Inhibition Of Porcine Reproductive And Respiratory Syndrome Virus Replication By RNA Interference Technology

Porcine reproductive and respiratory syndrome (PRRS) is characterized by a massive reproductive disorder of pregnant sows and respiratory distress of piglets. It is one of the most important causes of economic loss to swine industry worldwide. The causative agent PRRS virus (PRRSV) has restricted tropism for macrophages and its RNA genome is variable. The traditional vaccine strategies developed to control the disease are often unsuccessful. As a new effective antiviral tool, RNA interference (RNAi) has a different mechanism from classic vaccine which is based on the immunogenicity of proteins. It is a post-transcriptional mechanism of sequence-specific gene silencing that initiated by double-stranded RNA (dsRNA). In mammalian cells, short interfering RNA (siRNA) (21-25nt) can specifically silence the expression of the corresponding gene. It provides a new method in anti-virus research feilds.In this study, four (siRNA95, siRNA179, siRNA218 and siRNA294) directed against a well-conserved region of PRRSV ORF7 gene were selected. A non-specific siRNA sequence (siRNA-C) was designed as non-specfic control. Sense and antisense siRNAs encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes driven by mouse U6 promoter. The shRNA expression cassettes (shRNA95-PCR, shRNA 179-PCR, shRNA218-PCR, shRNA294-PCR and shRNA-C-PCR) were amplified by one-step PCR. Products of shRNA-PCR and N-EGFP fusion protein expression plamid pEN-ORF7 were co-transfected into 293T cells. Enhanced green fluorescent protein (EGFP) fluorescence intensity of transfected cells was monitored on an inverted fluorescent microscope. The effective siRNAs were selected rapidly. In addition, an N-EGFP stably expressing 293T cell line was selected and the inhibitory effect of the selected siRNA was demonstrated on it. In MARC-145 cell infected with PRRSV, the cytopathic effects (CPEs) induced by PRRSV was remarkablely reduced in cells treated with shRNA 179-PCR.In order to make shRNA long-lasting express and easily prepared, shRNA expression cassettes were inserted into pEGFP-N1 vector and shRNA expression vectors were constructed (pEN95-shRNA, pEN179-shRNA, pEN218-shRNA, pEN294-shRNA and pEC-shRNA). MARC-145 cells were transfected with shRNA expression vectors. The inhibitory effect was detected both at mRNA and protein expression levels. It was found pEN179-shRNA had the most effect in reducing PRRSV-induced CPEs. In indirect immunofluorescent assay (IFA) and Western blot, the cells treated with pEN179-shRNA had obviously less N protein positive cells and less N protein expression compared with controls. In fluorescence quantitative PCR (FQ-PCR) and virus titration, it was demonstred that the mRNA level of ORF7 was reduced by 96% and the titre of the virus titration was reduce 681-fold compared with controls. The inhibitory effect of pEN 179-shRNA was dose-dependent in a certain dosage range. In the same time, the mRNA levels of the minor proteins were detected. It was found that the mRNA levels of these proteins (GP2, GP3 and GP4)were reduced by 60%, 30% and 55%, respectively. It suggested that the replication of PRRSV was inhibited and the inhibitory effect of pEN179-shRNA was specific.In order to investigate the role of GP2, GP3 and GP4 in the viral replication, siRNAs directed to0RF2, 0RF3 and ORP4 were selected and shRNA expression vectors were constructed (21, 22, 23, 24, 31, 32, 33, 34, 41, 42, 43 and 44). Cells treated with shRNA expression vectors were infected by PRRSV. The effective shRNA expression vectors were selected by FQ-PCR. The virus titer of supernatant of the cells treated with effective shRNA expression vectors (23, 24, 31, 34 and 41) were reduced by 184 to 4.65 folds compared with that of controls. There was no obvious difference among shRNA expression vectors in IFA. It was suggested that the minor proteins played important roles in PRRSV infection.This study provided a primary material for anti-PRRSV infection by RNAi and provided a new approach to study of the function of viral proteins in PRRSV infection.