The Molecular Mechanism Underlying Host Innate Immune Response Against Avian Tembusu Virus Infection

Avian Tembusu virus(ATMUV)is a newly emerging flavivirus that belongs to Ntaya virus group.ATMUV is a highly pathogenic virus causing significant economic losses to Chinese poultry industry.The infected flocks are characterized by severe reduction in egg production,high fever,anorexia,diarrhea,weight loss and paralysis,with high morbidity and low mortality.Host innate immunity serves as the first line to defend against the infection of pathogens at early stages.However,little is known about the role of host innate immunity in defending the ATMUV infection.In this study,we investigated the innate immune signaling relevant to the host response against ATMUV infection.We found that ATMUV infection significantly up-regulated the expression of type I and type III interferons(IFNs)and some critical IFN-stimulated genes(ISGs)in vivo(chicken and ducklings)and in vitro(DEFs,CEFs,DF-1 and 293T cell lines).Such innate immune response was induced by genomic RNA of ATMUV.The expression of IFNs and ISGs was greatly up-regulated by transfection of CEF cells with either VG-RNA or viral RNA,whereas normal control cells RNA failed to stimulate IFNs and ISGs expression.Furthermore,we observed that ATMUV infection triggered the IFN response mainly through MDA5 and TLR3-dependent signaling pathways.We observed that disruption of TLR3 or MDA5 expression resulted in significant decrease in mRNA levels of type I and type III IFNs induced by ATMUV infection.Similarly,transfection of TLR3-siRNA and MDA5 shRNA significantly disrupted the expression of both TLR3 and MDA5,associated with lower IFN expression than those observed in cells silencing MDA5 only.Strikingly,shRNA-based disruption of IPS-1,IRF3 or IRF7 expression significantly reduced the production of IFNs in 293T cell model.Moreover,we observed that IKB-? protein levels were consistently reduced in CEF and 293T cells infected with ATMUV,suggesting that NF-?B is activated during the ATMUV infection.Inhibition of NF-?B signaling using BAY11-7082 also resulted in a significant decrease in expression of IFNs.The interferon-inducible transme mbrane proteins(IFITMs),a member of ISG superfamily,serve as critical effector molecules in host innate immune system and effectively restrict a wide range of pathogenic viruses.However,little is known about host IFITMs expression profile and antiviral ability in response to ATMUV infection.In present study,we explored the role of host IFITMs in defending against ATMUV infection.We observed that expression of IFITMs was significantly upregulated in DEF and DF-1 cells after infection with ATMUV.Similar results were obtained from in vivo studies using ATMUV-infected ducklings.Using DF-1 cell system,we found that knockdown of endogenous IFITM1 and IFITM3 by short hairpin RNA(shRNA)markedly enhanced ATMUV infection in host.However,silencing IFITM2 had no significant effect on ATMUV replication.Furthermore,overexpression of chicken or duck IFITMI and IFITM3 could impair the replication of ATMUV.Consistently,our experiments revealed that treatment with IFNs significantly impaired ATMUV replication in chicken cell.In addition,type I IFNs also exhibited promising antiviral activity against ATMUV replication in a human cell model.Taken together,these data indicated that ATMUV infection triggers host innate immune response through MDA5 and TLR3-dependent signaling that controls IFNs and ISGs production,and thereby induces an effective antiviral immunity.The induced expression of avian IFITMs and IFNs in response to ATMUV infection could effectively restrict the virus replication,and suggested that these increasing IFITMs and IFNs in host may be a useful strategy for control of ATMUV infection.