| Project/Area Number |
14370105
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | FUJITA HEALTH UNIVERSITY |
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Principal Investigator |
TANIGUCHI Koki Fujita Health University, School of Medicine, Professor, 医学部, 教授 (40094213)
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| Co-Investigator(Kenkyū-buntansha) |
SASAKI Jun Fujita Health University, School of Medicine, Assistant Professor, 医学部, 講師 (70319268)
MAENO Yoshimasa Fujita Health University, School of Medicine, Associate Professor, 医学部, 助教授 (70131191)
MORIGUCHI Kyoko Fujita Health University, School of Medicine, Research Associate, 医学部, 助手 (60298528)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥13,700,000 (Direct Cost: ¥13,700,000)
Fiscal Year 2004: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 2002: ¥4,300,000 (Direct Cost: ¥4,300,000)
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| Keywords | Human Rotavirus / Reverse Genetics / VP4 gene / Simian Rotavirus / Artificially Infectious Particle / Gastroenteritis / ヒトロタルス / ロタウイルス / リアソートメント / トリプシン依存性 / キメラウイルス |
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| Research Abstract |
At the final stage of the research project, we succeeded in the development of the system of reverse genetics of rotavirus, although we have to employ a helper virus for the system. The methodology for the development is the following. 1) We prepared a plasmid (termed as pT7/VP4-SA11-L2) which the VP4 gene from a trypsin-independent simian rotavirus SA11-L2 clone with the ability of efficient multiplicity in cultured cell was inserted. 2) Vaccinia virus expressing T7 RNA polymerase was infected into COST cells. 3) A plasmid pT7/VP4(SA11-L2) was transfected. 4) A human rotavirus strain KU as a helper virus was infected into COST cells, and the cells were cultured for two days. 5) The culture fluid from the virus-infected cells was infected to MA-104 cells, and the cells were maintained for two days in the presence of a neutralizing monoclonal antibody YO-2C2, which inhibits specifically the growth of a helper virus KU. 6) RNA pattern of the RNA extracted from the culture fluid was confirmed by polyacrylamide gel electrophoresis.
By these procedures, we succeeded in the preparation of infectious rotavirus particles which the VP4 gene was artificially inserted. Next, we prepared infectious rotaviruses with artificially mutated VP4 gene. The mutated VP4 genes are the one (pT7/VP4(SA11-L2)-Δ PstI) which a PstI restriction site on the gene was removed, and the other (pT7/VP4(SA11-L2)-Δ HaeII) whose a HaeII restriction site was removed by mutagenesis.
Thus, we first developed the reverse genetics system for rotavirus. We try to prepare the infectious rotavirus with the mutated VP7 gene or NSP1 gene, and to prepare the infectious rotavirus with the aid of only plasmid DNAs in near future.
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