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作为脑心肌炎病毒(EMCV)的天然宿主,感染小鼠能引发多种疾病,其中包括脑炎、心肌炎及糖尿病.本研究利用之前构建完成的EMCV分离毒株BJC3的野生型感染性克隆,采用定点突变方法构建了4个VP1第100位氨基酸突变的突变株感染性克隆(VP1第100位氨基酸分别突变为丝氨酸、丙氨酸、异亮氨酸和脯氨酸)并获得了拯救病毒.虽然各突变病毒及野生型亲本拯救病毒在BHK-21细胞上形成的噬斑大小有所不同,但各病毒在BHK-21细胞上的复制水平未见差异.通过对突变病毒致病性进行系统分析,探究了VP1第100位氨基酸在病毒致病性及感染后疾病表型中所起的重要作用.结果表明,异亮氨酸和脯氨酸突变病毒对小鼠的致死率降低,脑中病毒载量减少且脑组织损伤轻微,而丝氨酸和丙氨酸突变病毒表现了与野生型亲本病毒相似的高致病性.结果证实,EMCVVP1第100位苏氨酸在病毒的体内复制中起重要作用,其突变会导致病毒对小鼠致病性的改变.
As a natural host of encephalomyocarditis virus (EMCV), mice infected can cause various diseases, including encephalitis, myocarditis and diabetes.In this study, wild-type infectious clones of previously constructed EMCV isolates BJC3 were selected by site- Mutation method Four infectious clones (the 100th amino acid of VP1 was mutated to serine, alanine, isoleucine and proline respectively) were constructed and the rescue virus was obtained from four mutant strains with the mutation of amino acid 100 of VP1. The size of the plaques formed on the BHK-21 cells by the mutant virus and the wild-type parental rescue virus was different, but no difference was observed in the replication level of each virus on the BHK-21 cells.By systematic analysis of the pathogenicity of the mutant virus, And explored the important role of amino acid 100 of VP1 in the pathogenicity of virus and the phenotype of post-infection disease.The results showed that the lethality of isoleucine and proline mutant virus in mice decreased, Amount and brain damage slightly, while the serine and alanine mutants showed similar high pathogenicity to the wild-type parental virus.The results demonstrate that the threonine at position 100 of EMCV VP1 is involved in in vivo replication of the virus The important role that mutations can cause changes in viral pathogenicity in mice.