Mutation of lysine 396 of the fusion protein reduces its binding affinity with integrin αVβ1 and leads to attenuation of avian metapneumovirus subtype B

family and cause the most acute respiratory diseases in poultry and humans, respectively. Recently, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the USA, largely attributed to aMPV/B infections. Live-attenuated vaccines developed by the blind passage of virulent strains in tissue culture have been widely used to prevent aMPV/B infection. However, the mechanism of aMPV/B attenuation remains unclear. Here, we identified the F gene as a key determinant of the virulence of aMPV/B and confirmed that residue 396 in the F protein plays an important role in attenuating the virulence of aMPV/B. Importantly, we found that the K396R mutation decreased the binding affinity between the F protein and αVβ1 and reduced the replication ability of aMPV/B. This is the first study to identify the key virulence genes and amino acid residues of aMPV/B and elucidate the molecular mechanisms underlying the attenuation of virulence. Our work provides fundamental insights into aMPV/B pathogenicity and offers direction for guiding the rational design of novel and more effective vaccines against aMPV/B and, by extension, related pathogens, such as hMPV.