Infectious bursal disease virus induces GSDME-dependent pyroptosis via the mitochondrial caspase-9/3 pathway in DF-1 cells.

Infectious bursal disease virus (IBDV) is an important avian pathogen that damages the bursa of Fabricius and continues to threaten the poultry industry. Pyroptosis is an inflammatory form of programmed cell death, but its contribution to IBDV-induced cellular injury remains incompletely defined. Here, using DF-1 cells, we found that IBDV infection was associated with caspase-3 activation, cleavage of gasdermin E (GSDME), generation of the pore-forming N-terminal fragment (GSDME-N), increased lactate dehydrogenase release, plasma membrane damage, and enhanced inflammatory cytokine-associated readouts, including increased interleukin (IL)-1β secretion and elevated IL-18 messenger RNA (mRNA) expression. IBDV infection also induced mitochondrial dysfunction, as evidenced by loss of mitochondrial membrane potential (ΔΨm), increased reactive oxygen species production, and activation of caspase-9, supporting the involvement of a mitochondria-associated caspase-9/3 pathway. Pharmacological inhibition of caspase-9 or caspase-3 reduced pyroptosis-associated readouts, indicating that mitochondrial damage contributes to IBDV-induced lytic cell death under the tested conditions. Expression of viral protein (VP)2 or VP5 alone caused detectable cytotoxicity but did not robustly reproduce GSDME cleavage, suggesting that viral proteins may contribute to upstream cellular stress that is further amplified during infection. Together, these findings support that IBDV engages a mitochondria-associated caspase-9/3-GSDME axis to promote pyroptosis-like lytic cell death in DF-1 cells.