UC Riverside

Highly Pathogenic Influenza A Virus (HPAI) H5N1 strains have been reported in the United States, circulating in cattle farms and ultimately causing a spillover from cattle to humans. The emergence of this new strain has heightened public health concerns about its pathogenicity, transmissibility, and interactions with the innate immune system in humans. IAV employs multiple strategies to evade the host's innate immune system, including HA-mediated degradation of Type I Interferon Receptors during active infection. This study aims to compare and investigate whether the HA protein of the current circulating HPAI H5N1 IAV enhances IFN-1 receptor degradation in A549 cells and, if so, what mechanisms are being utilized to promote receptor degradation and enhanced viral pathogenicity. Protein sequence alignments of Texas (A/Texas/37/2024(H5N1), Washington (A/Washington/2148/2025(H5N5) H5 HA proteins, and California (A/California/07/2009(H1N1) H1 HA protein will be done to compare the conserved and divergent sequence regions of the IAV strains. In addition, the replication kinetics of HALo (H5N1) and NL09 (H1N1) IAV were conducted in A549 cells when exposed to IFN-β at different time points and concentrations using plaque assay analysis. Lastly, FLAG-tagged HA proteins will be transfected and used to compare their IFNAR1 degradation using Western blot analysis. The data suggest that the protein sequence alignments between the two H5 HA proteins showed highly conserved regions, while comparing both H5 HA proteins to the H1 HA protein demonstrated divergence and potential differences in biochemical functions and pathogenicity. As for the HALo (H5N1) and NL09 (H1N1) IAV, the replication kinetics experiments demonstrate distinct replication patterns between HALo and NL09; however, no statistically significant difference was seen following IFN-β at different time points and concentrations. Lastly, HA transfections were successful in 293T cells; however, due to nonspecific binding in the Western blot analysis, further investigation of IFNAR1 degradation was not explored. Collectively, while the findings are preliminary and require further exploration, this work serves as a foundation for future experiments and can help investigate the mechanisms underlying HA-induced IFNAR1 degradation and overall viral pathogenesis of the currently circulating HPAI H5N1 IAV.