Conserved deletion of two consecutive glycine residues in the 100K protein is a critical determinant of FAdV-11 pathogenicity

Fowl adenovirus serotype 11 (FAdV-11) causes inclusion body hepatitis, posing a persistent threat to poultry health. However, the genetic determinants governing its pathogenicity remain unclear. Comparative analysis of pathogenic and nonpathogenic FAdV-11 strains revealed that nonpathogenic strains exhibit a conserved deletion of two consecutive glycine residues (Δ2G) in the glycine-rich domain of the 100K protein. This deletion is associated with a corresponding two-glutamic-acid deletion (Δ2E) in the glutamic acid-rich domain of the 33K protein. Here, we investigated the functional significance of two consecutive glycine residues in the virulence of FAdV-11 using the reverse genetics system of a pathogenic FAdV-11 strain. A recombinant mutant, rFAdV-11-100K-Δ2G, was generated with a seamless deletion of 2G in 100K. The in vitro replication capacity of rFAdV-11-100K-Δ2G was significantly attenuated in LMH cells, with an 8-fold reduction in peak titer versus the wild-type FAdV-11. Mechanistically, co-immunoprecipitation revealed a 1.83-fold decrease in 100K-Δ2G-Hexon binding (p < 0.001), while confocal microscopy showed impaired cytoplasmic colocalization of 100K-Δ2G and Hexon (p < 0.01). Electron microscopy results showed that the assembly of the recombinant virus rFAdV-11-100K-Δ2G was significantly inhibited in LMH cells. The concurrent Δ2E in 33K led to suppressed late gene expression. In vivo, the mutant caused only 10% mortality in SPF chickens versus 100% for the wild-type, with attenuated pathology and viral load. These results identify the two glycine residues as critical virulence determinants of FAdV-11, essential for both viral assembly and late gene regulation, thereby providing potential targets for developing attenuated vaccines.