Effects Of N And H Proteins Of Canine Distemper Virus On IFN-β Expression

Canine distemper is a febrile,highly contagious infectious disease caused by canine distemper virus(CDV)of the genus Morbillivirus,family Paramyxoviridae.CDV usually invads the host via the respiratory tract and rapidly replicates in lymphoid tissue,causing severe immunosuppression in infected animals.In recent years,the natural host of CDV has continued to expand,including Canidae,Mustelidae,and Procyonidae.Even primate macaques can be infected and die.Since the 1950 s,the widespread use of live CDV vaccines has greatly controlled the prevalence of CD.The host innate immune system,especially the interferon(IFN)system,senses and protects it from infection by pathogenic microorganisms.However,several viruses,including CDV,have simultaneously evolved different strategies against innate immune recognition.At present,whether CDV structural proteins N and H can invade the host by antagonizing the innate immune response and the mechanism of their action have not been clarified.In order to investigate the interaction between CDV and host innate immune response,we used CDV wild strains LN(10)1,SD(14)7 and vaccine strains CDV3 to infect canine kidney cells(MDCK),respectively.The IFN-β promoter activity of MDCK at different time points was detected by a dual luciferase reporting system.Simultaneously,Quantitative RT-q PCR was used to detect IFN-β m RNA transcription level.LN(10)1 and SD(14)7 strains could not induce the activation of IFN-β promoter,while CDV3 strains could detect the activation of IFN-β promoter at 8 h after MDCK cells were infected.The RT-q PCR results showed that there was no significant change in transcription level of IFN-β m RNA in the wild strains LN(10)1,SD(14)7 and vaccine strains CDV3.To further explore the effect of wild strains and vaccine strains on IFN-β pathway,poly(I:C)was used in this study to activate IFN-β pathway to detect the influence of wild-type strains and vaccine strains on this pathway.The results of dual luciferase reporting system showed that both wild-type strains and vaccine strains had significant inhibitory effects on poly(I:C)-activated IFN-β promoter(P<0.05).RT-q PCR results also showed that both wild-type strains and vaccine strains downregulated the transcription level of IFN-β m RNA induced by poly(I:C)(P<0.05).In order to further investigate the molecular mechanism of CDV antagonism against IFN-β pathway,we constructed recombinant eukaryotic expression plasmids of N and H proteins of LN(10)1,SD(14)7wild strains and CDV3 vaccine strains.After the recombinant expression plasmid was transfected into293 T cells,the effects of different virulence N and H proteins on the Poly(I:C)-activated IFN-β promoter were detected using a dual luciferase reporter system,respectively.And the effect of different virulence N and H proteins on the transcription level of IFN-β m RNA induced by poly(I:C)was detected by RTq PCR.The results of dual luciferase reporting system showed that N and H proteins with different virulence could significantly inhibit the activity of poly(I:C)-activated IFN-β promoter(P<0.05),the inhibitory effect of vaccine strain N protein on poly(I:C)-activated IFN-β promoter was significantly lower than that of wild-type strain N protein LN(10)1,SD(14)7(P<0.05).The RT-q PCR results showed that both N and H proteins with different virulence could significantly downregulate the transcription level of IFN-β m RNA induced by Poly(I:C)(P<0.05).In order to determine the key amino acid regions of N protein inhibiting IFN-β pathway,we compared the amino acid sequences of N protein in wild strains LN(10)1,SD(14)7 and vaccine strains CDV3.Compared with the classical strain LN(10)1,the mutant strain SD(14)7 had only two amino acid mutations in the N protein,while the vaccine strain CDV3 had 16 amino acid mutations.Three N protein truncated mutants were constructed using LN(10)1 strain of N protein plasmid as the template.The dual luciferase reporting system and RT-q PCR were used to detect the difference in the effect of different N protein truncated mutants on IFN-β expression.We found that the deletion of amino acid 442-480 in the N-protein region resulted in the loss of the N-protein’s ability to antagonize the IFN-β pathway.In this study,the preliminary study of the differences in the antagonistic IFN-β signaling pathway of different virulence CDV strains provides a theoretical basis for further understanding of the interaction between CDV and host protein and the escape mechanism.