Systematic identification of chicken type I and III interferon-stimulated genes with antiviral potential against fowl adenovirus serotype 4

Fowl adenovirus serotype 4 (FAdV-4) is a highly destructive pathogen that causes hepatitis-hydropericardium syndrome (HHS), leading to substantial economic losses in the global poultry industry. Interferon-stimulated genes (ISGs) play a crucial role in controlling viral infections; however, their protective potential against FAdV-4 infections remains largely unknown. In this study, we systematically identified 482 type I and 107 type III ISGs with potential anti-FAdV-4 activity in chickens using RNA sequencing (RNA-Seq), of which 98 were shared by both types, and the expression level of the two types of ISGs was different, such as interferon alpha inducible protein 6 (IFI6). Functional analysis using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes revealed that these ISGs are involved in multiple essential biological processes, metabolic activities, signaling pathways, and immune regulations, suggesting their roles in host-virus interactions and cellular regulatory mechanisms. Heatmap analysis of gene expression profiles showed significant differences in ISG expression between interferon-alpha (ChIFN-α) and interferon-lambda (ChIFN-λ) treatment groups following FAdV-4 infection. Protein interaction analysis indicated that IFI6 is highly connected to multiple immune-related proteins. Notably, IFI6 exhibited the highest expression among the co-expressed genes. Functional validation demonstrated that IFI6 overexpression significantly suppressed FAdV-4 replication, while interference with IFI6 expression enhanced viral propagation. This study provides a comprehensive ISG resource for investigating antiviral responses in chickens and is the first to identify the antiviral activity of IFI6 against FAdV-4. These findings support future antiviral development and prevention and control measures for FAdV-4 infections in poultry.