Stage-specific antiviral roles of IFIH1, IRF7, and MAVS in the interferon-mediated response to DHAV-3 infection.

Duck hepatitis A virus genotype 3 (DHAV-3) is the predominant causative agent of duck viral hepatitis in East Asia, yet its mechanisms of immune evasion remain unclear. In this study, primary hepatocytes were collected at 6, 12, and 24 h post-infection (hpi). The results showed that overall IFN-I levels were significantly elevated after infection, with a particularly pronounced upregulation of IFN-β. Silencing of IFN-I expression markedly promoted DHAV-3 replication, indicating its distinct antiviral role. Furthermore, eight innate immune molecules (IFIH1, IRF1, IRF7, MAVS, RIG-I, TBK1, TLR3, and TLR4) related to IFN-I were investigated in primary duck liver cells using interference and overexpression assays. At 6 hpi, silencing IRF7 and TLR4 markedly promoted replication, while TLR3 inhibition suppressed it. IFIH1 and MAVS displayed stable antiviral activity, with overexpression consistently restricting replication and preferentially enhancing IFN-α. At 12 hpi, IRF7 and TLR4 knockdown reduced replication, whereas MAVS and IRF1 overexpression unexpectedly promoted replication despite increasing IFN levels, while IFIH1 and TLR3 maintained its inhibitory effect and further upregulated IFN-I gene expression. At 24 hpi, silencing IRF7, MAVS, RIG-I, TBK1, TLR3, and TLR4 enhanced replication, whereas overexpression of IFIH1 and IRF7 remained inhibitory, accompanied by a marked increase in IFN-β expression. Collectively, these results highlight IFIH1, MAVS, and IRF7 as central regulators in DHAV-3 infection, with IFIH1 acting as a stable antiviral effector, while MAVS and IRF7 undergo stage-specific functional shifts that balance host defense and viral exploitation.