Synergistic virulence of H9N2 AIV and APEC co-infection in laying hens involves a critical role for macrophage-mediated hyper-inflammation via the MAPK/NF-κB axis.

Abstract

H9N2 avian influenza virus (AIV) is a low-pathogenic subtype virus that is widely prevalent in poultries worldwide. Although H9N2 infection alone typically causes mild or asymptomatic disease in chickens, it facilitates secondary bacterial infections, such as those caused by avian pathogenic Escherichia coli (APEC). Such co-infections often result in severe inflammation of the reproductive tract in laying hens and significant reductions in egg production. However, the underlying immune mechanisms driving this exacerbated inflammatory response remain poorly understood. To address this, we established both in vivo and in vitro models of infection, including laying hens and HD11 macrophage cells, subjected to single or co-infection with H9N2 and APEC. In the animal model, co-infected hens exhibited more severe oviduct tissue damages and enhanced immune responses. Immunological analyses demonstrated activation of all three complement pathways, increased infiltration of CD4⁺ and CD8⁺ T cells, and elevated serum levels of cytokines including IL-6, IL-8, IFN-α, IFN-β, and TNF-α. In the HD11 cell model, transcriptomic analysis revealed that combined stimulation with H9N2 and bacterial LPS induced a broader spectrum of immune-related gene expression compared to either pathogen alone. Further mechanistic studies using pathway-specific inhibitors identified the NF-κB and MAPK signaling pathways as central regulators of macrophage activation and inflammatory cytokine production. This study provides new mechanistic insights into virus-bacteria co-infection within the avian reproductive system, offering a foundation for improved strategies to prevent and control associated poultry diseases.