猪繁殖与呼吸综合征病毒Nsp9影响病毒复制保真性的作用分析

Porcine reproductive and respiratory syndrome (PRRS) is an economically important infectious swine disease, caused by porcine reproductive and respiratory syndrome virus (PRRSV). The non-structural protein 9 (Nsp9) of PRRSV encodes RdRP which is essential for viral replication. It was reported that the Ribavirin resistant strains of PV, FMDV or Coronavirus, had higher replication fidelity than that of wild-type strains, and increased fidlelity were related with viral RdRP. There is no report of fidelity research on PRRSV yet. In this project, we plan using DNA shuffling to create two PRRSV libraries in which the viruses contain different chimeric Nsp9 genes, and then the Ribavirin-resistant strains can be screened out from the libraries. The replication fidelity of Ribavirin-resistant strains will be further evaluated and the sequences of their Nsp9 genes are going to be analyzed to determine if the mutation of RdRP confer the increased fidelity. The extra genes such as GFP and MYC tag will be inserted into the genome of Ribavirin-resistant strains and the rescued viruses will be serially passaged on MARC-145 cells to compare the stability of extra genes inserded to Ribavirin-resistant strains backbone and wildtype backbone. In all, the aim of this project is to analyze the corelations between PRRSV Nsp9 and replication fidelity. Considering the high fidelity of replication contributes to the stability of vaccine strains which can reduce the risk of virulent reverse and maintain the new advanced characterization such as neutralizing epitopes, cross protection against heterologous stains or extra gene tags, the results will not only contribute to understanding the mechanisms of PRRSV replication, they will also provide a clue to rational designation of next generation vaccine.

猪繁殖与呼吸综合征是严重危害我国生猪生产的传染性病毒病，病原为PRRSV。其非结构蛋白Nsp9编码病毒复制必需的RdRP。RdRP缺少3'-5'的校验功能，故RNA病毒复制保真性低于DNA病毒。对PV、 FMDV和冠状病毒的研究表明，具有核苷酸类似物Ribavirin抗性的毒株复制保真性更高，且该特性与RdRP相关。而PRRSV复制保真性的研究尚无报道。本项目将利用分子杂交技术构建含Nsp9突变体的病毒文库，经Ribavirin抗性筛选获得复制保真性较高的突变毒株，并对其进行复制保真性综合评价，外源插入基因传代稳定性分析，Nsp9序列分析以及反向遗传学验证，以期解析PRRSV复制保真性与Nsp9基因的相关性以及其相应分子机制。由于高复制保真性可降低疫苗毒力返强风险，促进新特性稳定存在，故该研究不但具有重要科学意义，还为疫苗开发提供了新思路。

选取9株代表性PRRSV毒株为亲本毒株，通过DNA shuffling 技术构建并成功拯救出含有nsp9 shuffling片段的嵌合病毒库，为后期nsp9与复制保真性及致病性的相关性研究提供了重要平台。其次，利用致突变药物ribavirin敏感性实验和病毒基因组深度测序系统分析了高低致病性PRRSV毒株的复制保真性，发现高致病性毒株JXwn06较低致病性毒株HB-1/3.9复制保真性更低，具有更高的易突变性；经反向遗传学构建nsp9互换的嵌合病毒并比较其复制保真性，证实高低致病性毒株间的复制保真性差异与nsp9基因相关。同时，将高致病性毒株JXwn06和HB-1/3.9在存在ribavirin培养基的条件下在细胞中连续传代 60代得到耐药性毒株，序列分析发现nsp9区域内氨基酸突变H→Y（566）和R→K（597）可能与复制保真性的升高相关。研究结果不但为从准种丰富度的角度分析病毒致病性差异的机制提供了重要依据，而且还为解释当前我国HP-PRRSV毒株在临床流行过程中易变异，以及PRRSV疫苗致弱株易发生毒力返强的机制提供了重要线索。