Avian influenza A virus (IAV) is an important threat to agriculture and poses a risk for cross-species transmission to humans. The authors profiled the interferon-induced response in avian cells and used that information to perform a functional genetic screen to identify ISGs that restrict IAV replication in chicken cells. The pooled CRISPR knockout screen was applied against two IAV strains: the mouse-adapted PR8 and a low pathogenicity avian isolate, WF10. For PR8, IFITM3 emerged as the dominant restrictor, with several other ISGs producing modest effects. For WF10, IFITM3 was again identified, and two additional hits—BAG5 and OTUD4—were detected. These results demonstrate species- and virus-specific differences in interferon-mediated antiviral activity and establish a screening platform for avian ISG functional studies.
Interferon responses and the collection of interferon-stimulated genes (ISGs) induced by the host are major determinants of susceptibility to viral infection and to cross-species transmission events. While the human innate immune response to IAV has been extensively characterized, the avian interferon response and the specific ISGs that limit IAV replication in bird hosts are less well defined. The authors sought to catalog ISG induction across avian species and to use those data to functionally identify ISGs that reduce IAV replication in chicken cells.
The study reports ISG induction data from chicken, quail, and duck cells and notes that the set of genes induced by interferon is largely species-specific. This divergence in ISG repertoires among bird species underscores that antiviral defenses and the molecular effectors used by different avian hosts can vary substantially, which may influence host range and the outcome of viral infection.
Using the avian ISG expression data, the authors designed a pooled CRISPR knockout screen targeting ISGs to identify genes that when disrupted result in increased replication of influenza A virus in chicken cells. Two viral strains were included to capture potential differences in restriction depending on virus lineage and adaptation: PR8, a mouse-adapted laboratory strain, and WF10, a low pathogenicity avian IAV isolate.
The screen against PR8 identified IFITM3 as the dominant antiviral factor restricting replication in the chicken cell system. Several additional ISGs were identified with modest effects on PR8 replication; the magnitude and number of these secondary hits were consistent with previous observations from mammalian systems in which IFITM3 plays a central role in restricting certain IAV strains.
When screened against the avian WF10 isolate, IFITM3 was again among the genes whose knockout enhanced viral replication. In addition to IFITM3, two other genes were identified as hits specifically for WF10: the HSP70 co-chaperone BAG5 and the deubiquitinase OTUD4. These WF10-specific hits were not detected in the PR8 screen, indicating strain-dependent differences in which ISGs are functionally restrictive.
The combined findings reinforce that interferon-mediated antiviral responses in birds are divergent between species and that the set of ISGs that effectively restrict influenza replication can differ depending on the virus strain. The prominence of IFITM3 in restricting both PR8 and WF10 aligns with its known antiviral role, while the identification of BAG5 and OTUD4 for the avian isolate highlights additional cellular pathways—chaperone/co-chaperone and ubiquitin processing—that may contribute to avian-specific antiviral activity.
The authors present a functional screening system that pairs avian ISG expression data with pooled CRISPR knockout to identify genes that limit IAV replication in avian cells. This approach can be applied to different avian species and viral strains to map species- and virus-specific antiviral effectors. Such knowledge may inform understanding of host range barriers and contribute to efforts to predict or mitigate spillover risk.
This report is a preprint and has not been peer reviewed. Details beyond what is presented in the manuscript (for example, quantitative screen metrics, validation experiments, or broader datasets) were not reported in the source abstract. Funding sources declared include the National Heart Lung and Blood Institute and the National Institute of Allergy and Infectious Diseases. The authors declared no competing interests.