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

(1)成功从cDNA中恢复仙台病毒（SeV）分离株。我们确定了仙台病毒（SeV）毒野分离株Hamamatsu株的全基因组核苷酸序列，并成功地从其基因组cDNA中恢复了活病毒。(2)通过鸡蛋通道鉴定与滨松病毒毒力衰减相关的突变在我们的滨松病毒在有胚的鸡蛋中的连续传代实验中，我们已经显示出病毒对小鼠的毒力衰减。我们进一步测序了三个SeV克隆的全基因组。E15cl2是在卵代15处分离得到的减毒克隆，其先导区有两个核苷酸突变，L蛋白有一个氨基酸突变，这表明这些突变是导致MLD_<50>的165倍衰减的原因。E30cl2在卵细胞传代30时，在L蛋白和HN蛋白中有额外的突变。另一方面，通过15次小鼠传代获得的E30cl2毒性逆转克隆与先导蛋白、L和HN蛋白的真正逆转以及L蛋白的第二位点逆转相关。(3)前导突变在毒力衰减中的作用我们从cDNA中恢复了具有前导序列突变的活病毒。具有先导序列U_<20>A或U_<24>A突变的突变病毒的致病性略低于亲本病毒，而具有这两种突变的双突变病毒的毒力减弱了25倍，同时病毒在小鼠肺中的复制率显著降低。在小鼠肺上皮细胞的原代培养中，领导突变病毒的复制也受到损害，但在鸡胚成纤维细胞中没有。这些发现表明，SeV的先导突变通过改变病毒的宿主依赖性复制来影响病毒的发病机制。

(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.