| Project/Area Number |
12670283
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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 | Hiroshima University |
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Principal Investigator |
SAKAGUCHI Takemasa Hiroshima University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (70196070)
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| Co-Investigator(Kenkyū-buntansha) |
KIYOTANI Katsuhiro Hiroshima University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (00106824)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Sendai virus / virus recovery from cDNA / pathogenesis / leader sequence / infection to mice / the Hamamatsu strain |
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| Research Abstract |
(1) Success of virus recovery of a Sendai virus (SeV) isolate from cDNA
We have determined nucleotide sequence of the entire genome of a virulent field isolate of SeV, the Hamamatsu strain, and succeeded in recovery of a live virus from its genomic cDNA.
(2) Identification of mutations associated with attenuation of virulence of the Hamamatsu strain by egg passage
In our serial passage experiment of the Hamamatsu strain through embryonated chicken eggs, we have shown attenuation of virulence of the virus to mice. We further sequenced entire genomes of three SeV clones. E15cl2, an attenuated clone isolated at egg-passage 15, possessed two nucleotide mutations in the leader region and one amino acid mutation in the L protein, suggesting that these mutations are responsible for the 165-fold attenuation in MLD_<50>. E30cl2 at egg-passage 30 possessed additional mutations in the L protein and in the HN protein. On the other hand, a virulent revertant clone obtained by 15 mouse-passages of E30cl2, was associated with true reversions in the leader and in the L and HN proteins as well as second-site reversions in the L protein.
(3) Involvement of the leader mutations in attenuation of virulence
We recovered live viruses possessing mutatiops in the leader sequence from cDNA. A mutant virus possessing either a mutation of U_<20>A or U_<24>A in the leader sequence showed a slightly lower pathogenicity than that of the parental virus, while a double mutant virus possessing both of the mutations showed 25-fold attenuated virulence, accompanying a significantly lower virus replication in the mouse lung. Replications of the leader mutant viruses were also impaired in a primary culture of mouse pulmonary epithelial cells but not in chick embryo fibroblasts. These findings suggest that leader mutations of SeV affect virus pathogenesis by altering virus replication in a host-dependent manner.
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