Early innate immune response to highly pathogenic avian influenza and functional characterisation of the chicken IRF1 promoter.

Highly pathogenic avian influenza (HPAI) virus outbreaks in poultry farming impose a substantial economic burden and present a significant threat to animal welfare, with the H5N1 subtype representing a potential candidate for a future pandemic strain. Advancing the current understanding of the host immune response and viral dynamics following HPAI H5N1 infection is critical for improving prevention and treatment strategies. Using the chicken embryo fibroblast DF-1 cell line, this study aimed to investigate the initial cell-intrinsic innate immune response and corresponding viral transcription following challenge with HPAI H5N1 through RNA sequencing. Significant enrichment of genes associated with interferon signalling and the p53 pathway was observed at four and 8 h post infection, with sustained upregulation of interferon regulatory factor 1 (chIRF1) observed throughout the time course. Leveraging this finding, the proximal promoter region of chIRF1 was functionally characterised to investigate its transcriptional regulation following exposure to viral stimuli. Motif-based analysis identified putative regulatory elements within the -200 bp proximal promoter region, including an NF-κB binding motif, an gamma interferon-activated site (GAS), a CCAAT box, and several GC boxes. Luciferase reporter assays employing 5' deletion constructs and targeted disruption of the GAS element demonstrated that this motif is required for full induction of the chIRF1 promoter in response to poly(I:C) stimulation and HPAI H5N1infection.