H5Ny highly pathogenic avian influenza viruses (HPAIV) cause significant mortality in birds and have led to fatal cases in humans and other mammals, though sustained transmission in mammals is rare, except in seals and sea lions. This changed in March 2024, when clade 2.3.4.4b H5N1 HPAIV was detected in dairy cattle in the United States, and the virus spread to other animals and humans. As of May 09, 2025, 1,053 dairy herds across 17 states have been affected (https://www.cdc.gov/bird-flu/situation-summary/mammals.html) and 41/70 human cases of bird flu were linked to exposure to dairy cattle since 2024 (https://www.cdc.gov/bird-flu/situation-summary/index.html). While no fatal human infections from clade 2.3.4.4b bovine H5N1 HPAIV have occurred, concerns remain that the virus could mutate, increasing its infectivity and pathogenicity in humans.

Here, we studied host cell entry of bovine H5N1 HPAIV and investigated whether seasonal influenza virus vaccines can induce cross-neutralizing antibodies. In the absence of a virus isolate, we employed pseudoviruses bearing influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA) proteins, since pseudoviruses are a frequently used surrogate system for the analysis of IAV neutralization. Further, neutralization titers obtained from pseudovirus particles bearing IAV HA and NA have been demonstrated to correlate well with neutralization titers obtained by hemagglutination inhibition and microneutralization assays assays carried out with authentic virus.