靶向HP-PRRSV ORF7基因的siRNA重组伪狂病病毒的体外复制抑制效果

Inhibition of PRRSV replication by recombination pseudorabies virus mediated RNA interference in vitro

为了评价靶向高致病性猪繁殖与呼吸综合征病毒（HP-PRRSV）ORF7的siRNA重组伪狂犬病病毒（rPRV）对HP-PRRSV复制的体外抑制效果。将500MOI的siRNA rPRV：rPRV gG-/siRNA N1、rPRV gG-/siRNA N2、rPRV gG-/siRNA N3分别感染Marc-145细胞,24h后再分别接种10 MOI PRRSV,感染病毒48h后,观察其抑制HPPRRSV HN1株复制效果;待细胞感染病毒72h后,应用间接免疫荧光试验（IFA）、实时荧光定量PCR及Western blot检测重组rPRV介导的siRNA对HP-PRRSV在细胞中复制的抑制作用。结果显示,与表达突变siRNA的rPRV gG-/siRNA Neg及接毒对照组相比,HP-PRRSV在细胞中的复制均可被3个siRNA rPRV有效抑制,其中重组病毒rPRV gG-/siRNA N2抑制效果最佳;进一步检测siRNA重组rPRV在Marc-145细胞中抑制HP-PRRSV HN1株的复制效果,结果显示,与对照组相比,重组病毒rPRV gG-/siRNA N1和rPRV gG-/siRNA N3抑制作用不明显,而rPRV gG-/siRNA N2不但能够有效抑制HP-PRRSV引起的细胞病变,而且能够消减HP-PRRSV N蛋白的表达,明显降低ORF7mRNA的表达水平（P〈0.05）,且其抑制作用具有剂量依赖性,其抑制效果至少可持续5d,但随着时间的延长逐渐减弱。结果提示siRNA rPRV在体外能抑制HP-PRRSV的复制。

Abstract：Three recombinant pseudorabies viruses mediated siRNA targeting ORF7 of highly path- ogenic porcine reproductive and respiratory syndrome virus（HP-PRRSV）were detected for the in hibition of HP-PRRSV replication in vitro. Marc-145 cells were inoculated with 500 MOI recombi- nant pseudorabies viruses （PRV）expressing siRNAs against ORF7 gene of HP-PRRSV （rPRV gG-/siRNANI,rPRV gG-/siRNAN2 and rPRV gG-/siRNAN3）respectively, 24 h later,the cells were challenged with 10 MOI PRRSV. After 48 h,the inhibition effect of rPRV gG-/siRNAN1, rPRV gG-/siRNAN2,and rPRV gG-/siRNAN3 on the replication of high pathogenic HN1 train were detected in Marc-145 cells. After 72 h,the cells and the supernatants were collected and virus yields were examined by TCID50,and PRRSV N protein and ORF7 mRNA were detected by indi rect immunofluorescence（IFA）and Western blot or real time PCR. The results indicated that all of rPRV-mediated siRNA could significantly inhibit the replication of HP PRRSV in Marc-145 cells. Moreover,the inhibition of the rPRV gG-/siRNAN2 at both the protein and ORF7 mRNA level was the best compared with rPRV gG-/siRNAN1 and rPRV gG-/siRNAN3（P〈0. 05）, The inhibitory effect of the rPRV gG-/siRNAN2 was dose-dependent and sustained for at least 120 h,but the in- hibition effect gradually decreased as a delayed time went on. These results indicated that siRNAs mediated by recombinant PRV could effectively suppress the replication of HP PRRSV in vitro.