经典和高致病性PRRSV疫苗株嵌合病毒的构建及其免疫原性分析

The construction and immunogenic analysis of chimeric virus between attenuated classical and highly pathogenic porcine reproductive and respiratory syndrome virus vaccine strains

为研究经典和高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)疫苗株间的生物学差异,本实验以经典PRRSV疫苗株CH-1R的全长感染性克隆p CH-1R为骨架,利用反向遗传操作技术分别将其ORF1a、ORF1b和ORF2-7替换为HP-PRRSV Hu N4-F112的相应片段,构建3个全长c DNA嵌合质粒。将构建的3个重组c DNA质粒经体外转录后转染BHK-21细胞,并于Marc-145细胞中传代,拯救出3种嵌合病毒,分别命名为r CH-1R-T1a、r CH-1R-T1b和r CH-1R-T27。病毒一步生长曲线结果表明,3种嵌合病毒在Marc-145细胞中的生长特性与亲本病毒CH-1R基本一致;动物实验结果显示,嵌合病毒实验组血清中PRRSV N蛋白抗体阳转率分别为3/3(r CH-1RT1a)、2/3(r CH-1R-T1b)和1/3(r CH-1R-T27),而CH-1R免疫组血清N蛋白抗体检测始终为阴性。以上结果表明,HP-PRRSV Hu N4-F112非结构蛋白在N蛋白抗体产生中起关键辅助或协同作用。

To investigate the biological differences between attenuated classical porcine reproductive and respiratory syndrome virus （PRRSV） and highly pathogenic PRRSV （HP-PRRSV） vaccine strains, based on the infectious clone of attenuated classical PRRSV of pCH-1R, three chimeric full-length cDNA plasmids were constructed, in which the ORFla, ORFlb and ORF2-7 genes of pCH-1R were replaced by the corresponding genes from HP-PRRSV vaccine strain HuN4-F112, respectively. The full-length RNAs were prepared from the three infectious eDNA clones by in vitro transcriptions and transfected into BHK-21 cells for the virus packaging and then passaged in Marc-145 cells to rescue the chimeric virus, correspondingly, designated rCH-1R-T1a,rCH-1R-T1b and rCH-1R-T27. The growth kinetics revealed that three chimeric viruses showed similar growth patterns to their parental virus CH-1R. Then, the animal experiment results showed that the seroconverted percentages of the antibodies against the PRRSV N protein from the inoculated piglets were 3/3 for rCH-1R-T1a, 2/3 for rCH-1R-T1b and 1/3 for rCH-1R-T27, but no anti-N protein antibody was detected in the CH-1R inoculated piglets. Our results indicated that the nonstructural protein of HuN4-F112 plays an important role in the procedure of inducing the specific antibodies of N protein in vivo.