Mutations in viral polymerases and virulence changes associated with cultured cell adaptation of negative strand RNA viruses

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Measles is an acute febrile infectious disease with high mortality and morbidity.Measles virus (MV), classified in the negative-stranded RNA virus, is the causative agent of the disease. Live attenuated vaccines are available for the disease, but molecular bases for the virus attenuation is poorly understood. In this study, we analyzed molecular bases for the high virulence of MV and attenuation mechanisms of MV vaccine strains, by using reverse genetics techniques, by which MV can be engineered at will by site directed mutagenesis and genetic recombination. One of focuses of this study is to elucidate roles of viral polymerases in determining MV virulence.MV genome possesses six genes, N, P, M, F, H and L genes. Among them, P and L genes encode small and large subunits of viral polymerase, respectively. We have generated various recombinant MVs possessing chimera genomes between the virulent IC-B and attenuated Edmonston vaccine strains. Reporter protein genes (enhanced green fluorescent protein or luciferase) were inserted into the recombinant virus genomes for efficient monitoring of virus growth or quantification of viral gene expression. A variety of cell lines, including epithelial and immune cell lines, were infected with recombinant MVs possessing various chimera genomes. Signaling lymphocyte activation molecule (SLAM) is a receptor for MV. We have also established a small animal model of measles, by generating SLAM-knockin mice, in which mouse SLAM gene was replaced with the human SLAM gene. Recombinant MVs were also inoculated into SLAM-knockin mice to analyzed virus virulence in vivo.Our data indicated that the P and L genes are major determinants of MV virulence, and both the P and L genes of the Edmonston vaccine strains greatly contribute to its attenuation phenotype. These data provide useful information to develop vaccines against negative-strand RNA viruses.