Anti-PRRSV Effect Of RAd-Delivered 3’-Untranslated Region-Targeted Artificial MicroRNA

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease characterized by reproductive failures of sows and respiratory syndromes of pigs of all ages. Porcine reproductive and respiratory syndrome virus (PRRSV) has the properties of quick mutation, heterogenous viral strains and weak protective immune responses. As a consequence, the present vaccines failed to provide the substantial protection against heterogenous viral strains and new vaccine development faces great challenges. RNA interference is a quickly developing novel strategy against viral infections, but its application is limited by low delivery efficiencies of interference RNA. By transfection of Marc-145 cells with recombinant plasmids, our research group has found that the artificial microRNA (amiRNA) targeting the 5’or 3’ un-translated region (UTR) has strong effect against PRRSV replication. However, the in vivo application of this RNA interference strategy is limited by low plasmid transfection efficiencies of macrophages.First, the expression cassette of PRRSV 3’UTR-targeted amiRNA (amiR3UTR) or control amiRNA (amiRcon) was subcloned into adenoviral shuttle vector pShuttle-GFP, and the recombinant vector pShuttle-amiR3UTR-GFP or pShuttle-amiRcon-GFP was transformed into E. coli containing adenovirus genes for homologous recombination. After antibiotic selection, the recombinant vectors were identified and transfected into AAV-293 cells, and the resultant recombinant adenovirus (rAd) was called rAd-amiR3UTR-GFP or rAd-amiRcon-GFP. Porcine alveolar macrophage (PAM) and its cell line 3D4/163 were transduced with different doses of rAd-amiR3UTR-GFP, and analyzed for GFP-positive cells by fluorescent microscopy 24 h after transduction. The results showed that, although the two types of macrophages could be transduced by the rAd in a dose-dependent manner, the transduction efficiency of primary PAM was significantly lower than that of 3D4/163 cells, requiring multiplicity of infection (MOI) of 100 for efficient transduction. At 48 h after transduction, the cells were harvested for RNA extraction and the sequence-specific amiRNA was detected by quantitative RT-PCR. The results showed that the amiRNA of expected size was detected in rAd-transduced macrophages in a dose-dependent manner and the amiRNA expression level in PAM was significantly lower that in 3D4/163 cells. At different time points after transduction. the amiRNA was detected quantitatively by RT-PCR. The result showed that the amiRNA expression was maintained for at least 72 h. These data suggest that porcine macrophages could be transduced by rAd and transduced cells could express sequence-specific amiRNA.Next, primary PAM and 3D4/163 cell line was transduced with different doses of rAd-amiR3UTR-GFP or rAd-amiRcon-GFP. At 48 h after transduction, cells were infected with a same dose of VR2332 strain PRRSV and harvested for RNA extraction 24 h after infection. By using the ORF7-specific primer pair, quantitative RT-PCR showed that the copy number of PRRSV genomic RNA in rAd-amiR3UTR-GFP-transduced cells was significantly lower than that in rAd-amiRcon-GFP-transduced cells. The viral genomic RNA was decreased in a dose-dependent manner, which was more significant in 3D4/163 cells than that in primary PAM. The similar inhibitory effect of rAd-expressed amiR3UTR on PRRSV N protein expression was detected using Western blotting. Then, primary PAM and 3D4/163 cell line was transduced with a same dose of rAd-amiR3UTR-GFP or rAd-amiRcon-GFP. After 48 h after transduction, cells were infected with different doses of PRRAV. At 24 h after infection, the similar and dose-dependent anti-PRRSV effect of rAd-delivered amiR3UTR was detected by quantitative RT-PCR and Western blotting. At different time points after infection, PRRSV was harvested and titrated on Marc-145 cells. The result showed that the anti-PRRSV effect of rAd-delivered amiR3UTR lasted for 72 h. At 48 h after rAd transduction, cells were infected with different PRRSV strains and the similar anti-PRRSV effects against 3 different viral strains were detected by viral titration 24 h after infection. These data suggest that rAd could serve as amiRNA transfer vector for porcine macrophages and the delivered amiR3UTR had potent anti-viral effect against different PRRSV strains.