Research On The Effect Of Caprine Parainfluenza Virus Type3 Replication By Interferon Stimulated Genes(ISGs)

Caprine parainfluenza virus type 3?CPIV3?belongs to the Paramyxoviridea family,Paramyxovirinea family,and Respirvirus genus,which is a negative-strand RNA virus.The same members also include human parainfluenza virus type 3?HPIV3?,human parainfluenza virus type 1?HPIV1?,bovine parainfluenza virus type 3?BPIV3?and Sendai virus?Se V?.At present,research on HPIV3,HPIV1,BPIV3,and Se V is relatively mature at home and abroad,but CPIV3 is a newly discovered virus,research on CPIV3 is still in its infancy,and lack of in-depth research on CPIV3 replication,infection,pathogenic mechanism and immune response.Therefore,it is of great significance to study the pathogenic mechanism of CPIV3and the host cell's antiviral immune response.Innate immunity is the first barrier for the body to resist the invasion of pathogens,and the antiviral effect mediated by interferons?IFN?is one of the most important links.Interferon stimulated genes?ISGs?induced by IFN play a very important role in the host's resistance to viral infection.In previous studies,it was found that when CPIV3 JSHA-2014-1 was inoculated into MDBK cells,interferons?IFN-?,INF-??and IFN upstream genes My D88,MAVS,MDA5,IRF2,IRF3,IRF7 and IRF9 m RNA expression levels were expressed.There was no significant difference in the interferon pathway downstream ISGs:IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 m RNA were all up-regulated to varying degrees,suggesting that host cells interfere with CPIV3 proliferation by producing antiviral factors.In this study,we established SYBR Green?quantitative detection methods for IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 genes,along to investigate the up-regulation levels of 7 ISGs transcription in MDBK cells infected with CPIV3.And constructed overexpression vectors for IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 genes.Meanwhile,study on the antiviral effect of seven ISGs on CPIV3,with a view to clarifying the pathogenic mechanism of CPIV3 and laying the foundation for the development of antiviral drugs.The main research include:1.Establishment and application of SYBR Green I fluorescent quantitative PCR assay for seven ISGsIn order to study the antiviral immune response of MDBK cells infected with CPIV3,this study designed specific primers for seven interferon stimulated genes?ISGs?:IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2.After optimization,the SYBR Green I fluorescene quantitative PCR method was established.The m RNA transcript levels of seven ISGs were detected by the established method for 24 h,48 h and 72 h of MDBK cells infected with CPIV3 JS2013 strain.The results showed that the longer the infection time,the greater the transcriptional up-regulation of the seven ISGs,and the transcriptional upregulation of ISG15,Mx1,Mx2 and RSAD2 was consistently higher than that of IFI6,OAS1Y and OAS1Z.The results showed that CPIV3 JS2013 strain can stimulate the massive transcription of these seven ISGs in MDBK cells,providing a theoretical basis for further study of its antiviral function.2.Construction and expression identification of seven ISGs overexpression vectorsConstruction of IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 genes overexpression vectors and high expression in MDBK cells.In this study,seven ISGs genes overexpression vectors were constructed,and their expression in MDBK cells was detected by RT-q PCR,IFA and Western blot.The results showed that seven recombinant plasmids were constructed correctly:p EGFP-N1-Flag-IFI6,p EGFP-N1-Flag-ISG15,p EGFP-N1-Flag-OAS1Y,p EGFP-N1-Flag-OAS1Z,p EGFP-N1-Flag-Mx1,PEGFP-N1-Flag-Mx2 and p EGFP-N1-Flag-RSAD2.RT-q PCR results showed that compared with the control group,the transcription level of seven ISGs in the recombinant plasmid group was significantly up-regulated.IFA results showed that compared with the control group,there were obvious green fluorescent signals in the transfected seven recombinant plasmids,while the control group had no fluorescent signal,indicating that these seven recombinant plasmids were successfully expressed in MDBK cells.At the same time,the Western blot test detected seven recombinant proteins:Flag-IFI6,Flag-ISG15,Flag-OAS1Y,Flag-OAS1Z,Flag-Mx1,Flag-Mx2 and Flag-RSAD2 fusion expression.This provided theoretical support for the subsequent research on its antiviral effect on CPIV3.3.Effects of IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 on CPIV3 replicationStudy the antiviral effect of IFI6,ISG15,OAS1Y,OAS1Z,Mx1,Mx2 and RSAD2 on CPIV3.In this study,seven recombinant plasmids transfected into MDBK cells and inoculated with CPIV3 JS2013 virus solution 24 h after transfection.The cell culture fluid was harvested at 24 h and 48 h,respectively.The proliferation of CPIV3 in MDBK cells was detected by RT-q PCR and virus proliferation titer.RT-q PCR results showed that the load of CPIV3 RNA in seven ISGs overexpressing cells was significantly down-regulated compared with the control group.The results of TCID₅₀ showed that the viral supernatant titer of the transfected recombinant plasmid group was lower than that of the empty plasmid group and the control group.Preliminarily indications are that seven ISGs have an exert inhibitory effect on CPIV3.Based on the gene sequences of seven ISGs,seven si-RNAs were synthesized:si-IFI6,si-ISG15,si-OAS1Y,si-OAS1Z,si-Mx1,si-Mx2 and si-RSAD2.Seven si-RNAs transfected into MDBK cells and inoculated with CPIV3 JS2013 virus solution 24 h after transfection.The cell culture fluid was harvested at 24 h and 48 h,respectively.RT-q PCR results showed that compared with the control group,the concent of CPIV3 RNA was significantly up-regulated in the group transfected with seven si-RNAs.The results of TCID₅₀showed that the virus supernatant titer of seven si-RNAs transfected groups was significantly higher than that of the control group.This further confirmed the inhibitory effect of seven ISGs on CPIV3.This provided a new method and medicine target for drug study.