Inhibitory Effects Of CD163-, CD169- And 3’ Untranslated Region-targeted Artificial Micrornas Against Porcine Reproductive And Respiratory Syndrome Virus Infection Of Alveolar Macrophages

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases which causes heavy losses to the pig industry worldwide. Presently, vaccination is the main strategy for PRRS control. However, the current inactivated vaccines have poor immune protective effects and the live vaccines have the potential risk of virus spreading. RNA interference is the post-transcriptional gene silencing and natural anti-viral mechanism of eukaryotes, which has developed as a novel anti-viral strategy. It has been recognized that the vector-encoded artificial microRNAs (amiRNAs) are more effective than the conventional small interfering RNAs (siRNAs), and the exosomes derived from human and mouse cells can serve as miRNA transfer vector.PRRSV targets the cells of porcine monocyte-macrophage lineage, porcine alveolar macrophage (PAM) in particular. The main receptors involved in PRRSV infection of PAM include CD 163 and sialoadhesin (Sn)/CD169, which are the core receptor and accessory receptor, respectively. The 5 and 3 untranslated regions (UTRs) of PRRSV RNA genome contain conserved cis-regulatory elements, which play important roles in viral replication and viral RNA synthesis. To develop the novel strategies against PRRSV, in this study we constructed recombinant adenoviruses (rAds) expressing CD163-, Sn- or 3’UTR-targeted amiRNA and investigated the inhibitory effects of amiRNAs against PRRSV infection and/or replication.To investigate the inhibitory effect of CD 163- and Sn-targeted amiRNAs against PRRSV infection, the amiRNA expression cassettes were cloned into Ad transfer vector and two rAds were generated by transfecting AAV-293 cells with the recombinant vectors. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD 163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration. The results showed that maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD 163 receptor was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment a mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAM with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains. These data sugges that Sn- and CD 163-targeted amiRNAs has an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.To investigate further the exosome-mediated amiRNA transfer efficiencies across different types of pig cells, a rAd expressing the 3’UTR-targeted amiRNA was constructed. Quantitative RT-PCR showed that the sequence-specific amiRNA was expressed in and secreted via exosomes from the rAd-transduced pig kidney cell line PK-15, PAM cell line 3D4/163, kidney fibroblast cells (PFC) and endometrial endothelial cells (PEEC) with different secretion efficiencies. Fluorescent microscopy revealed that the dye-labeled amiRNA-containing exosomes of different cell origins were efficiently taken up by all of the five types of pig cells, including primary PAM. Quantitative RT-PCR showed that the amiRNA-containing exosomes of different cell origins were taken up by primary PAM in time- and dose-dependent manners. Both quantitative RT-PCR and viral titration assays showed that the exosome-delivered amiRNA had potent anti-viral effects against three different PRRSV strains. These data suggest that the exosomes derived from pig cells could serve as an efficient miRNA transfer vehicle, and that the exosome-delivered amiRNA had potent anti-viral effects against different PRRSV strains.