Analyses Of Prometics In CDV-infected Mv.1.Lu Cells And The Impact Of TLR3 On CDV Replication And Proliferation

Canine distemper(CD)caused by canine distemper virus(CDV)is one of the most lethal disease in mink,resulting in huge economic loss.There is no effective treatment for the disease,but prevention by vaccine.However,increasing numbers of reports have suggested that vaccinated animals can still be infected.Effective prevention and control of the CDV infection has always been a hot topic for researchers.The balance and restriction of the virus-host interactions determines the outcome of viral infection.Therefore,a better understanding of these host-virus interactions may lead to the identification of potential drug targets.Based on iTRAQ,the present study analyzed the changes in the mink proteome upon CDV infection in Mv.1.Lu cells,for the first time.Further,the impact of TLR3 on CDV replication and the mechanisms involving in were explored.It may provide theoretical basis for effective protection of mink from CDV infection.To reveal the interactions between CDV and mink cellular proteins,a quantitative proteomic analysis was performed to identify differentially expressed proteins(DEPs)in Mv.1.Lu cells infected with CDV PS strain at 24 hpi.Totally,520 DEPs were identified.Among them,151 and 369 proteins were markedly up-regulated or down-regulated,respectively.GO enrichment were conducted using DAVID and UniProt databases.The result showed that DEPs were associated with various biological processes.Network and KEGG pathway analyses revealed that the DEPs mainly involved in NF-?B signaling pathways,and were also related to NLRs and TLRs signaling.Phosphorylation and nuclear accumulation of the NF-?B P65 proteins were subsequently detected by Western Blot and under confocal microscopy,which verified the activation of NF-?B signaling induced by CDV infection.To confirm whether TLRs participated in the immune response induced by CDV infection,and to further study whether TLRs involved in NF-?B activation during CDV infection in Mv.1.Lu cells,the mRNA expressions of TLR3,TLR7 and TLR8 were detected using qRT-PCR method.In addition,the influence of TLRs on NF-?B luciferase activity during CDV infection was analyzed after inhibition of MYD88 and TLR3 proteins expression by respective inhibitors.The results indicated that TLRs indeed participated in CDV infection and were also associated with NF-?B activation induced by CDV challenge.Based on the above findings,the impact of TLR3 on CDV replication and the possible mechanisms involved in the process were explored.Firstly,the full length of mink TLR3 gene was cloned using RACE method,eukaryotic expression plasmid named pCMV-myc-TLR3 was then successfully constructed.Additionally,CDV replication was detected in TLR3 over-expressing cells and cells challenged by TLR3 inhibitor.Overexpression of TLR3 leaded to decreased viral mRNA and viral protein synthesis,reduction of TLR3 expression conversely enhanced CDV replication.To further clarify the involving mechanism,the effect of TLR3 on IFN-?,Mx1 and NF-?B expressions and the effect of inhibition of NF-?B expression on CDV replication were analyzed.The resulted indicated that TLR3 enhanced the expressions of IFN-?,Mx1 and NF-?B,while,inhibition of NF-?B expression resulted in elevated CDV proliferation.Taken together,our observation strongly suggested anti-CDV effects of TLR3 in Mv.1.Lu cells after PS infection,probably depending on contributing to IFN-? and Mx1 production and NF-?B activation as well.The present study started with comparison of the changes in mink proteome upon CDV infection in Mv.1.Lu cells to thoroughly analyze the virus-host cell interactions,thereby selected the critical proteins reacting violently to CDV infection and tightly linking to immune response.Subsequently,the research concentrated on identification of the impact of target gene on CDV infection and clarification of the possible mechanisms.Our results contributed to understanding of the molecular mechanism involved in innate immune response against CDV infection,which provided experimental basis for potential antiviral agents and further effective protection of mink from CDV infection.