Isolation And Identification Of Seneca Valley Virus And The Molecular Mechanism Of Antagonizing Interferon Production By 3C Protease

Seneca valley virus(SVV),belongs to the Senecavirus genus of the family Picornavirus,has the closest genetic relationship with members of the myocarditis virus family.In recent years,SVV has been found to be associated with new pathogens such as idiopathic vesicular disease and epidemic transient neonatal losses.The clinical manifestations of infected pigs were claudication,blisters,and depression and anorexia,and the mortality rate of newborn piglets was as high as 30%~70%.Since 2014,cases of PIVD caused by SVV infection have been reported in many countries around the world.The SVV infection was reported for the first time in a pig farm in Guangzhou,China,followed by SVV infections in Hubei,Heilongjiang,Henan,Fujian and Guangdong.Currently,as a new infectious disease of pigs,the interaction between SVV and its host is yet to be fully characterized.How SVV causes morbidity in pigs is urgently needed to analyze its pathogenic mechanism from all levels.Therefore,in this study,the isolation and identification of the virus,the use of reverse genetic manipulation to obtain SVV infectious clones,and the regulation of SVV on the innate immunity of the host against virus were studied.The details are as follows: 1.Isolation and identification of Seneca valley virusBlister fluid is collected from piglets with idiopathic vesicular disease.Pathogen-specific PCR was used to detect the important pathogens causing vesicular disease in pigs,such as vesicular stomatitis virus,foot-and-mouth disease virus and porcine vesicular disease virus.The results showed negative for VSV,FMDV and SVDV infections.Subsequently,the VP1 specific PCR primers of the newly infected SVV were used for the detection,and the result was positive for SVV infection.Virus isolation was carried out in a BHK-21 cell culture system.The suspicious SVV virus which can cause the typical cytopathic effect of BHK-21 cells was obtained.Immunofluorescence assay and Western blot analysis were conducted using homemade mouse polyclonal anti-SVV VP1 antibody,and the results were shown to be positive for VP1.The virus characteristics of SVV were studied by virus plaque assay,and the results showed that that after 48 hours of SVV infection BHK-21 cells could produce virus plaque with a diameter of 1-3mm.Finally,the c DNA template was obtained by reverse transcription of total RNA from SVV infected cells,and SVV-specific primers were used to perform genome-wide multistep PCR to obtain multiple fragments of the full-length SVV-containing genome,and subclones were sequenced with DNA in p Easy-Blunt vector.The results showed that we obtained a full length genome of SVV virus with a length of 7300 bp,including a 668-nt 5'UTR,an open reading frame(ORF)encoding a 2181 amino acid polyprotein and a 86-nt 3'UTR cantains a partial poly(A)sequence.The isolated virus strain was named SVV HB-CH-2016 strain,and the complete genome was submitted to Gene Bank.The accession number was KX377924.2.Establishment a DNA-launched infectious clone of SVVIn this study,the infectious clone plasmid of SVV HB-CH-2016 strain,which was mediated by CMV promoter,was constructed with p Bluescript SK ? as a vector and named as p SK?-CMV-SVV/HB.Cytopathic effects of SVV-like virus infection on cells were observed after transfection of plasmid p SK?-CMV-SVV/HB in 293 T or BHK-21 cells 36 hours after transfection.The supernatants of transfected cells were collected by centrifugation and the supernatants of cultured cells were continuously used for virus passage.The results of immunofluorescence,Western blot and specific PCR showed that we obtained the rescue virus r SVV.The results of virus plaque detection showed that the size of the plaque produced by r SVV on BHK-21 cells was similar to that of the wild type SVV.The CMV driven SVV infectious clone was successfully established,avoiding the cumbersome in vitro transcription and RNA transfection process,providing a convenient basis for subsequent SVV related virus characterization,viral vectors and novel virus vaccine research.3.SVV infection negatively regulates the production of type ? interferonIn this study,the dual luciferase activity reporter system and real-time fluorescence quantitative PCR were used to investigate the effect of SVV infection on interferon production.It was found that SVV infection could not activate the IFN-? promoter activity and IFN-? m RNA production.Through the detection of IRF3 activity also found that SVV can not effectively induce the phosphorylation and nuclear translocation of IRF3.In addition,IFN-? treatment experiments found that IFN-? can significantly inhibit the proliferation of SVV.Viral titer assay results showed that IFN-? can significantly inhibit the proliferation of SVV,but SVV infection does not trigger the response of host type ? interferon.The above results show that SVV is an interferon-sensitive virus.SVV may escape the host's antiviral immune response through some mechanism.4.SVV 3Cpro negatively regulates the production of host type ? interferonThe expression plasmid of SVV protein was constructed,and the effect of SVV infection on interferon production was explored using a dual luciferase activity reporter system and real-time fluorescence quantitative PCR.The results showed that SVV VP2,3Cpro and 3D inhibited the activation of IFN-b promoter in different degrees,and the 3Cpro inhibitory effect was extremely significant(P < 0.001).The negative regulation of SVV 3Cpro on IFN-? production was confirmed by real-time fluorescence quantitative PCR assay,nuclear transport of IRF3,and phosphorylated IRF3 protein levels.These results suggest that SVV 3Cpro can effectively antagonize the production of host type ? interferon.5.Molecular Mechanism of SVV 3Cpro Targeting Antagonism to Type ? Interferon ProductionThe promoter activity of IFN-? was detected by the dual luciferase system and it was found that the SVV 3Cpro effect range was located between the RIG-1/MDA-5 pathway and the IRF3 upstream.293 T cells were co-transfected with SVV HA-3C and the corresponding linkers of the innate immune signaling pathway.Western blot results showed that SVV 3Cpro specifically cuts MAVS,TRIF and TANK.The inhibitors of protein degradation pathways MG132,NH4 Cl and Z-VAD-FMK did not inhibit the cleavage of MAVS,TRIF and TANK by SVV 3Cpro.The enzyme activity deletion mutant results show that SVV 3Cpro is dependent on its protease activity for MAVS,TRIF and TANK cleavage effects.Immunolocalization(Co-IP)and intracellular laser confocal microscopy analysis showed that SVV 3Cpro interacts with MAVS,TRIF and TANK.A series of potential cleavage site mutants of MAVS,TRIF and TANK were constructed on the basis of the recognition site of polypeptide protein of SVV 3Cpro cleavage virus.Western blot was used to detect the cleavage of each protein.The results show that SVV 3Cpro cleaves MAVS and TRIF at amino acid residues Q148 and Q159,respectively,while TANK is cleaved twice at E272 and Q291.These results indicated that the cleavage site mutants of MAVS,TRIF and TANK could resist the cleavage effect of SVV 3Cpro and maintain its activation to IFN-b.However,any cut product of MAVS and TANK lost the activation of IFN-b.The N-terminal product of MAVS and TANK lost the activation of IFN-b,but the C-terminal product was still active.In addition,SVV infection can upregulate the transcription of NF-kB signaling pathway-dependent inflammatory cytokines IL-6 and TNFa m RNA.The expression of SVV 3Cpro can enhance the activation of NF-kB signaling pathway by Sev infection.SVV 3Cpro-mediated TANK cleavage promotes TRAF6-induced activation of the NF-kB signaling pathway.In summary,this study isolated a SVV from clinically suspected cases of PIDV;established an invasive cloning platform for SVV;confirmed that SVV 3Cpro specifically cleaves MAVS,TRIF and TANK,which are important innate immunity related host molecules of the host,and are negatively regulated the production of type ? interferons,thereby escaping the host's antiviral immunity.The cleavage of TANK by 3Cpro also promotes the activation of NF-kB signaling pathway mediated by TRAF6,which is associated with the SVV infection activating the host NF-kB signaling pathway and inducing the production of inflammatory cytokines.