Screening Of Microrna Inhibiting PRRSV Replication

porcine reproductive and respiratory syndrome virus (PRRSV) have9open reading frames (ORFs). The viral genome has a size of15kb. Since it was reported, porcine reproductive and respiratory syndrome has caused serious economic lost in the swine industry. MicroRNAs (miRNAs) are a class of noncoding RNAs of lengths from18to23nucleotide (nt). As regulation factors, miRNA widely participate in biological processes including growth and development of host.Now, with the development of research, scientists found that more and more miRNAs have anti-viral functions. The reason why PRRSV is hard to control is its genetic diversity. Tradition vaccines can not provide enough protection between different strains of PRRSV, but miRNA can bind to the conservative region of distinct PRRSV strains. In our study, we wanted to detect whether some miRNAs can targets the PRRSV genome and suppresses the proliferation of PRRSV. So that, this study might provide some theoretical basis for the study of prevention of PRRSV. The details of this research are as follows:1. The prediction of candidate miRNAs that can bind to genome of PRRSV and construction of reporter plasmidWe used the on-line software targetscan to analysis the genome of PRRSV, selected and chemosynthesized63miRNAs of the candidates according to the structure and binding energy of miRNA-mRNA duplexes. At the same time, we successfully cloned all the open reading frames (ORFs) and2untranslated regions (UTRs) of PRRSV, and constructed them into the vector pMIR-report.2. screening the miRNAs that can inhibit the proliferation of PRRSVWe cotransfected the candidate miRNA mimics and their accordant reportor plasmid into the HEK-293cells, and detected it by the dual luciferase system24h later. Then, we found that there were6miRNA mimics including miR-let-7g、 miR-let-7a、miR-3125、 miR-379、 miR-3165and miR-127-3p may suppress their report plasmid pMIR-WUH3-NSP2、 pMIR-WUH3-ORF5、 pMIR-WUH3-3’UTR、 pMIR-WUH3-5’UTR and pMIR-WUH3-ORF2a. miR-let-7g and miR-3125can bind to the NSP2gene(2037-2061bp) and ORF5gene(14104-14136bp) which are conserved region of distinct PRRSV strains. The western-blotting and plaque result showed that miR-let-7g and miR-3125can suppress the proliferation of PRRSV in Marc-145cells.3. miR-let-7g and miR-3125do not regulate the expression of beta-interferonIn order to explore whether miR-let-7g and miR-3125can regulate the expression of beta-interferon, we used the dual luciferase system to detect whether miR-let-7g and miR-3125regulate the promoter of beta-interferon gene or not. The result show that miR-let-7g and miR-3125did not up-regulate the promoter of beta-interferon gene. Further more, the result of VSV-GFP proliferation test show that miR-let-7g and miR-3125did not enhance the secretion of beta-interferon. So miR-let-7g and miR-3125did not regulate the excretion of beta-interferon.4. Infection of PRRSV inhibit the expression of miR-let-7g and miR-3125In order to detect whether Infection of PRRSV affect the expression of miR-let-7g and miR-3125, we used the real-time PCR to detect their expression at different time points after PRRSV infected in Marc-145cells. Comparison to the non-infected control, the expression of miR-let-7g and miR-3125are significantly decrease in the first12h, the expression of miR-let-7g recovered in36h. The expression of miR-3125is more than non-infected control24h later. So infection of PRRSV can significantly inhibit the expression of miR-let-7g and miR-3125in the early phage in Marc-145cells.