猪繁殖与呼吸综合征病毒复制保真性差异影响病毒变异特性的分子基础

Porcine reproductive and respiratory syndrome (PRRS) is an economically important infectious viral disease that seriously hinders the development of pig industry in China. The causative agent porcine reproductive and respiratory syndrome virus (PRRSV) is characterized as genetic variation and great diversity in field strains. The variants, recombined strains and reversion to virulence from modified live vaccines increase the difficulty of disease prevention and control. The viral replication fidelity is closely related to the viral diversity, adaptability and pathogenicity, however, the variation in viral replication fidelity among different PRRSV strains is less reported. As well the molecular basis of fidelity difference and its impact on viral variation, recombination efficiency and efficiency of reversion to virulence are worth to investigated. In this proposal, “what is molecular basis of PRRSV replicating fidelity differences affecting on viral mutation characteristics" is the key scientific questions that guides the study from three following aspects: (1) Evaluation of mutagenic susceptibility and in vitro replication variability among different field strains and modified live vaccine strains; (2) Identifying the gene and mutation sites contributing to the variation of replication fidelity; (3)Analysis on the role of increased replication fidelity on viral pathogenicity, tissue tropism, efficiency of virus recombination and reversion to virulence, as well as, verifying whether the increased replication fidelity is sufficient to reduce the risk of reversion of virulence. The results of this study are able to provide some new clues for exploring the mechanism of PRRSV mutation and recombination, meanwhile it will contribute to designation on next generation live vaccine with improved stability and safety.

猪繁殖与呼吸综合征是严重危害我国养猪业发展的重要传染病，其病原PRRSV存在易变异性和广泛的毒株多样性，变异、重组毒以及疫苗返强毒的不断出现是导致其难以防控的重要原因。病毒复制保真性与病毒的准种丰富度、适应性以及致病性密切相关，而不同毒株间复制保真性是否存在差异以及该差异的分子基础和对病毒变异、重组效率以及毒力返强的影响尚无报道。本研究以“PRRSV复制保真性差异影响病毒变异特性的分子基础”这一关键科学问题为引导，拟从三个方面进行研究：（1）从致突变剂敏感性和体外复制变异率两方面评价不同毒株复制保真性的差异；（2）结合反向遗传学解析病毒基因组中导致该差异的关键基因和位点；（3）分析复制保真性提高对毒株致病性、组织嗜性、变异率以及毒株间重组效率的影响,并评测复制保真性提高对减少毒力返强风险是否足够有效。以期为PRRSV变异重组机制的研究提供新线索以及为提升疫苗稳定性和安全性提供新思路。

猪繁殖与呼吸综合征病毒（PRRSV）是严重危害生猪健康养殖的重要病原，具有毒株多样易变异以及疫苗对异源毒株交叉保护不足的特征，田间毒株的变异重组以及致弱活疫苗的毒力返强，导致该病防控的复杂化。为解析PRRSV复制保真性差异影响病毒变异特性的分子基础，本研究首先系统比较了不同类型的代表性毒株以及疫苗株的复制保真性高低，发现类NADC30毒株以及HP-PRRSV来源的疫苗株具有较低的复制保真性；其次，完成Ribavirin耐药毒株nsp9和nsp10内突变位点与其复制保真性的相关性分析，明确了nsp9上H566Y、R597K两处氨基酸突变与PRRSV的Ribavirin敏感性相关性；同时，通过对毒株进行不同路径的Ribavirin筛选与耐药评估以及基于深度测序和生物信息学分析的突变特性评估，发现了PRRSV对致突变剂Ribavirin耐药一种潜在机制；并基于RdRP特殊基序比对以及结构模拟分析了PRRSV RdRP中Motif G/B中重要位点对病毒增殖复制能力，正负链基因组和亚基因组合成的影响，以及与 CVB3和MHV RdRP中复制保真性关键位点相对应区域位点对PRRSV复制增殖能力及复制保真性的影响；最后结合复制保真性评估结果，明确了不同复制保真性毒株的致病、重组特征以及在遗传演化中对密码子以及密码子对的使用偏好性。相关研究不但揭示了毒株复制保真性改变的分子基础和突变规律，而且加深了对我国PRRSV毒株演化和变异规律的了解。