Screening And Function Study Of Tongcheng Pigs IFN-? Induced Genes To Inhibite PRRSV

Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease,which is induced by porcine reproductive and respiratory syndrome virus(PRRSV).The persistent infection,which was induced by PRRSV,can induce host immunosuppression and increase the probability of other bacterial or viral disease.PRRSV is a single-standed positive RNA virus.The high variability of its genome takes many difficulties in controlling PRRSV infectious by traditional vaccine.It would be of great significance to control PRRS in genetic level through deeply understanding PRRSV pathogenic mechanism and the role of host genes on PRRSV.Our team used Tong Cheng pigs and Large White pigs was used to infect PRRSV.We found that the level of interferon gama(IFN-y)significantly increased at 5dpi and 7dpi in Tong Cheng pigs.However,there was no change in Large White pigs after infection.Transcriptome sequencing was used to find the differerntially expression genes of two breed pigs by RNA-Seq from Porcine alveolar macrophages(PAMs).We focuse on IFN-y signaling pathway to find many IFN-y induced genes,which is as the condational genes to be studied in MARC-145 cells.The major studying result of this paper is as follows:1 The RNA-Seq data from infected and control PAMs,which came from artificial infected Tong Cheng and Large White pigs at 7th day post-infection PRRSV,was used to analysis.RNA-Seq data analyzing results from PAMs of IFN-? treatment(NCBI:SRR1047809)were used to be reference.Eighteen IFN-y relative DEGs of Tong Cheng pigs were screened,which were as anti-PRRSV candidate genes.The relative expression of ISG12A and OASL mRNA were detected in PAMs of two breeds by qRT-PCR.Consistent with RNA-Seq data,they were up-regulated after infection.It demonstrates the RNA-Seq data and Bioinformatics analysis results are reliable.ISG12A and OASL were used to be the target genes in this study.2 The multiple sequence alignment result of pig ISG12A,together with human and monkey,show that pig ISG12A has a special sequence at 5' terminal.Their C terminal of ISG12A amino acid sequence has the ISG12 motif.So it is sure that they are homologous gene.Evolutionary tree was built for ISG12A.We found that the pig ISG12A is distant to monkey ISG12A,and has the closest relative with sheep and cow ISG12A.ISG12A located in mitochondrial by sub-cellular lacation assay.The expression of pig ISG12A mRNA was detected in spleen?lung and lymphoid tissues.The results showed that the expression of ISG12A mRNA was up-regulated in Tong Cheng pigs post infection,but they were insignificant.Large White pigs had the same tendency,and the expression was significant up-regulated in spleen and lung tissues post infection.ISG12A was overexpressed or silenced in MARC-145 cell for 24h,and then infected PRRSV.The cells were harvested at different post infection times.qRT-PCR results show that the relative expression of PRRSV ORF7 mRNA in overexpressed MARC-145 cell is significantly lower than the control group.The relative expression of PRRSV ORF7 mRNA is significantly higher in siRNA MARC-145 cells than control group.Immunofluorescence assay(IFA)was used to detect the level of PRRSV-N protein in MARC-145 cells of pig ISG12A overexpression and control group at 36h post infected PRRSV.The results show that the level of PRRSV-N protein in overexpression or silencing ISG12A MARC-145 cells is significantly lower than the control group.It means that ISG12A could significantly suppress the replication of PRRSV in MARC-145 cells.Overexpression of ISG12A could significantly reduce the G2/S stage numbers of MARC-145 cells.As is reported in previously study,PRRSV infects the MARC-145 cells easier in its G2/S stage.So SG12A can inhibit the replication of PRRSV by changing the cell cycle stage of MARC-145 cells.This study successfully constructed three different eukaryotic expression vectors,including pRK-flag-pOASL?pRK-flag-mOASL?pRK-flag-rOASL(including pig OAS-like domain and human UBL domain).They are used to study the function of OASL gene.Three plasmids were transiently transfected into MARC-145 cells respectively,vector(pRK-flag)as control.And then cells were harvested at Oh,12h,24h,36h,48h post infected PRRSV.qRT-PCR results showed that the relative expression of PRRSV ORF7 mRNA was significantly lower in overexpression monkey OASL than control group,beginning 12h post PRRSV infection,and as well as pig OASL and recombinant OASL beginning 24h.IFA was used to detect the level of PRRSV-N protein after 36h post PRRSV infection in MARC-145 cells overexpressed different plasmids.The results showed that the level of PRRSV-N protein in three overexpression MARC-145 cell were significantly lower than control group.It indicates that OASL could significantly suppress the replication of PRRSV in MARC-145 cells.Pig OASL may have a different mechanism on aggainst virus replication which is independent on ubiquitin-like domain.It need to be the deeply studied.This study screens the IFN-y relative DEGs of Tong Cheng pigs after PRRSV infection through RNA-Seq.MARC-145 cells were used to study the IFN-y stimulated genes.ISG12A and OASL can suppress the replication of PRRSV.This study provides the basis for subsequent research.