Chicken DDX21 inhibits H9N2 avian influenza virus replication by modulating type I interferon signaling pathway

H9N2 avian influenza virus (AIV) is one of the major pathogens causing respiratory disease in chickens and poses a threat to public health by serving as a gene donor for zoonotic avian influenza strains. The DEAD-box RNA helicase DDX21 is known to play a critical role in innate immunity in mammals during influenza virus infection. However, the function role and regulatory mechanisms of its avian ortholog, chicken DDX21 (chDDX21), remain poorly understood. In this study, the chicken DDX21 (chDDX21) gene was cloned and analyzed using bioinformatics tools. Phylogenetic analysis revealed that chDDX21 clustered within the avian lineage and shared a close relationship with DDX21 from other avian species. Notably, a widespread distribution of chDDX21 was observed in various tissues in healthy chickens. Following infection with H9N2 AIV, chDDX21 mRNA expression was significantly upregulated in tissues of chickens compared to the control group. Similarly, in CEF cells, chDDX21 transcript levels increased post-infection, peaking at 24 h, a trend that corresponded with the kinetics of viral replication. Furthermore, overexpression of chDDX21 activated the IFN-β promoter, and its DEXDc and HELICc domains were identified as critical for this activity. Co-transfection of chDDX21 with chTLR3 or chTRIF in CEFs resulted in significant upregulation of immune-related gene expression, suggesting that chDDX21 acted as a modulator of the chicken TLR3-mediated type I interferon (IFN) signaling pathway. Additionally, overexpression of chDDX21 significantly increased the expression of antiviral immune factors and concurrently suppressed H9N2 AIV replication. Consequently, these results demonstrated that chDDX21 inhibited H9N2 AIV replication by positively regulating the TLR3-mediated type I IFN signaling pathway. This study provides mechanistic insights into the interaction between the avian innate immune system and H9N2 AIV, highlighting chDDX21 as a key regulator of antiviral response in chickens.