Strain-specific responses of avian influenza virus to disruption of solute carrier family 35 member A1 (SLC35A1) in chicken cells

Authors: Liu, Heidi; CRUVINEL, JESSICA - University Of Missouri; WARREN, WESLEY - University Of Missouri; MA, WENJUN - University Of Missouri; Chen, Paula

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/24/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Outbreaks of avian influenza virus are of great concern to the poultry industry, resulting in major production and economic losses. The virus continues to overwhelm traditional control strategies, such as improved hygiene in poultry facilities; therefore, alternatives are needed to reduce or prevent outbreaks. Genome editing has the potential to result in a chicken with complete resistance to avian influenza virus. In the present study, we disrupted the function of a gene in chicken cells that is predicted to permit avian influenza virus infection. The genome edited cells were protected against infection by different avian influenza viruses, but the degree of protection depended on the viral strain. Thus, strain differences is another factor that needs to be considered when evaluating genome editing targets.

Technical Abstract: Avian influenza virus (AIV) poses a persistent threat to poultry health and food security, with conventional control measures offering limited protection. A promising alternative is the use of gene editing to generate host resistance by ablating viral entry receptors or cellular proteins that are required for completion of the viral life cycle. The solute carrier family 35 member A1 (SLC35A1) gene encodes a CMP-sialic acid transporter essential for the sialylation of cell surface glycoproteins. In this study, we used CRISPR/Cas9 to disrupt SLC35A1 in chicken DF-1 fibroblasts and evaluated the effect on sialic acid expression and susceptibility different strains of AIV. Lectin staining and flow cytometry confirmed a significant reduction in a2,3-linked sialic acids in SLC35A1 knockout cells, while a2,6-linked sialic acids were absent in the cells regardless of genotype. Infection experiments with three avian influenza virus strains (H5N1 PR8, H5N2, and H7N1) revealed that SLC35A1 knockout reduced viral replication in a strain-dependent manner. Knockout cells infected with H5N1 PR8 showed the greatest dependence on SLC35A1-mediated sialylation with decreased viral load at 24 hours post-infection (hpi) and 48 hpi compared to wildtype cells and no observable viral growth between the timepoints. Infection of knockout cells with H5N2 resulted in a more modest decrease in viral load at both timepoints as well as absence of viral growth. On the other hand, infection of knockout cells with H7N1 resulted in decreased viral load only at 48 hpi compared to wildtype cells, but the amount of virus in knockout cultures increased from 24 hpi to 48 hpi. These results demonstrate that SLC35A1 is a key host factor that supports AIV entry via a2,3-linked sialic acids; however, viral dependency on this host factor may be confounded by strain.