乙型脑炎病毒NS2A-C60A位点突变对其生物学特性影响研究*

目的:旨在探索Ⅰ型日本乙型脑炎病毒传代致弱后基因组突变NS2A-C60A对乙脑病毒生物学特性的影响。方法:首先通过对传代致弱及原始乙脑毒株基因组序列进行测序比对、结构预测分析并利用Western blotting(WB)确定了目标研究位点NS2A-C60A;然后使用反向遗传定点突变技术构建拯救了包含NS2A-C60A单点突变的病毒株;最后利用噬斑形态观察、生长曲线、双萤光素酶分析,WB以及炎性因子检测和动物实验研究了该单点突变对于乙脑病毒生物学特性的影响。结果:首次研究发现Ⅰ型乙脑病毒传代致弱会导致NS1'蛋白表达的显著下降以及可能的相关位点NS2A-C60A,并成功拯救获得了NS2A-C60A单点突变毒株rJEV-C60A,研究发现NS2A-C60A突变对乙脑病毒的生长特性及噬斑形成没有显著影响,但是能够显著降低乙脑病毒NS1'蛋白的表达,并且该位点突变能够轻微阻碍乙脑病毒对细胞炎性因子表达的抑制,动物实验结果显示NS2A-C60A点突变病毒与原毒株具有相似的神经毒力,说明该位点突变不是影响乙脑病毒毒力致弱的关键位点。结论:新发现的NS2A-C60A位点突变能够显著减少乙脑病毒NS1'蛋白的表达,但是对其增殖、诱导炎症及神经毒力等生物学特性没有显著影响。

The Study on the Effect of NS2A-C60A Site Mutation of Japanese Encephalitis Virus on Its Biological Characteristics

Objective: In order to explore the effect of the NS2A-C60A gene mutation in the attenuation process on the biological characteristics of Japanese encephalitis virus. Method: First of all, the target research site NS2A-C60A that may be related to the expression of NS1' and virulence weakening was determined by sequencing and comparison of the genomes of the JEV attenuated strain and the original strain, prediction of the secondary structure of the 5'-terminal sequence of NS2A gene, and changes in NS1' protein expression; then, the NS2A-C60A single point mutation virus strain rJEV-C60A was rescued by using reverse genetic site-directed mutation technology; Finally, the influences on the biological characteristics for JEV were studyed by the observation of plaque morphology, growth curve, dual luciferase analysis, WB, inflammatory factor detection and animal experiments. Results: The research found for the first time that the passage attenuated of type Ⅰ JEV would lead to a significant decrease in NS1' protein expression and the related site NS2A-C60A, and successfully rescued the NS2A-C60A single point mutant strain rJEV-C60A. Plaque and growth curve results show that the NS2A-C60A mutation have no effect on the growth characteristics of JEV, but dual luciferase analysis and WB results reveal that NS2A-C60A mutation can significantly reduce the expression of viral NS1' protein. The results of inflammatory factors suggeset that the NS2A-C60A site mutation can slightly hinder the JEV suppression of inflammatory factors expression, but animal experiments show that the NS2A-C60A point mutation virus has similar neurovirulence to the original strain, indicating that the site mutation is not a key site that affects the virulence of JEV. Conclusion: The novel mutation NS2A-C60A can significantly reduce the expression of the JEV NS1' protein, but it has no significant effects on its other biological characteristics, like proliferation, induced inflammation and neurovirulence.