Study On The Suppression Of Porcine Reproductive And Respiratory Syndrome Virus Replication By Celluar MiRNAs

Porcine reproductive and respiratory syndrome(PRRS) is characterized by late term abortions and respiratory disease, particularly in young pigs. Porcine reproductive and respiratory syndrome virus(PRRSV), as the causative agent of PRRS, can mutate rapidly and cause persistent infection and immunosuppression. In recent years, there is a growing body of evidence that cellular micro RNAs(mi RNAs) are important regulators of innate and adaptive immune responses and the intricate networks of host-pathogen interactions. Mi RNAs also reveal big roles in viral infections by binding directly to viral RNA or by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection.To identify host celluar mi RNAs important to controlling PRRSV infection, we screened 15 mi RNAs that were previously implicated in innate immunity or antiviral functions. Over-expression of the mi R-26 family strongly inhibited PRRSV replication in vitro, as shown by virus titer assays, Western blotting, and q RT-PCR assays. Mi R-26 a inhibited the replication of both type 1 and type 2 PRRSV strains. Mutating the seed region of mi R-26 restored viral titers. Luciferase reporters showed that mi R-26 a does not target the PRRSV genome directly but instead affects the expression oftype I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection. Our study reveals an example of a mi RNA that affects viral propagation and highlights a host factor that may be important for future control measures against PRRS.Then we performed computational prediction analysis of the HP-PRRSV strain v JX143 and the classic PRRSV strain v APRRS using Vi Ta and mi Randa database to predict mi RNA target sites in order to find more potent PRRSV inhibitors. Prediction results indicated that mi R-130 could target the sites(bp 155 to 162) in viral genomic RNA through seed base pairing. It was pleasantly surprising that the target region was highly conserved in type 2 PRRSV, which now circulates in most commercial swine industries through out the world, but absent in type 1 PRRSV. As expected, delivered mi R-130 family mimics, especially mi R-130 b inhibited PRRSV replication in vitro according to the results of virus titer assays, IFA, Western blotting and q RT-PCR assays. We also found that the anti-PRRSV effect of mi R-130 is confirmed in mutiple type 2 PRRSV strains but not in v SHE, a classical type 1 strain. Moveover, over-expression of mi R-130 did not induce IFN-alpha or TNF-alpha expression in either mock- or PRRSV-infected PAMs. Luciferase reporter assays showed that relative luciferase activity produced a 70 % decrease in p GL3-5UTR while no decrease in relative luciferase activity was observed in any other vectors. The results above indicated that mi R-130 directly targets PRRSV RNA. As mi RNAs are small molecules without antigenic properties, they are considered to have potential efficacy in antiviral therapeutic applications. Finally, to address whether mi R-130b-mediated inhibition of PRRSV replication can be used in therapy, we performed the animal expriment and the results showed that intranasal delivery of mi R-130 b significantly inhibited v JX143 replication in piglets. The average virus load in serum was about 1000-fold lower than that in v JX143-infected group. Surprisingly, in 3 of the 4 mi R-130b-treated pigs, it appeared that PRRSV replication could be controlled in 21 dpi. Overall, these results demonstrate the important role of mi R-130 family in modulating PRRSV infection and also support the possibility of using host mi R-130 b to achieve RNAi-mediated antiviral therapeutic strategies.In the last part of the article, we performed deep sequencing to construct small RNA expression profiles from in vitro cultured PRRSV-infected MARC-145 cells to indentify the impact of PRRSV infections on the cellular mi RNAome. According to the expression of different mi RNA in MARC-145 cells, we chose six mi RNAs of differential expression as study objects. Based on PRRSV full-length c DNA clone p APRRS, a range of mi RNA reverse complement sequences inserted mutants in PRRSV 3’UTR were constructed, which provided artificial engineered perfect complementary base-pairing target sites of mi RNAs, and transfected into MARC-145 cells. As a result, we found that no fluorescent signal was detected in the case of high expression mi RNA target mutants, and no infectious particles were produced even after three further passages. All the low expression mi RNA target viruses were successfully rescued and showed similar growth property to v APRRS. Interestingly, fluorescent signal was detected at 24 h post transfection in the case of the medium expression mi RNA target mutants, but there is no infectious particle at the first 60 h post transfection. To assess their genetic stability, all rescued viruses were serially passaged five times to establish virus stocks of P 1-5 virus. The spontaneous mutations only existed in the mi RNA target sites in the case of the medium expression mi RNA target rescued virus, which indicated the reason of early repression of virus replication. This approach provides a general micro RNA-based gene silencing strategy for inhibiting PRRSV replication by harnessing endogenous mi RNAs.In summary, our work provides evidence for the important role played by host mi RNAs in modulating PRRSV replication both under celluar and natural conditions and also implies that cellular mi RNAs can be considered therapeutic tools for antiviral therapy in the future.