Studies On The Molecular Mechanism That The Duck Tembusu Virus Activates NF-κB And Induces IFN-β

Since 2010, the newly emerged flavivirus genus virus, Duck Tembusu virus(DTMUV), had caused serious economic losses in the duck industry. Infected ducks are characterized by loss of appetite, weakness, diarrhea and decreased egg production. The pathogenic mechanisms associated with DTMUV infection are unclear. The DTMUV infection could be sensed by the pattern recognition receptors(PRRs) leading to the activation of signaling pathways to suppress virus replication by IFN-β production. However, the high-level IFN-β may be associated with high cytokines and chemokines concentrations, resulting in the cytokine storm and serious pathological damage.In this study, we firstly established a platform to support the further research on DTMUV through researching on the role of duTRAF6 in duck innate immune response. Then the molecular mechanisms of IFN-β production activated by DTMUV were studied to reveal the mechanism of immune responses and cytokine storm induced by DTMUV, and details are as follows. 1. Molecular characterization and functional analysis of duTRAF6We cloned the full-length duTRAF6 cDNA for the first time(GenBank accession number KJ461514.1). The amino acid sequences alignment and evolutionary tree analysis revealed that duTRAF6 had a close evolutionary relationship to other birds. Real-time PCR analysis showed the most abundant expression of duTRAF6 protein is in colon, spleen and muscle. Dual luciferase reporter assay showed that over-expression of duTRAF6 could strongly activate NF-κB pathway in a does-dependent manner. Furthermore, deletion mutant analysis indicated that the zinc fingers and coiled-coil domain of duTRAF6 were essential for the NF-κB activation. In accordance with the above results, siRNA interference also suggested that duTRAF6 plays an important role in duck innate immune response. 2. DTMUV infection activated NF-κB, IRF1 and induced IFN-β expression through RIG-I、MDA5、TLR3 and MAVSTo determine whether DTMUV could induce IFN-α/β/γ and cytokine gene expression in DEFs in which DTMUV replicated, RT-PCR analysis was performed and IFN-α/β/γ, IL-1β, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18 and TNF-α gene expression were readily detected. These results suggested that DTMUV contributed to innate immune responses in DEFs. Furthermore, the luciferase reporter assay showed that DTMUV infection dramatically activated NF-κB, IRF1 and induced IFN-β in a time- and dose-dependent manner. We also found that RIG-I, MDA5, TLR3 and MAVS mediated the DTMUV induced IFN-β production through over-expressed the domain negative regulatory mutants of RIG-I and MDA5 and knockdown of TLR3 and MAVS. 3. The NS5 protein coded by DTMUV could significantly activate the NF-κB and induce IFN-βTo further investigate the effect of DTMUV proteins on the induction of IFN-β, we found NS5 protein significantly activated the NF-κB, IRF1 and induced IFN-β in a timeand dose-dependent manner. However, the substitution mutations generated in the GDD motif(D668A) and YADD(D536A) motif, which inactived RdRp catalytic activity, completely lost the ability to activate the IFN-β promoter compared to the full-length and two truncated mutations namely NS5(aa45-905) and NS5(aa224-905), suggesting that the RdRp activity was essential for IFN-β expression. 4. MDA5, MAVS, MyD88 and TRIF were involved in IFN-β inducing by NS5 protein in DF1 cells.To confirm the proteins mediated NS5-induced IFN-β expression signalling pathways, the participation of MDA5, MAVS, MyD88 and TRIF in NS5-mediated IFN induction was examined by synthetized certain siRNAs targeting chicken MDA5, MAVS, MyD88 and TRIF. Inhibition of the genes expression by siRNA significantly decreased NS5-induced activation of IFN-β. These results indicated that MDA5, MAVS, MyD88 and TRIF were necessary for IFN-β induction by NS5 protein in DF1 cells.In conclusion, our results revealed duTRAF6 plays a key role in duck innate immune response for the first time; We found that DTMUV activated IFN-β through the RIG-I, MDA5 or TLR3 mediated signaling pathways and discovered the ability of NS5 protein coded by DTMUV activated the IFN-β is associated with its RdRp activity and the IFN-β activation is mediaded by MDA5, TRIF, MyD88 and MAVS. This study provides some technical methods and certain theoretical basis for further study on the DTMUV.