Identification of duck type II interferon-stimulated genes and revelation of duIFI35 inhibition of H5N6 AIV replication by promoting apoptosis.

H5N6 highly pathogenic avian influenza virus (HPAIV) poses a serious threat to both poultry and public health due to its ability to cross species barriers. Although interferon-stimulated genes (ISGs) are key components of the host's antiviral defense, a systematic identification and functional characterization of duck ISGs has not yet been conducted. In this study, we identified 815 potential duck ISGs induced by type II interferon (IFN-γ) in duck embryo fibroblasts (DEFs). The majority of these type II ISGs were enriched in immune-related pathways, including "cytokine-cytokine receptor interaction" and "influenza A". Functional validation using siRNA-mediated knockdown demonstrated that six ISGs, including duIFI35, promote H5N6 AIV replication when silenced. Through TUNEL assay, flow cytometry, and apoptotic pathway analysis, Our analysis revealed that H5N6 AIV infection markedly upregulates apoptotic genes such as Fas, FADD, caspase-8, BAK, cytochrome c, APAF1, caspase-9, and caspase-3 (P < 0.05), thereby promoting apoptosis in DEFs. In investigating the antiviral mechanism of duIFI35, it was found that overexpression of duIFI35 further enhanced H5N6-induced apoptosis, as evidenced by increased transcription of these apoptotic genes, whereas duIFI35 knockdown had the opposite effect. Importantly, the antiviral effect of duIFI35 was significantly diminished upon treatment with the caspase inhibitor Z-VAD-FMK (20 μM) (P < 0.05), indicating that its antiviral activity is mediated through apoptosis induction. Collectively, this study provides the first systematic identification of type II ISGs in ducks and reveals duIFI35-mediated apoptosis as a critical antiviral mechanism, offering foundational insights into ISG-driven innate immunity against AIV in waterfowl.