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Analysis of pathogenesis of a virulent field isolate of Sendai virus by using a virus recovery from cDNA

利用 cDNA 回收病毒分析仙台病毒强毒现场分离株的发病机制

基本信息

批准号：
12670283
负责人：
SAKAGUCHI Takemasa
金额：
$ 1.66万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670283/
关键词：
Sendai virus virus recovery from cDNA pathogenesis leader sequence infection to mice the Hamamatsu strain

项目摘要

(1) Success of virus recovery of a Sendai virus (SeV) isolate from cDNAWe 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 passageIn 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 virulenceWe 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.

(1) 成功从 cDNA 中回收仙台病毒 (SeV) 分离株我们已经确定了 SeV 强毒现场分离株滨松株的整个基因组的核苷酸序列，并成功从其基因组 cDNA 中回收了活病毒。 (2) 通过卵传代鉴定与滨松株毒力减弱相关的突变在我们对滨松株进行的连续传代实验中，通过对含胚鸡蛋的研究，我们发现病毒对小鼠的毒力减弱。我们进一步对三个 SeV 克隆的整个基因组进行了测序。 E15cl2是在第15代卵传代中分离的减毒克隆，其前导区具有两个核苷酸突变，L蛋白具有一个氨基酸突变，这表明这些突变是MLD_<50>165倍减毒的原因。卵子传代 30 时的 E30cl2 在 L 蛋白和 HN 蛋白中具有额外的突变。另一方面，通过15次小鼠E30cl2传代获得的强毒回复克隆与前导序列、L和HN蛋白的真实回复以及L蛋白的第二位点回复相关。(3)前导突变参与毒力减弱我们从cDNA中回收了前导序列中具有突变的活病毒。前导序列中具有U_ 20 A或U_ 24 A突变的突变病毒显示出比亲本病毒稍低的致病性，而具有这两种突变的双突变病毒显示出25倍的毒力减弱，伴随着小鼠肺中病毒复制的显着降低。在小鼠肺上皮细胞的原代培养物中，先导突变病毒的复制也受到损害，但在鸡胚成纤维细胞中则没有。这些发现表明，SeV 的前导突变通过以宿主依赖性方式改变病毒复制来影响病毒发病机制。