猪繁殖和呼吸综合征病毒HuN4株nsp9、nsp10、nsp11对毒力影响的研究

EFFECTS OF NONSTRUCTURAL PROTEINS 9、10 AND 11 ON THE VIRULENCE OF HIGHLY PATHOGENIC PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

为研究高致病性猪繁殖和呼吸综合征病毒非结构蛋白对病毒毒力的影响,本研究通过反向遗传操作技术将高致病性猪繁殖与呼吸综合征病毒HuN4株的非结构蛋白编码区nsp9、nsp10、nsp11分别置换弱毒疫苗株HuN4-F112中的相应片段,经间接免疫荧光鉴定拯救的重组病毒,分别命名为rHuN4-F112-nsp9、rHuN4-F112-nsp10、rHuN4-F112-nsp11,测序证实HuN4强毒nsp9、nsp10、nsp11片段正确替换入HuN4-F112骨架。拯救病毒在Marc-145细胞上的生长曲线显示,rHuN4-F112-nsp9和rHuN4-F112-nsp10在前24 h的复制速度明显高于rHuN4-F112-nsp11和拯救的母源毒rHuN4-F112,36 h后无明显差别。将拯救病毒以105TCID50剂量接种仔猪,对体温、临床症状、病毒血症、组织器官病变情况进行观察和检测。结果显示,拯救病毒rHuN4-F112-nsp9、rHuN4-F112 nsp10、rHuN4-F112 nsp11在致病性、发热以及病毒血症强度上与rHuN4-F112无显著差异,提示单独nsp9、nsp10、nsp11对病毒致病性以及病毒在体内的复制无影响。

In order to investigate if nonstructural proteins were necessary for the virulence of the highly pathogenic Porcine reproductive and respiratory syndrome virus （HP-PRRSV）, we generated three full length infectious cDNA clones by using reverse genetic manipulation, in which the three coding regions for nsp9, nspl0 and nspll of the attenuated live PRRSV HuN4-Fll2 were respectively substituted by the corresponding regions of HP-PRRSV HuN4-F5. The three recombinant viruses （rHuN4-F112- nsp9, rHuN4-F112-nsp 10 and rHuN4-F112-nsp11） were rescued and confirmed in indirect immunofluorescence assay and genomic sequencing. The results from growth studies on MARC-145 cells demonstrated that rHuN4-Fll2-nsp9 and rHuN4-F112-nsp10 showed significantly higher titers than rHuN4-F 112-nsp 11 and rHuN4-F 112 at the first 24 hours and then turned to no difference. After inoculation into PRRSV-negative piglets, no differences in body temperature, clinical signs, viremia and lesions were observed between recombinant viruses rHuN4-F 112-nsp9, rHuN4-F 112-nsp 10, rHuN4-F 112-nsp 11 and rHuN4-F 112. Therefore, we concluded that nonstructural proteins nsp9, nsp 10 and nsp 11 were not individually related to the virulence of HPPRRSV.