猪繁殖与呼吸综合征病毒嵌合感染性克隆的构建和应用

Construction and Application of Chimeric Infectious Clones of Porcine Reproductive and Respiratory Syndrome Virus

摘要:

高致病性猪蓝耳病近年来在我国广泛流行, 目前尚无高效疫苗用于该病的防制。本研究以PRRSV弱毒感染性克隆-pAPRRS作为主要骨架, 将高致病性猪蓝耳病病毒江西分离株(JX143)的主要结构蛋白基因(ORF4-7)以及3￠UTR区域替换入pAPPRS相应编码区域, 构建了pSX12(ORF4-7-3￠UTR)、p5NX12(ORF5-7-3￠UTR)以及p56N12(ORF5-6)三个PRRSV全长嵌合克隆。经DNA转染Marc-145细胞后拯救出嵌合病毒, 并对拯救病毒进行了病毒生物学特性的分析; 选取拯救病毒v56N12(ORF5-6)和vSX12(ORF4-7-3￠UTR)免疫29日龄猪进行免疫原性试验, 结果表明, 以v56N12免疫后抗体水平相对较低, 故免疫原性相对较差; 而vSX12病毒免疫后14 d血清ELISA抗体达到阳性值(IDEXX ELISA值 s/p≥0.4), 免疫后28 d中和抗体效价从原来的1:5以下上升到1:15以上, 说明vSX12具有良好的免疫原性并可进行免疫监测;免疫后28 d以强毒JX143株攻毒, 结果vSX12免疫组未见发病, 而未免疫攻毒对照组全部发病并有1头死亡, vSX12免疫猪攻毒后病毒血症持续6 d后消失, 而对照组攻毒后至少持续13 d, 说明vSX12可对高致病性猪蓝耳病提供有效的免疫保护。本研究构建的三个嵌合病毒为开发同时抗经典和变异株PRRSV感染的二价疫苗, 以及探寻高致病性猪蓝耳病病毒的毒力因子奠定了基础。

Abstract:

In recent years, mass outbreaks of highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) have spread all over the Chinese swine industry. Based on the first infectious cDNA clone of HP PRRSV strain pJX143 and that of an attenuated PRRSV, pAPRRS, constructed in our group, we constructed several chimeric clones with various substitutions of structural protein genes (ORF4-7) and 3￠ UTR between attenuated pAPRRS and virulent pJX143.Upon transfection of MA-104 cultured cells, all chimeric constructs pSX12, p5NX12, and p56N12 were rescued. The rescued viruses maintained the similar virological properties, based on the results of the growth curve of the rescued viruses. To test if the chimeric viruses can be used as a vaccine candidate, vSX12 and v56N12 vaccinated pigs were challenged with the HP PRRSV JX143 strain. As a result, the vSX12 vaccinated pigs were all seroconverted by 14-day-post vaccination, while v56N12 vaccinated pigs showed poor antibody response. Upon challenge, the vSX12-vaccinated group showed no signs of clinical PRRS syndrome, and virema period was shorten to 6 days post-challenge. Our results demonstrated that 1) vSX12 chimeric virus is a good vaccine candidate; 2) the virulence determinants of HP PRRSV probably located in coding regions other than ORF3-7 and 3￠ UTR, as our chimeric viruses were proved to be attenuated.