Duck STING Mediates Innate Immune Response Induced by H5N8 Subtype Avian Influenza Viruses

Background: The H5N8 highly pathogenic avian influenza virus (HPAIV) poses a significant threat to global poultry and public health due to its high mortality rate and potential for cross-species transmission. As natural H5N8 hosts, Anatidae (e.g., ducks) drive transmission and evolution. STING, a key innate immunity regulator, activates IFN-β via signaling to combat infection. While STING's anti-AIV role is known, its mechanisms in ducks during H5N8 infection remain unclear. This study analyzes STING pathway dynamics in SPF ducks post-infection, revealing its core antiviral role in waterfowl. Findings support HPAIV control strategies. Materials, Methods & Results: This study utilized sixty 4-week-old specific pathogen-free (SPF) ducks, randomly divided into H5N8-infected (n = 30) and PBS control (n = 30) groups. The experimental group was inoculated via intranasal and oropharyngeal routes with 106 EID50/0.2 mL of A/whooper swan/Henan/SMQ9/2020 (H5N8) viral suspension, while the control group received an equivalent volume of PBS. Euthanasia was performed at 0.25, 1, 2, 3, 5, 7, 10, and 14 days post-infection (dpi) [n = 3-4 per time point], with collection of lung, spleen, and rectal tissues. Clinical observations revealed mild respiratory symptoms in infected ducks, including coughing and nasal secretions. Two SPF ducks died at 3 dpi and 5 dpi. Necropsy demonstrated mucus accumulation in the trachea and pulmonary hemorrhages in the infected group. The control group exhibited no clinical symptoms or histopathological lesions throughout the experimental period. Absolute quantitative fluorescent PCR detected peak viral loads in tissues at 2-3 dpi, with pulmonary viral loads significantly exceeding those in other organs, followed by a gradual decline after 5 dpi. qRT-PCR revealed time-dependent differential changes in the expression of STING pathway immune factors (STING, TBK1, IRF7) as well as TLR7 and IL6. During the early stages of infection, the expression of these immune factors was significantly upregulated, peaking at 2-3 dpi, followed by a gradual decline in expression from 5 dpi onward. Notably, the expression trends of TBK1, IRF7, and IFN-β exhibited a striking similarity to that of STING. SPSS correlation analysis demonstrated a positive correlation between STING and IFN-β expression in the lungs, spleen, and rectum. These results indicate that STING regulates the expression of multiple immune factors and promotes IFN-β production, playing a critical antiviral role in SPF ducks during the early phase of H5N8 avian influenza virus infection. Discussion: This study elucidates the pivotal role of the STING signaling pathway in coordinating the innate immune response of SPF ducks against H5N8 HPAIV. Our findings demonstrate that the expression of STING and associated immune factors peaked at 2-3 dpi before gradually declining from 5 dpi onward. The STING-mediated TBK1-IRF7 signaling axis was found to promote IFN-β production, as supported by significant positive correlations (SPSS analysis) between STING and IFN-β expression levels in the lung and spleen. These results suggest that enhancing STING activity or stabilizing its expression could augment host antiviral defenses, thereby curbing viral replication during early-stage infections. Collectively, our work establishes a molecular foundation for developing STING-targeted immunomodulatory strategies, such as novel vaccine adjuvants, to improve avian influenza control. This approach not only holds potential to mitigate economic losses in poultry industries but may also reduce zoonotic transmission risks. Keywords: H5N8, AIV, ducks, STING, IFN-β.