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

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(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.