Screening And Functional Domain Study Of Bovine Viral Diarrhea Virus Proteins Activating NF-?B Signaling Pathway

Bovine viral diarrhea-mucosal disease,(BVD-MD)is a febrile infectious disease of cows caused from bovine viral diarrhea virus(BVDV),showing a worldwide distribution.OIE lists it as the type B animal epidemic disease.In China it belongs to Class II infectious disease.With diversified clinical symptoms,the major manifestations of this disease are the inflammatory reactions like fever,diarrhea,inflammation,erosion and necrosis of digestive tract mucosa,pneumonia and it causes cattle's persistent infection,seriously affecting cattle's production performance.It is one of the major infectious diseases harming the cattle-feeding industry development and caused huge economic loss.Nuclear factor ?B(NF-?B)is widely found in tissue cells.As one of the important gene expressions of transcription regulator to regulate gene expression of multiple cytokines and inflammatory media,it participates in inflammatory reaction process.In addition,as a significant member of the NLRs family,the NLRP3 inflammasome plays an important role in the onset and development of the inflammatory diseases.Is the inflammatory reaction caused from BVDV infection related to the activation of NLRP3 inflammasome via NF-?B? Which protein plays a key role? These need further research.Based on the consideration of the above issues,this study examined the effects of BVDV on the activation of NF-?B signaling pathway and NLRP3 inflammasome at the viral level,further screened the main viral proteins that activated NF-?B signaling pathway and identified their key functional domains,which provides clues for the analysis of the mechanism of inflammatory response induced by BVDV infection.The first part was using primary BT cells as model cells.It detected the activation effect of BVDV infection on NF-?B signaling pathway and the activation situation of NLRP3 inflammasome at the virus level.The specific content and results are as follows: The effects of BVDV on NF-?B signaling pathway after it was infected by BT cells were detected by dual luciferase reporting system.The result showed that BVDV infection significantly activated cell's NF-?B signaling pathway.And the activation effect was positively correlated with virus titer;the localization of p65 inside cells after BT cells were infected by BVDV was further detected by adopting the indirect immunofluorescence.The result showed that BVDV infection promoted p65 transfer to the nucleus;the RT-q PCR method was adopted to detect the m RNA expressions of the components of inflammasomes NLRP3,Caspase-1 and ASC after the NF-?B signaling pathway was activated.The result showed that gene expressions of NLRP3,Caspase-1 and ASC were significantly increased,showing the NLRP3 inflammasome was activated.Finally,the inflammation-associated cytokines(IAC)related factors like IL-6,IL-8 and IL-1?were detected by adopting RT-q PCR method.The result showed gene expressions of the above inflammasomes were significantly increased.In the second part,the BVDV protein having significant activation of NF-?B signaling pathway activity was screened.The specific content and results were as follows: Eukaryotic expression vectors of C,E0,E1,E2,Npro,p7,NS2,NS3,NS4 A,NS4B,and NS5 B NS5A(it is classified as segment a and segment b)of BVDV protein were built respectively to transfect BT cells and 293 T cells respectively.The protein expression was detected by Western blot method and IFA.The result showed that the target protein was successfully expressed in both BT and 293 T cells;the activation of NF-?B signaling pathway by viral proteins in BT cells and 293 T cells was detected with the dual luciferase reporting system.The result showed BVDV protein C,E0,NS4 B and NS5 A significantly activated NF-?B signaling pathway in BT cells,and E0,NS4 B,NS5A significantly activated NF-?B signaling pathway in 293 T cells.In the third part,the E0 and NS5 A proteins that both significantly activate NF-?B signaling pathway activity in BT and 293 cells were selected to analyze their effects on key factor I?B? and p65 in the NF-?B signaling pathway,and identified key amino acid regions in which they activated NF-?B signaling pathway.The specific content and result are as follows: After the BT cells were transfected with eukaryotic plasmids expressing E0 and NS5 A proteins,the Western blot and IFA methods were adopted respectively to detect the effects of E0 and NS5 A proteins on I?B? and p65 in the cells.The result showed that E0 and NS5 A proteins promoted I?B? degradation as well as p65 phosphorylation and nuclear transfer,showing E0 and NS5 A proteins activate NF-?B signaling pathway via classic route,where 1-94 amino acid of E0 protein and 212-496 amino acid of NS5 A protein were the main function areas for activating NF-?B signaling pathway.In summary,BVDV infection activates NF-?B signaling pathway,thus mediates the activation of NLRP3 inflammasomes,where the BVDV structural protein E0 and non-structural protein NS5 A play a major role in activating the NF-?B signaling pathway.The research findings provide basis for deep interpretation of the pathogenic mechanism of inflammatory reaction caused from BVDV infection.