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

麻疹是一种急性发热性传染病，发病率高，死亡率高，麻疹病毒（MV）是麻疹的病原体，属于负链RNA病毒。减毒活疫苗可用于该病，但对病毒减毒的分子基础知之甚少。本研究利用反向遗传学技术，通过定点突变和基因重组，对MV高毒力的分子基础和MV疫苗株的减毒机制进行了分析。MV基因组含有N、P、M、F、H和L六个基因，它们是决定MV毒力的关键基因。其中，P和L基因分别编码病毒聚合酶的小亚基和大亚基。我们已经产生了各种重组MV之间的毒性IC-B和减毒Edmonston疫苗株嵌合体基因组。将报告蛋白基因（增强型绿色荧光蛋白或荧光素酶）插入重组病毒基因组中，用于有效监测病毒生长或定量病毒基因表达。用具有各种嵌合体基因组的重组MV感染各种细胞系，包括上皮细胞系和免疫细胞系。信号淋巴细胞活化分子（SLAM）是MV的受体。我们还建立了麻疹的小动物模型，通过产生SLAM-敲入小鼠，其中小鼠SLAM基因被替换为人SLAM基因。结果表明，P和L基因是决定MV毒力的主要基因，Edmonston疫苗株的P和L基因对MV的减毒表型有重要作用。这些数据为开发针对负链RNA病毒的疫苗提供了有用的信息。

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.