| Project/Area Number |
17590632
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Gastroenterology
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| Research Institution | Mie University |
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Principal Investigator |
WATANABE Shozo Mie University, Center for physical and mental health, Professor (20134934)
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| Co-Investigator(Kenkyū-buntansha) |
KAITO Masahiko Mie University, Graduate School of Medicine, Associate Professor (70214244)
KONISHI Masayoshi Mie University, Center for physical and mental health, Associate Professor (00397514)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,480,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥180,000)
Fiscal Year 2007: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2006: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2005: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | hepatitis C virus / in vitro multiplication / IMY-N9 cell / Huh7.5 cell / COS cell / immunoelectron microscopy / COS7細胞 / IMY細胞 |
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| Research Abstract |
Full genome of HCVcDNA(genotype 1b) was induced into IMY-N9 cells, hybrid cells derived from human hepatocytes and HepG2 cells, by using transfection-infection method. High level of HCV replication in this system was demonstrated by Western blot analysis for expression of HCV-specific structural and nonstructural proteins in the cells and real time RT-PCR quantification for HCVRNA in the cell culture media. Furthermore, the cell culture media were fractionated by sucrose density gradient centrifugation and HCV particles were detected by immunoelectron microscopy in the fractions with high level of HCVRNA and HCV core protein.
In order to evaluate the in vitro infectivity of HCV produced in transfection-infection system, the cell culture media containing HCV was inoculated to IMY-N9 cells, Huh7.5 cells of human hepatoma cell line, or COS7 cells of monkey kidney cell line. Although intracellular HCV replication was not apparently detected, HCVRNA in the cell culture media were positive until 48 and 24 hours, respectively after HCV inoculation in IMY-N9 cells and Huh7.5 cells, but in contrast, negative as early as 24 hours after exposure to the inoculum in unsusceptible COS7 cells.
In conclusion, complete HCV virions are produced and secreted by the transfection-infection system using IMY-N9 cells and human hepatocyte derived cells appear to be susceptible to secreted HCV in this system, but more susceptible cell lines are required to develop more efficient in vitro HCV infection-multiplication system and elucidate the HCV life cycle.
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