Generation of DFNA5-deficient chickens reveals a species-specific role of pyroptosis in avian antiviral immunity.

The global burden of viral diseases in both humans and agricultural animals is exacerbated by dysregulated inflammatory cell death and cytokine storms. Pyroptosis, executed by gasdermin family proteins, is a key driver of this pathology, yet its functional conservation and therapeutic targeting in avian species remain critically unexplored. Here, we establish the first germline-edited heterozygous DFNA5-mutant chicken model using primordial germ cell mediated genome editing. We demonstrate that genetic disruption of DFNA5 markedly attenuates Raptinal-induced pyroptosis and reduces IL-1β and IL-18 secretion. Using an optimized adenoviral dual-sgRNA delivery system, we successfully generated chimeric chickens with stable germline transmission of a frameshift deletion in DFNA5. Notably, DFNA5 deficiency is associated with reduced expression of the viral gp85 gene following avian leukosis virus subgroup J (ALV-J) infection. Mechanistically, transcriptome analysis reveals that downregulated genes in ALV-J-infected DFNA5-mutant cells are enriched in apoptosis and NOD-like receptor signaling pathways, while upregulated genes are associated with cell adhesion, extracellular matrix organization, and homeostatic pathways, suggesting a possible shift from pyroptotic inflammation toward cellular integrity and repair. This work provides genetic evidence that a possible association between DFNA5-mediated pyroptotic responses and establishes a genetic strategy for poultry disease resistance research and for understanding host-virus evolution and viral-induced inflammatory pathologies across vertebrates.