Duck Tembusu Virus Infection Promotes the Expression of Duck Interferon-Induced Protein 35 to Counteract RIG-I Antiviral Signaling in Duck Embryo Fibroblasts.

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that has caused a substantial drop in egg production and severe neurological disorders in domestic waterfowl. Several studies have revealed that viral proteins encoded by DTMUV antagonize host IFN-mediated antiviral responses to facilitate virus replication. However, the role of host gene expression regulated by DTMUV in innate immune evasion remains largely unknown. Here, we utilized a stable isotope labeling with amino acids in cell culture (SILAC)-based proteomics analysis of DTMUV-infected duck embryo fibroblasts (DEFs) to comprehensively investigate host proteins involved in DTMUV replication and innate immune response. A total of 250 differentially expressed proteins were identified from 2697 quantified cellular proteins, among which duck interferon-induced protein 35 (duIFI35) was dramatically up-regulated due to DTMUV infection in DEFs. Next, we demonstrated that duIFI35 expression promoted DTMUV replication and impaired Sendai virus-induced IFN-ß production. Moreover, duIFI35 was able to impede duck RIG-I (duRIG-I)-induced IFN-ß promoter activity, rather than IFN-ß transcription mediated by MDA5, MAVS, TBK1, IKKϵ, and IRF7. Importantly, we found that because of the specific interaction with duIFI35, the capacity of duRIG-I to recognize double-stranded RNA was significantly impaired, resulting in the decline of duRIG-I-induced IFN-ß production. Taken together, our data revealed that duIFI35 expression stimulated by DTMUV infection disrupted duRIG-I-mediated host antiviral response, elucidating a distinct function of duIFI35 from human IFI35, by which DTMUV escapes host innate immune response, and providing information for the design of antiviral drug.

Characterization of duck IκB kinase ß involved in innate immunity.

IκB kinase ß (IKKß), a catalytic subunit of the IKK complex, is involved in a wide array of biological processes, particularly in inflammation and innate immunity. Although extensive studies have been carried out to explore the roles of mammalian IKKßs in innate immune response, the function of IKKß in avian innate immunity is largely unknown. Here, we cloned and characterized the duck IKKß (duIKKß) gene for the first time. DuIKKß encoded 755 amino acids and displayed high sequence similarity to pseudopodoces and haliaeetus IKKßs. DuIKKß transcripts were widely distributed in all tested tissues, especially with high expression in the thymus and bursa of Fabricius. Overexpression of duIKKß promoted NF-κB activation and initiated the downstream cytokines expression including IFN-ß, ZAP, PKR, IL-8, and CCL5 in duck embryo fibroblasts. Furthermore, knockdown of endogenous duIKKß significantly reduced LPS-, poly(I:C)- or SeV-induced NF-κB activation. Finally, we demonstrated that duIKKß showed antiviral activity against Duck Tembusu virus infection. Our findings provide insights into the roles of duIKKß in avian innate immunity.

Molecular characterization and functional analysis of duck IKKα.

IκB kinase α (IKKα) is a vital component of the IKK complex, which is involved in innate immune response, inflammation, cell death and proliferation. Although the functional characteristics of IKKα have been extensively studied in mammals and fish, the roles of IKKα in avian remain largely unknown. In this study, we cloned and characterized the duck IKKα (duIKKα) gene for the first time. DuIKKα encoded a protein of 757 amino acid residues and showed high sequence identities with the goose IKKα. The duIKKα was expressed in all tested tissues, and a relatively high expression of duIKKα mRNA was detected in liver and heart. Overexpression of duIKKα dramatically increased NF-κB activity and induced the expression of duck cytokines IFN-ß, IL-1ß, IL-6, IL-8 and RANTES in DEFs. Knockdown of duIKKα by small interfering RNA significantly decreased LPS-, poly(I:C)-, poly(dA:dT)-, duck enteritis virus (DEV)-, or duck Tembusu virus (DTMUV)-induced NF-κB activation. Moreover, duIKKα exhibited antiviral activity against DTMUV infection. These findings provide important insights into the roles of duIKKα in avian innate immunity.