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.