Investigation On Antiviral Effect Of Porcine 2’-5’ Oligoadenylate Synthetases On Jev Replication

Japanese encephalitis(JE) is a zoonotic disease that caused by the Japanese encephalitis virus(JEV).JEV is a member of the genus Flavivirus in the family Flaviviridae. Pigs are an important reservoir for this virus in nature. JEV is prevalent in much of Southeast Asia and bring serious harm to human and animal.2’-5’ oligoadenylate synthetases(OAS) is an antiviral protein induced by interferon and plays an important role in innate immune system of mammal. Intracellular OAS/RNase L signaling pathway plays an important role in host innate immune response by controlling flavivirus, reovirus and encephalomyocarditis virus.In recent years, studies have confirmed that RNase L-independent pathway is exist during antiviral effect of the OAS family. Since it is identified that the murine Oas1b gene were responsible for restricting West Nile virus (WNV) replication, OAS gene displays antiviral activity against EMCV, VSV, RRV and so on. The studies have found that the human OAS gene shown to be associated with susceptibility to HCV and SARS.OAS genes are found to be biological marker genes for resistant to flavivirus infection among mice, horses and human beings.The homology between porcine OAS1 protein and human OAS1 protein is 73%, and their enzymology characteristics are similar. Therefore, further research about whether the OAS gene is the risk marker gene of pig about JEV infection is necessary. This study,we transfected pOAS1a,pOAS1b,pOAS2 and pOASL plasmids to PK-15 cells to analyze the anti-JEV effect of porcine OAS gene family.Then we compared the difference of OAS1 exon 1 to 6 gene sequence between JEV-infected porcine and healthy ones to analyze whether OAS1 polymorphisms are associated with susceptibility to Japanese encephalitis in porcine. All these studies were listed as below:1. Preparation of porcine OAS2 polyclonal antibodiesBased on the published porcine OAS2 gene sequence, we designed primers and used the method of RT-PCR to amplify porcine OAS2 gene from PK cells, its length is 2124bp. The pOAS2 genes were cloned into the prokaryotic expression vector pET-28a (+) and verified by enzyme digestion and DNA sequencing.Then the recombinant plasmids were transformed into E.coli Rosetta2(DE3) respectively and induced them by IPTG.The results of SDS-PAGE showed that we obtained a size of 70kDa pOAS2 protein as expected.The pOAS2 protein was separated in gel slices and used to immunize rabbits.Antiserums were prepared and specificity analyzed by Western blot. High titer of antiserums against porcine OAS2 proteins were obtained.The successful expression of pOAS2 proteins in E.coli Rosetta2(DE3) and preparation of pOAS2 specific rabbit antiserum laid foundation for the further study on the antiviral mechanism.2. The antiviral effect of porcine OAS gene family to the JEV infectionThis study analyzed the effect of porcine interferon a to JEV proliferation and the change of pOAS expression level.It was showed that the interferon significantly inhibited JEV proliferation and induced pOAS expression in PK15 cell.The pOAS expression could be significantly improved after JEV virulent strain stimulating PK15 cell 24h,but this was lower than the interaction of JEV and interferon.The pFlag-pOASlb and pcDNA-pOAS2 were successfully constructed.we overexpressed these recombinant plasmids together with pcDNA-pOASla and pFlag-pOASL from our lab conserved in PK15 cell.The results showed that the pcDNA-pOAS 1 a,pcDNA-pOAS2 and pFlag-pOASL have anti-JEV effect but the pFlag-pOAS1b have not.lt confirmed that the pOAS gene family anti-JEV pathway is being in PK15 cell. 3. Characteristics of single nucleotide polymorphisms of the porcine OAS1 coding regionBased on the porcine exons sequences,we analyzed the exons 1 to 6 of pOAS1a and pOAS1b.The results showed that the homology of exon 1 to 4 of pOAS1a and pOAS1b is 96.9%,exon 5 to 6 is 66.5%,it suggested that procine OAS1 selective splicing of exons 5 and 6 formed the two splice variant.We analyzed the gene sequence of porcine OAS1 exon 1 to 6 form porcine infected JEV and healthy ones, native and introduced species.The results showed that the porcine OAS1a have 22 SNPs and the porcine OAS1b have 28 SNPs.Comparing porcine OAS1a and OAS1b SNPs sites of the porcine infected JEV and healthy ones,we found that the porcine infected JEV has high mutation rate at A→T SNP on the position of 114/105nt.Through the study of protein structure.we found that this site is closely related to OAS binding with RNA and activation.mutation of this site will affect the activation of OAS protein.Finding of this site lay the foundation to find the risk site of porcine infected with JEV.