Signaling Pathways Of Inflammatory Response In Swine Testis Cells Infected By Japanese Encephalitis Virus

Japanese encephalitis virus(JEV)is a member of the Flaviviridae family of Flaviviridae,a single-stranded positive-stranded RNA virus with a full-length genome of 11 kb,encoding a total of 3 structural proteins(C,E,Pr M)and 7 Non-structural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,NS5)are classified into five genotypes(GI-GV)according to the E gene of Japanese encephalitis virus.Japanese encephalitis virus is mainly transmitted by mosquitoes,pigs and birds.Culex is the main transmission medium of Japanese encephalitis virus.Pigs are prone to form high titers and persistent viremia after infection,and are the main amplifying host of the virus.Japanese encephalitis is more prevalent in southeastern Asia,and it is also one of the epidemic encephalitis in my country.Vaccination of people in endemic areas,control of vector density and enhanced control of live pigs can effectively curb the spread of Japanese encephalitis virus.Japanese encephalitis virus strain(JEV-mosq-YN-2016)isolated and preserved in our laboratory.The target fragment of 1751 bp was amplified by PCR.After sequencing,the Meg Align software was used for comparison and analysis.The results showed that the JEV-mosq-YN-2016 strain was a genotype I Japanese encephalitis virus,with the highest homology of 98.30% with the Yunnan JEV strain(Gen Bank: KT229573.1)in China.The potential mechanism of Japanese encephalitis virus on porcine reproductive organs was explored by non-standard quantitative proteomics technology.Swine testis cells were infected with genotype I JEV-mosq-YN-2016 strain,and the inoculation test was carried out at 2MOI.Statistical analysis of samples by mass spectrometry(LC-MS)technology,searching for sample-specific proteins according to the Uni Prot protein database,and then performing peptide and protein level quality control analysis on the database search results,and performing common functional database for the identified proteins.Annotation and quantitative analysis of proteins.Differential screening was performed according to the quantitative results.When the P value was ≤ 0.05,the change in the differential expression was more than 1.5 as the threshold for significant up-regulation and less than 1/1.5 as the threshold for significant down-regulation.A total of 595 differential proteins in the 24h-0h group were counted,and analyzed by GO secondary classification,COG/KOG classification,KEGG pathway and subcellular structure annotation.Based on the statistical results obtained by different classification methods,Fisher’s exact test method was used for enrichment analysis,and it was found that the relevant differences were mainly concentrated in the immune response,signal transduction,biosynthesis and metabolism,etc.The functional relationship of differential proteins at different time points was compared by enrichment cluster analysis,and then specific differential proteins BCL10,NF-κBp65,ICAMI,etc.were screened by volcano plot.The differential proteins were verified by Western blot method,and the results were as follows:(1)Proteomic screening of the target protein BCL10: The expression of BCL10 protein was significantly up-regulated in the 6h and 12 h groups of Japanese encephalitis virus-infected swine testis cells,and the histone expression at 24 h of exposure The amount of protein was significantly down-regulated,and the protein expression was significantly up-regulated 24h-0h after inoculation,which was the same as the expected result of proteomics.(2)NF-κB signaling pathway: The downstream signaling pathway was found according to the significant difference in the expression of the protein BCL10,and the NF-κB signaling pathway was located.The NF-κBp65,IKBα,and IKKα proteins in the NF-κB signaling pathway were verified according to Western blot.The results it was shown that the protein expressions of IKKα,NF-κBp65and IKBα were significantly up-regulated in the 6h inoculation group.(3)Toll-like receptor signaling pathway: Exploring the upstream signaling pathway of NF-κB showed that the My D88-dependent and My D88-independent pathway proteins of the Toll-like receptor signaling pathway had significant changes.In the My D88-dependent pathway,the expression of TLR4,My D88,IRAK-4,IRAK-1,TRAF6,TAB1,and TAK1 proteins was significantly up-regulated in the 6h inoculation group.In the My D88-independent pathway,TLR3,TRAF3,and TBK1 proteins were significantly up-regulated in the 6h inoculation group.The cytokines were validated at the genetic level by q PCR,and the results were as follows:(1)In the My D88-dependent pathway of Toll-like receptor signaling pathway,the cytokines interleukin β(IL-1β),interleukin 12(IL-12)and tumor necrosis factor α(TNF-α)were detected,the expressions of cytokines IL-1β,IL-12 and TNF-α were significantly up-regulated in the 6h and 12 h inoculation groups.(2)In the My D88-dependent pathway of Toll-like receptor signaling pathway,cytokines interferon alpha(IFN-α)and interferon beta(IFN-β)were detected,and cytokines IFN-α and IFN-βwere significantly up-regulated in the 6h,12 h and 24 h inoculation groups.In this study,by verifying TLR4,My D88,IRAK4,TRAF6 and other proteins and cytokines in the Toll-like receptor signaling pathway,it was confirmed that the My D88-dependent pathway mediated by the Toll-like receptor signaling pathway TLR4 was activated,thereby promoting the NF-κB signaling pathway.Involved in immune response and inflammatory response,My D88-independent pathway mediated by Tolllike receptor signaling pathway TLR3 is activated and type I interferon is produced to participate in immune regulation.