Avian metapneumovirus subtype B (aMPV/B) infections significantly affect the global poultry industry. However, the virulence determinants and attenuation mechanism remain unknown. Here, a series of chimeric and mutant viruses was constructed, and their pathogenicity was evaluated in a specific-pathogen-free (SPF) chicken model. First, substitutions in different genes (N, P, F, SH, G, and L) and amino acid sites (323rd, 396th, and 522nd residues in F protein) in virulent (LN16-V) and attenuated (LN16-A) viruses revealed that the residue 396th in F protein is closely related to replication ability in vitro and in vivo and is a critical determinant of aMPV/B virulence in chickens. Further studies revealed that the K396R mutation in the F protein decreases the adsorption of aMPV/B to DF-1 cells, which reduces the binding of the F protein with αVβ1 integrin. Structural and surface plasmon resonance analysis indicated that the K396R mutation primarily reduced the electrostatic potential of the RDD motif of the F protein that binds with αVβ1 and decreased the binding between the F protein and αVβ1, which is a critical determinant of the replication ability of aMPV/B. Collectively, these findings not only contribute to a better understanding of the aMPV attenuation mechanism but also offer novel strategies for developing Metapneumovirus members live vaccines.IMPORTANCEBoth aMPV and hMPV belong to the Metapneumovirus 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.

Keywords: attenuation mechanism; avian metapneumovirus subtype B; binding; virulence; αVβ1 integrin.