The Investigation Of Inhibiting Senecavirus A Replication By Porcine 2′-5′Oligoadenylate Synthetase

2’-5’oligosyladenosine synthase（2’-5’oligoadenylate synthetase,OAS）is an interferon-induced antiviral protein,which plays an antiviral role through the typical OAS/RNase L-dependent pathway and OAS/RNase L-independent pathway,and is an important component of mammalian innate immune system.Numerous studies have shown that the antiviral effect of OAS gene family in human and mice on virus replication.The knowledge of porcine OAS gene of the antiviral activity is limited.Porcine idiopathic vesicular disease（PIVD）caused by Senecavirus A（SVA）were first reported in Canada in 2007.An increasing number of cases have been reported worldwide,seriously threatening the development of the pig industry since then.In order to investigate the antiviral activity of porcine OAS in vitro,PK-15 cells and SVA were selected as research objects in this study.The p OAS（p OAS1 p OAS2 p OASL）and p RNase L genes were amplified from PK-15 cells stimulated by poly I:C.The results showed that the p OAS1 and p OAS2 have the structural domain of OAS,and p OASL lack the UBL structural domain.p RNase L has the arginine（R）and histidine（H）which is important for its activity of catalysis and tyrosine（Y）and phenylalanine（F）which is important for the RNA binding and degradation;p OAS m RNA levels were up-regulate in PK-15 cells stimulated with poly I:C or infected with SVA.The m RNA levels of p OAS1,p OAS2 and p OASL peaked at 12 h,24 h and 48 h after poly I:C stimulation,and 48 h,24 h and 24 h after SVA infection,respectively.In order to study the effect of overexpression of p OAS and p RNase L on virus replication,p OAS and p RNase L eukaryotic plasmids with flag tags were constructed.IFA and Western blot were used to verify the expression of the target gene.The virus replication was measured by TCID₅₀in supernatants harvested for SVA infected cells transfected with the eukaryotic plasmids.The results of IFA showed that p OAS and p RNase L proteins were successfully expressed in transfected cells.The results of Western blot also showed the p OAS1 and p OASL proteins were successfully expressed in cells,while p OAS2 and p RNase L proteins were not.The viral in the cells overexpressed with p OAS and p RNase L were lower than those in the control group,indicating that both the overexpressed p OAS and p RNase L could inhibit SVA replication.To investigate the effect of the knockout and knockdown of these genes on SVA replication,virus titers were was measured by TCID₅₀in supernatants harvested for SVA infected cells which were transfected with interfere plasmids.The results showed that the titer of SVA genes knockout group was higher than that of the control group,indicating that p OAS and p RNase L gene knockout promoted the replication of SVA in PK-15 cells.PK-15 cells lines with p OAS1,p OAS2,p OASL or p RNase L knockout were constructed and then to infected with 0.1 MOI SVA.The results showed that the titer of SVA virus p OAS1-KO,p OAS2,p OASL-KO and p RNase L-KO cell lines was significantly higher than that of control group.The results showed that p OAS1,p OAS2,p OASL and p RNase L gene knockout significantly promoted SVA replication.This study demonstrated the antiviral effect of porcine OAS on SVA and provided some basic but important data for further study the interaction between host and SVA.The development of PK-15 cell lines with OAS or RNase L knockout provide material for the biological and immunologic functions of porcine OAS and RNase L.