS5Aキメラウイルスを用いた第二世代NS5A阻害剤の抗ウイルス活性の評価 (2013)
  • Author(s): 村山麻子、杉山奈央、脇田隆字、加藤孝宣
  • Organizer: 第61回日本ウイルス学会学術集会
  • Place of Presentation: 神戸
• [Presentation] Evaluation of second-generation NS5A inhibitors against hepatitis C virus by using NS5A replaced JFH-1 viruses and full-length infectious clones other than JFH-1 (2013)
  • Author(s): Murayama A, Sugiyama N, Wakita T, Kato T
  • Organizer: 64th Annual Meeting of the American Association for the Study of Liver Diseases
  • Place of Presentation: Washington D.C., USA
• [Presentation] Assessment of antiviral activities of NS5A inhibitors on recombinant HCV replaced with NS5A from genotypes 1 and 2 strains (2013)
  • Author(s): Murayama A, Sugiyama N, Masaki T, Wakita T, Kato T
  • Organizer: 20th International Symposium on Hepatitis C and Related Viruses
  • Place of Presentation: Melbourne, Australia
• [Presentation] Identification of cell-culture adapted hepatitis C virus JFH-1 variants by single virus isolation with end-point dilution method (2013)
  • Author(s): Sugiyama N, Murayama A, Suzuki R, Shiina M, Wakita T, Kato T
  • Organizer: 20th International Symposium on Hepatitis C and Related Viruses
  • Place of Presentation: Melbourne, Australia
• [Presentation] NS5A領域置換HCVキメラ株を用いたNS5A阻害剤の株特異的抗ウイルス活性の評価 (2012)
  • Author(s): 村山麻子、杉山奈央、岡本有加、政木隆博、脇田隆字、加藤孝宣
  • Organizer: 第35回日本分子生物学会年会
  • Place of Presentation: 福岡
• [Presentation] C型肝炎ウイルス遺伝子型2b株とJFH-1株のキメラウイルスを用いた抗ウイルス薬評価系の樹立 (2012)
  • Author(s): 村山麻子、加藤孝宣、杉山奈央、脇田隆字
  • Organizer: 第60回日本ウイルス学会学術集会
  • Place of Presentation: 大阪
• [Presentation] Efficient hepatitis C virus production associated with enhanced virus assembly and evasion of cell cycle arrest (2012)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara-Sugano M, Wakita T, Mishiro S, Kato T
  • Organizer: 63rd Annual Meeting of the American Association for the Study of Liver Diseases
  • Place of Presentation: Boston, USA
• [Presentation] Infectious Virus Production with Hepatitis C Virus Genotype 2b Genome Harboring Minimal Regions of JFH-1 (2012)
  • Author(s): Murayama A, Kato T, Sugiyama N, Wakita T
  • Organizer: 19th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Venice, Italy
• [Presentation] C型肝炎ウイルス産生効率の良いHuH-7細胞サブクローンの同定と解析 (2012)
  • Author(s): 村山麻子、三代俊治、脇田隆字、加藤孝宣
  • Organizer: 第19回肝細胞研究会
  • Place of Presentation: 札幌
• [Presentation] Efficient hepatitis C virus production associated with enhanced virus assembly and evasion of cell cycle arrest. (2012)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara-Sugano M, Wakita T, Mishiro S, Kato T
  • Organizer: 63rd Annual Meeting of the American Association for the Study of Liver Diseases
  • Place of Presentation: Boston, USA
• [Presentation] C型肝炎ウイルス産生効率の良いHuH-7細胞サブクローンの同定と解析. (2012)
  • Author(s): 村山麻子、三代俊治、脇田隆字、加藤孝宣
  • Organizer: 第19回肝細胞研究会
  • Place of Presentation: 札幌
• [Presentation] C型肝炎ウイルス遺伝子型2b株とJFH-1株のキメラウイルスを用いた抗ウイルス薬評価系の樹立. (2012)
  • Author(s): 村山麻子、加藤孝宣、杉山奈央、脇田隆字
  • Organizer: 第60回日本ウイルス学会学術集会
  • Place of Presentation: 大阪
• [Presentation] HuH-7 subclone that supports high HCV production due to high virus assembly (2011)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara- Sugano M, Wakita T, Kato T
  • Organizer: International Union of Microbiological Societies 2011 Congress. XV International Congress of Virology
  • Place of Presentation: Sapporo, Japan
• [Presentation] Strain Specific Susceptibility to The Hepatitis C Virus NS5A Inhibitor (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Nomoto A, Wakita T, Kato T
  • Organizer: International Union of Microbiological Societies 2011 Congress. XV International Congress of Virology
  • Place of Presentation: Sapporo, Japan
• [Presentation] Efficient HCV production system using HuH-7 subclone with high virus assembly efficiency (2011)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara- Sugano M, Wakita T, Kato T
  • Organizer: 18th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Seattle, USA
• [Presentation] Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2 : virus production and susceptibility to NS5A inhibitor (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Wakita T, Kato T
  • Organizer: 18th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Seattle, USA
• [Presentation] 国内献血検体を用いたC型肝炎ウイルス陽性血漿パネルの作製とウイルス量測定法の評価 (2011)
  • Author(s): 加藤孝宣、村山麻子、政木隆博、相崎英樹、脇田隆字
  • Organizer: 第４７回 日本肝臓学会総会
  • Place of Presentation: 東京
• [Presentation] Efficient HCV production system using HuH-7 subclone with high virus assembly efficiency. (2011)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara- Sugano M, Wakita T, Kato T.
  • Organizer: 18th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Seattle, USA
• [Presentation] Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2: virus production and susceptibility to NS5A inhibitor. (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Wakita T, Kato T.
  • Organizer: 18th International Symposium on Hepatitis C Virus and Related Viruses
  • Place of Presentation: Seattle, USA
• [Presentation] HuH-7 subclone that supports high HCV production due to high virus assembly. (2011)
  • Author(s): Murayama A, Sugiyama N, Yoshimura S, Ishihara- Sugano M, Wakita T, Kato T.
  • Organizer: International Union of Microbiological Societies 2011 Congress. XV International Congress of Virology.
  • Place of Presentation: Sapporo, Japan.
• [Presentation] Strain Specific Susceptibility to The Hepatitis C Virus NS5A Inhibitor. (2011)
  • Author(s): Okamoto Y, Masaki T, Murayama A, Nomoto A, Wakita T, Kato T.
  • Organizer: International Union of Microbiological Societies 2011 Congress. XV International Congress of Virology.
  • Place of Presentation: Sapporo, Japan.
• [Presentation] Identification and analysis of envelope N-glycans required for HCV lifecycle. (2011)
  • Author(s): Watanabe N, Murayama A, Date T, Kato T, Aizaki H, Wakita T.
  • Organizer: International Union of Microbiological Societies 2011 Congress. XV International Congress of Virology.
  • Place of Presentation: Sapporo, Japan.
• [Presentation] 25-hydroxy- vitamin D inhibits hepatitis C virus replication and production of the infectious viruses. (2011)
  • Author(s): Matsumura T, Kato T, Tasaka-Fujita M, Murayama A, Masaki T, Wakita T, Imawari M.
  • Organizer: The 62nd Annual Meeting of the American Association for the Study of Liver Diseases
  • Place of Presentation: San Francisco, USA.
• [Presentation] C型肝炎ウイルス粒子の産生効率の良いHuH-7細胞サブクローンの分離と同定 (2011)
  • Author(s): 村山麻子、三代俊治、脇田隆字、加藤孝宣
  • Organizer: 第１５回日本肝臓学会大会
  • Place of Presentation: 福岡
• [Patent(Industrial Property Rights)] C型肝炎ウイルスJ6CF株ゲノム由来の変異体レプリコン(MUTANT REPLICON DERIVED FROM GENOME OF HEPATITIS C VIRUS J6CF STRAIN) (2012)
  • Inventor(s): 脇田隆字、村山麻子、加藤孝宣
  • Industrial Property Rights Holder: 脇田隆字、村山麻子、加藤孝宣
  • Industrial Property Rights Type: 特許
  • Filing Date: 2012-05-31
  • Overseas
• [Patent(Industrial Property Rights)] C型肝炎ウイルスJ6CF株ゲノム由来の変異体レプリコン (MUTANT REPLICON DERIVED FROM GENOME OF HEPATITIS C VIRUS J6CF STRAIN) (2012)
  • Inventor(s): 脇田隆字、村山麻子、加藤孝宣
  • Industrial Property Rights Holder: 脇田隆字、村山麻子、加藤孝宣
  • Industrial Property Rights Type: 特許
  • Filing Date: 2012-05-31
  • Overseas
• [Patent(Industrial Property Rights)] 培養細胞で感染性ウイルス粒子産生可能なＣ型肝炎ウイルスJ6CF株変異体 (2011)
  • Inventor(s): 脇田隆字、村山麻子、加藤孝宣
  • Industrial Property Rights Holder: 脇田隆字、村山麻子、加藤孝宣
  • Filing Date: 2011