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，病死率高，属于负链RNA病毒，是该病的病原体。目前已有针对这种疾病的减毒活疫苗，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，分析了MV高毒力的分子基础和MV疫苗株的致弱机理，通过定点突变和遗传重组的方法，实现了对MV的随意工程。本研究的重点之一是阐明病毒聚合酶在决定MV毒力中的作用。MV基因组有N、P、M、F、H和L基因6个基因。其中，P基因和L基因分别编码病毒聚合酶的大亚基和小亚基。我们已经在强毒株IC-B和弱毒疫苗株Edmonston之间产生了各种具有嵌合体基因组的重组MVS。报告蛋白基因(增强型绿色荧光蛋白或荧光素酶)被插入到重组病毒基因组中，用于有效监测病毒生长或量化病毒基因表达。用含有不同嵌合体基因组的重组MVS感染多种细胞系，包括上皮细胞系和免疫细胞系。信号转导淋巴细胞激活分子(SLAM)是MV的受体。我们还建立了一个麻疹的小动物模型，通过产生SLAM敲门小鼠，其中小鼠SLAM基因被人SLAM基因取代。结果表明，P基因和L基因是决定病毒毒力的主要决定因素，埃德蒙斯顿疫苗株的P基因和L基因对病毒的减毒表型有很大贡献。这些数据为研制针对负链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.