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2011 Fiscal Year Research-status Report

次世代シークエンサーによるC型肝炎ウイルスゲノム多様性解析法の確立

Research Project

Project/Area Number 23659407
Research InstitutionNational Institute of Infectious Diseases

Principal Investigator

脇田 隆字  国立感染症研究所, ウイルス第二部, 部長 (40280789)

Project Period (FY) 2011-04-28 – 2013-03-31
KeywordsHCV / ウイルス / 次世代シーケンス / quasispecies / インターフェロン
Research Abstract

C型肝炎ウイルス(HCV)は我が国に多くのキャリアが存在する重要な感染症の原因ウイルスである。HCVはRNAウイルスでありそのゲノムは多様性に富む(quasispecies)ことが知られている。このquasispeciesのHCV感染症における意義は十分に解明されていないが、持続感染あるいは病原性に関わっていることが想定されている。ウイルスゲノムのquasispeciesに関してはこれまでのシークエンス技術では十分に解析ができなかった。本研究では飛躍的に遺伝子解析能力が向上した次世代シークエンサーを用いてウイルスゲノムのquasispecies解析法を確立する。さらに、今後対応が必要な薬剤耐性ウイルス解析への応用を検討する。最近の次世代シークエンス技術の開発により、ウイルスゲノムのquasispeciesに対する理解が大きく変化する可能性がある。次世代シークエンスを従来のPCR法と組み合わせ、HCVゲノムの多種性をウイルスゲノム全長にわたり解析する手法を開発した。その結果、HCV陽性血清1検体から、少なくとも3種類の独立したウイルスゲノムRNA配列を特定した。C型慢性肝炎患者の標準治療法であるインターフェロン/リバビリン(IFN/RBV)併用療法の感受性を決定する因子は複数報告されているが、その相関関係は明らかになっていない。今年度の解析で複数の感受性因子が一種類の配列上に共存していることが初めて確認された。さらに同一患者由来の経時サンプルを解析し、複数のウイルスゲノムに生じる変化と相関を解析した。また、従来の手法で決定したコンセンサス配列との比較を行った。

Current Status of Research Progress
Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

次世代シーケンサーによるウイルスゲノム解析法を樹立し、解析を進めている。

Strategy for Future Research Activity

同定した3種類の代表的なウイルスゲノムが、実際に患者血清中に存在することを、ファージライブラリー法を用いて確認する。決定した一患者血清内に存在する配列をそれぞれクローニングし、培養細胞あるいはキメラマウスでの増殖が可能か検証する。可能であればそれぞれの株の相関や感染性を解析する。治療感受性やウイルス遺伝子型の異なる患者群で解析をおこない、ウイルスゲノム存在様式との関係を解析する。

Expenditure Plans for the Next FY Research Funding

今年度解析方法を確立したので、来年度はその手法を用いてデータの解析をすすめる。今年度は解析方法の確立に集中したため、研究費の使用を予定よりも押さえたが、来年度はデータ取得と解析を加速するために博士研究員レベルの研究員を雇用して解析をすすめたい。

  • Research Products

    (43 results)

All 2012 2011

All Journal Article (12 results) (of which Peer Reviewed: 11 results) Presentation (31 results)

  • [Journal Article] Replication and infectivity of a novel genotype 1b hepatitis C virus clone2012

    • Author(s)
      Date T, Morikawa K, Tanaka Y, Tanaka-Kaneko K, Sata T, Mizokami M, Wakita T
    • Journal Title

      Microbiol Immunol

      Volume: 56(5) Pages: 308-17

    • DOI

      10.1111/j.1348-0421.2012.00437.x.

  • [Journal Article] Detergent-induced activation of the hepatitis C virus genotype 1b RNA polymerase.2012

    • Author(s)
      Weng L, 他
    • Journal Title

      Gene.

      Volume: 496(2) Pages: 79-87

    • Peer Reviewed
  • [Journal Article] Potent and selective inhibition of hepatitis C virus replication by novel phenanthridinone derivatives2011

    • Author(s)
      Salim MT, 他
    • Journal Title

      Biochem Biophys Res Commun.

      Volume: 415(4) Pages: 714-9.

    • Peer Reviewed
  • [Journal Article] Genetic Variation of the IL-28B Promoter Affecting Gene Expression.2011

    • Author(s)
      Sugiyama M, 他
    • Journal Title

      PLoS One.

      Volume: 6(10) Pages: e26620

    • Peer Reviewed
  • [Journal Article] Hepatitis C virus reveals a novel early control in acute immune response2011

    • Author(s)
      Arnaud N, 他
    • Journal Title

      PLoS Pathog

      Volume: 7(10) Pages: e1002289.

    • Peer Reviewed
  • [Journal Article] Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles2011

    • Author(s)
      Saeed M, 他
    • Journal Title

      J Biol Chem.

      Volume: 286(43) Pages: 37264-73

    • Peer Reviewed
  • [Journal Article] Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2.2011

    • Author(s)
      Okamoto Y, 他
    • Journal Title

      Biochem Biophys Res Commun.

      Volume: 410(3) Pages: 404-9.

    • Peer Reviewed
  • [Journal Article] Structural requirements of virion-associated cholesterol for infectivity, buoyant density and apolipoprotein association of hepatitis C virus.2011

    • Author(s)
      Yamamoto M, 他
    • Journal Title

      J Gen Virol.

      Volume: 92(Pt 9) Pages: 2082-7.

    • Peer Reviewed
  • [Journal Article] Production and characterization of HCV particles from serum-free culture.2011

    • Author(s)
      Akazawa D, 他
    • Journal Title

      Vaccine.

      Volume: 29(29-30) Pages: 4821-8

    • 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, 他
    • Journal Title

      Hepatology.

      Volume: 54(2) Pages: 425-33.

    • Peer Reviewed
  • [Journal Article] Malnutrition impairs interferon signaling through mTOR and FoxO pathways in patients with chronic hepatitis C.2011

    • Author(s)
      Honda M, 他
    • Journal Title

      Gastroenterology.

      Volume: 141(1) Pages: 128-40

    • Peer Reviewed
  • [Journal Article] Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV).2011

    • Author(s)
      Aly HH, 他
    • Journal Title

      PLoS One.

      Volume: 6(6) Pages: e21284.

    • Peer Reviewed
  • [Presentation] Hepatitis C virus replication in vitro and persistent infection in vivo: mechanistic analysis and antiviral development2012

    • Author(s)
      T Wakita
    • Organizer
      Challenges to overcome Emerging Infectious Diseases in South-eastern Asia(招待講演)
    • Place of Presentation
      Siran Kaikan, Kyoto Uninversity. Kyoto
    • Year and Date
      2012 Jan 13
  • [Presentation] Hepatitis C virus replication models and anti-viral development2012

    • Author(s)
      T Wakita
    • Organizer
      The 1st International Symposium on Latent TGF-beta Activation Reaction, RIKEN Kobe Inst
    • Place of Presentation
      Ctr. For Delop Biol, Auditorium, Kobe
    • Year and Date
      2012 Feb 25
  • [Presentation] Identification of novel NS5A-associated proteins in the host-cell membrane fraction and their role in HCV life cycle2011

    • Author(s)
      K Goto, T Kimura, K Watashi, R Suzuki, S Yamagoe, T Miyamura, K Moriya, H Yotsuyanagi, K Koike, T Suzuki, T Wakita, H Aizaki
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Does a vaccine derived from a single HCV strain elicit broadly cross-neutralising antibodies in humans?2011

    • Author(s)
      J Law, D Hockman, S Frey,R Khoshy, T Wakita, J Bukh, C Rice, M Houghton
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Isolation of a highly infectious hepatitis C virus with adaptive mutations2011

    • Author(s)
      M Fukasawa, Y Shirasago, K Saito, Y Murakami, H Fukazawa, T Suzuki, R Suzuki, T Wakita, K Hanada, J Chiba
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Development of purification method for HCV particles using chromatographic technique2011

    • Author(s)
      H Yokokawa, D Akazawa, M Moriyama, N Nakamura, T Kato, K Ishii, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Serum and trypsin inhibitors inhibit the early step of hepatitis C virus infection2011

    • Author(s)
      M Esumi, S Kikuta, H Yamaguchi, S Nakajima, M Ishibashi, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Immunological memory response to induce neutralizing immunoglobulin in HCV particles-immunized mice2011

    • Author(s)
      M Moriyama, D Akazawa, H Yokokawa, K Nishimura, N Nakamura, H Mochizuki, T Kato, K Ishii, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Identification of small molecules affecting late steps of hepatitis C virus life cycle2011

    • Author(s)
      K Watashi, N Uchida, R Suzuki, H Aizaki, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Halopemide inhibited a post-assembly step in hepatitis C virus life cycle2011

    • Author(s)
      N Uchida, K Watashi, R Suzuki, H Aizaki, J Chiba, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      20110908-12
  • [Presentation] Small molecules that elicit anti-HCV activity through down-modulation of HCV entry receptors2011

    • Author(s)
      Takebe, Y.,他
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      2011, Sep. 8-12
  • [Presentation] E2 binding peptide identified by RAPID system inhibited HCV infection2011

    • Author(s)
      N Watanabe, K Futai, H Suga, T Wakita
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      2011, Sep. 8-12
  • [Presentation] IDENTIFICATION AND CHARACTERIZATION OF SIGNAL PEPTIDASE COMPLEX 1 THAT INTERACTS WITH HEPATITIS C VIRUS NS2 PROTEIN AND IS INVOLVED IN THE VIRAL ASSEMBLY2011

    • Author(s)
      R Suzuki,他
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      2011, Sep. 8-12
  • [Presentation] Development of chimeric hepatitis C virus expressing NS5A from strains of genotypes 1 and 2: virus production and susceptibility to NS5A inhibitor2011

    • Author(s)
      Y Okamoto, T Masaki, A Murayama, T Wakita, T Kato
    • Organizer
      18th International Meeting on Hepatitis C and Related Viruses
    • Place of Presentation
      Seattle Sheraton Hotel, Seattle, USA
    • Year and Date
      2011, Sep. 8-12
  • [Presentation] Identification and analysis of envelope N-glycans required for HCV lifecycle2011

    • Author(s)
      N Watanabe,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Strain Specific Susceptibility to The Hepatitis C Virus NS5A Inhibitor2011

    • Author(s)
      Y Okamoto,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Identification of a host factor that interacts with hepatitis C virus NS2 protein and is involved in the viral assembly2011

    • Author(s)
      R Suzuki,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Efficient HCV production system using HuH-7 subclone with high virus assembly efficiency2011

    • Author(s)
      A Murayama,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Identification of lipid droplet-associated membrane proteins that are involved in HCV production2011

    • Author(s)
      H Aizaki,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Antiviral activity of glycyrrhizic acid against hepatitis C virus in vitro2011

    • Author(s)
      Y Matsumoto,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Identification of small molecules affecting late steps of hepatitis C virus life cycle2011

    • Author(s)
      K Watashi,他
    • Organizer
      XV International Congress of Virology.
    • Place of Presentation
      Sapporo Convention Center, Sapporo, Japan
    • Year and Date
      2011, Sep. 11-16
  • [Presentation] Screening of small molecules affecting the production of hepatitis B virus2011

    • Author(s)
      K Watashi,他
    • Organizer
      2011 International Meeting on Molecular Biology of Hepatitis B Viruses
    • Place of Presentation
      Holliday Inn Walt Disney World Resort, FL USA
    • Year and Date
      2011, Oct 9-12
  • [Presentation] Hepatitis C Virus Infection and Replication2011

    • Author(s)
      T Wakita
    • Organizer
      Annual meeting of Prof. Juei-Low Sung’s Research Foundation(招待講演)
    • Place of Presentation
      Taipei, Taiwan
    • Year and Date
      2011, 8. 6
  • [Presentation] HCV感染に伴う宿主細胞の脂質代謝の変化と代謝産物のメタボロミクス解析2011

    • Author(s)
      相崎英樹、鈴木哲朗、脇田隆字
    • Organizer
      第47回日本肝臓学会総会
    • Place of Presentation
      ホテルグランパシフィック
    • Year and Date
      2011, 6.2-3
  • [Presentation] 2種類のヒト肝細胞株を用いたHCV感染レポーターアッセイ系の開発2011

    • Author(s)
      武田緑、池田正徳、有海康雄、脇田隆字、加藤宜之
    • Organizer
      第47回日本肝臓学会総会
    • Place of Presentation
      ホテルグランパシフィック
    • Year and Date
      2011, 6.2-3
  • [Presentation] 1999年から2009年における日本のC型急性肝炎の発生状況2011

    • Author(s)
      相崎英樹、他
    • Organizer
      第47回日本肝臓学会総会
    • Place of Presentation
      ホテルグランパシフィック
    • Year and Date
      2011, 6.2-3
  • [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
      ホテルグランパシフィック
    • Year and Date
      2011, 6.2-3
  • [Presentation] 新型シーケンサで展開するウイルスゲノム研究2011

    • Author(s)
      脇田隆字
    • Organizer
      ランチョンセミナー、第34回日本分子生物学会年会(招待講演)
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2011, 12.14
  • [Presentation] HCV JFH-1株のチンパンジー感染実験で得られた適応変異株の機能解析2011

    • Author(s)
      加藤孝宣、政木隆博、脇田隆字
    • Organizer
      第15回日本肝臓学会大会
    • Place of Presentation
      福岡国際会議場
    • Year and Date
      2011, 10.20-21
  • [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
      福岡国際会議場
    • Year and Date
      2011, 10.20-21
  • [Presentation] Hepatitis C virus replication in vitro and persistent infection in vivo: mechanistic analysis and antiviral development2011

    • Author(s)
      T Wakita
    • Organizer
      Singapore-Japan Forum on Emerging Concepts in Microbiology(招待講演)
    • Place of Presentation
      National University of Singapore, Singapore
    • Year and Date
      2011 Nov 15-16

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Published: 2013-07-10  

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