Hemagglutinin‑inhibition (HI) assay and Microneutralization (MN) assay
Antisera were treated with cholera filtrate (Sigma-Aldrich, St. Louis, MO, USA) to remove nonspecific hemagglutination inhibitors before HI assay. HI assay was performed using 4 hemagglutination units (HAU) of H9N2 and 1% (v /v) chicken erythrocytes as we described previously (Peng et al., 2019).
MN assay was performed as previously described (H. Zhu et al., 2018). Briefly, the sera were serially diluted with 100 TCID50virus and incubated at 37 °C with 5% CO2 for 1 h. The serum-virus mixtures were added to MDCK cells and incubated for 1 h. After incubation, the serum-virus mixtures were removed. Serum-free DMEM containing 2 µg/mL TPCK‑ trypsin was added to each cell and incubated at 37°C and 5% CO2. After 72 h of incubation, culture supernatant was mixed with equal volume of 1% (v /v) chicken erythrocytes to confirm the existence of hemagglutination by virus. The MN titer was defined as the highest dilution of serum with absence of hemagglutination.