Enzyme‑linked immunosorbent assay (ELISA)
ELISA assay was performed as we described previously (Peng et al., 2019). Sucrose gradient-purified viruses were diluted in PBS and added to Nunc-Immuno MaxiSop 96-well plates (Corning, NY, USA) at 16 HAU per well. After incubation overnight at 4°C, samples in wells were blocked with PBS-nonfat dry milk. Antisera against the F/98 virus, the rF/HAS145N virus or the rF/HAA198V virus in chickens were then added in serial twofold dilutions with PBS containing 0.05% Tween 20, respectively, and incubated for 3 h at 37°C. After washing, goat anti chicken horseradish peroxidase antibody (Abcam, Cambridge, MA) was added and allowed to incubate for 1.5 h at 37 °C. After washing, TMB (3,3′,5,5′ Tetramethylbenzidine) (Sigma, St. Louis, MO, USA) substrate was added, and the reaction was stopped by adding H2SO4. Absorbance was recorded at 450 nm using an automated ELISA plate reader (model EL311SX; Biotek, Winooski, VT). The area under curve (AUC) of either virus was assessed for virus-Abs binding with GraphPad Prism 8 software (San Diego, CA) above that of the corresponding negative control.