| Project/Area Number |
22590429
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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 |
Virology
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| Research Institution | National Institute of Infectious Diseases |
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Principal Investigator |
KOJI Ishii 国立感染症研究所, ウイルス第二部, 室長 (40280763)
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| Co-Investigator(Kenkyū-buntansha) |
TIANCHENG Li 国立感染症研究所, ウイルス第二部, 主任研究官 (90370957)
清原 知子 国立感染症研究所, ウイルス第二部, 主任研究官 (70270642)
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| Project Period (FY) |
2010 – 2012
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| Project Status |
Completed (Fiscal Year 2012)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2011: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2010: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
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| Keywords | ウイルス病原性 / 治療薬 / スクリーニング / 分子生物学 / ウイルス / 病原性 / 肝臓病 |
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| Research Abstract |
Hepatitis E virus (HEV) causes acute and fulminant hepatitis E in humans. Infectious cDNA clones of HEV were established by several groups, and efficient HEV propagation in cultured cells has been recently developed using PLC/PRF/5 and A549 cells. However, our PLC/PRF/5 cells showed limited permissiveness for HEV. In this study, we performed single-cell cloning of PLC/PRF/5 cells and analyzed heterogeneity by using infectious HEV clone. We constructed an infectious cDNA clone of HEV from porcine liver. Then we performed single-cell cloning of PLC/PRF/5 cells by limited dilution. To analyze the limited permissiveness of our PLC/PRF/5 cells, cloned cells were either infected with HEV or transfected with an infectious HEV RNA and the production of HEV was measured by the detection of HEV capsid protein in culture supernatant by ELISA. Ninety-eight clones were obtained after single-cell cloning of parental PLC/PRF/5 cells. The cloned PLC/PRF/5 cells exhibited various levels of HEV virus infection efficiency, and some clones were not permissive. While the replication efficiencies measured by the transfection of infectious HEV RNA differed among the cloned PLC/PRF/5 cells, these efficiencies did not correlate with infectious permissibility. We cloned several PLC/PRF/5 cells that were nonpermissive for HEV. However, HEV could replicate in these nonpermissive cell lines, suggesting that these cell lines may have some deficiencies in the attachment or entry steps of HEV infection. Investigation of host factors responsible for these steps is in progress.
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