Study On The Mechanism Of Inhibition Of Porcine Reproductive And Respiratory Syndrome Virus Replication By Host MicroRNA-181

Porcine reproductive and respiratory syndrome (PRRS) also known as blue-ear pig disease is caused by PRRS virus (PRRSV) which continuously caused huge economic losses in our country or even throughout the world. Emerging evidence indicates that host microRNAs (miRNAs) can target viral RNAs during infections, resulting in inhibition of virus replication, which has been regarded as a new antiviral defense. Whether host miRNAs can target PRRSV RNA and be used to inhibit virus infection has not been well characterized. This dissertation work was focused on this question.As only a few of pig miRNAs have been identified, the miRNA expression profilings of porcine alveolar macrophages (PAMs) when infected with or without PRRSV were identified first by molecular cloning and deep sequencing. The results showed that PAMs could express over800distinct miRNAs, and most of them were down-regulated by PRRSV. The analysis of miRNA target prediction in PRRSV genomic RNA by two bioinformatic softwares (RegRNA and ViTa) showed that microRNA-181(miR-181) could target the site (nt13684to13692) in PRRSV JXwn06genomic RNA through seed base-pairing, and the target site was highly conserved (over96%) in type2PRRSV. Then our results showed that overexpressed miR-181indeed inhibited viral gene expression and PRRSV production in PAMs or Marc-145cells. Interestingly, all the four members of miR-181family (a, b, c and d) could inhibit PRRSV replication. Analysis of dynamics of PRRSV growth indicated that overexpressed miR-181could steadily inhibit PRRSV replication in PAMs and miR-181c was the strongest inhibitor of PRRSV. Direct targeting of the PRRSV genomic RNA by miR-181was then identified by luciferase reporter assays. Meanwhile, the viral RNA could compete with miR-181target site in luciferase vector to bind to miR-181, resulting in less inhibition of luciferase activity, which further demonstrated the specific interaction between miR-181and PRRSV RNA. Moreover, physical interaction of miR-181with viral genomic RNA in the miRNA-induced silencing complex (miRISC) was then tested by RNA immunoprecipitation (RNA-IP) assay, which provided stronger evidence for this model. Detection of miRNAs from different primary cells (including PAMs, peripheral blood mononuclear cells, microglia and peritoneal macrophages) and different tissues showed that miR-181and other potential PRRSV-targeting miRNAs (such as miR-206) were expressed much more abundantly in minimally permissive cells or tissues than in highly permissive cells or tissues. Finally, highly pathogenic PRRSV-infected pigs treated with miR-181c showed substantially decreased viral loads in blood and relief from PRRSV-induced fever, supporting that miR-181could inhibit PRRSV replication in vivo.Taken together, these results indicate that miR-181can inhibit PRRSV replication by directly targeting viral genomic RNA and the expression of miR-181is negatively correlated with viral replication capacity. These findings suggest an important role of host miRNAs in modulating PRRSV infection and replication, and viral pathogenesis. They also support the idea that host miRNAs could be useful for antiviral therapeutic strategies.