2.4 Solid-phase direct binding assay
Solid-phase glycan‐coated ELISA with the synthetic sialylglycopolymers Neu5Acα2-3Galb1-4GlcNAcb1 (3’SLN)-PAA-biotin and Neu5Acα2-6Galb1-
4GlcNAcb1 (6’SLN)-PAA-biotin (GlycoTech) was performed to evaluate the receptor binding properties of H7N9 AIV isolates as described previously (Li et al., 2014). An early poultry isolate A/mallard/Huadong/S/2005 [HD/05(H5N1)] with specificity for SAα‐2,3Gal, and a human isolate A/California/04/2009 [CA04(H1N1)] with specificity for SAα‐2,6Gal, were used as controls, respectively (Li et al., 2014; H. Sun et al., 2016).
2.5Thermal stability
In order to understand the thermal stability of H7N9 AIV isolates, virus-containing allantoic fluids were incubated at 56°C in a water bath for various time intervals (0, 5, 10, 15, 30, 60, 90, 120, 150, 180 and 210 min) according to a previously described protocol (Imai et al., 2012). Then, HA activity was analyzed by HA test against 1% chicken erythrocytes in duplicate, while infectivity of viruses was assessed by 50% tissue culture infective dose (TCID50) on MDCK cells (O’Donnell, Vogel, Matsuoka, Jin, & Subbarao, 2014).