Chicken DDX60 potentiates dsRNA analog-induced IFN-β signaling through synergistic interaction with MDA5

Double-stranded RNA (dsRNA) viruses cause devastating economic losses to the global poultry industry by triggering severe diseases in chickens; however, the anti-dsRNA innate immune mechanism in this species remains poorly defined. Critical limitations include the lack of the mammalian retinoic acid-inducible gene I (RIG-I) receptor (a core dsRNA sensor) and the unelucidated role of DEAD/H-box helicase DDX60 (a key antiviral factor in mammals) in chicken antiviral immunity. To address these knowledge gaps, we investigated the function and mechanism of chicken DDX60 (chDDX60) in anti-dsRNA innate immunity using chicken embryo fibroblast cells (CEFs) and DF-1 cells as in vitro models, combined with gene overexpression/knockdown, luciferase reporter assays, co-immunoprecipitation (Co-IP), immunofluorescence, molecular docking, and stimulation with the dsRNA analog polyinosinic-polycytidylic acid (poly I:C). Our results showed that chDDX60 is highly expressed in immune-relevant tissues (small intestine and spleen), induces IFN-β and downstream antiviral genes (ISG15, MX-1, PKR) via activating IRF7, interacts with chMDA5 to synergistically enhance IFN-β activation, acts upstream of chMDA5 to trigger the chMDA5-chMAVS-chSTING-chIRF7-IFN-β signaling axis, and is essential for establishing an antiviral state against dsRNA viruses under poly I:C stimulation. Taken together, our findings identify chDDX60 as a positive regulator of dsRNA analog-induced IFN-β signaling in chicken cells and provide new insight into the regulation of RIG-I-like receptors (RLR)-mediated innate immune signaling in RIG-I-deficient avian species. Further studies using infectious RNA virus models are needed to determine whether chDDX60 contributes to antiviral defense against dsRNA and/or ssRNA viruses.IMPORTANCEViral RNA sensing is essential for host defense, but chickens naturally lack retinoic acid-inducible gene I (RIG-I), a major RNA sensor in mammals. This study shows that chicken DDX60 helps activate IFN-β signaling in response to the synthetic double-stranded RNA (dsRNA) analog polyinosinic-polycytidylic acid (poly I:C) by working together with melanoma differentiation-associated gene 5 (MDA5). Mechanistically, chDDX60 functions upstream of chMDA5 and promotes activation of the chMDA5-chMAVS-chSTING-chIRF7-IFN-β signaling axis. These findings help explain how chicken cells regulate RNA-triggered innate immune signaling despite the absence of RIG-I and provide insight into avian-specific antiviral immune regulation. Because this study did not use infectious RNA viruses, future studies using dsRNA and ssRNA virus models will be needed to define the antiviral function and virus-type specificity of chDDX60.