Receptor binding assay
Receptor binding assay was performed as previously described (Chambers,Parkhouse,Ross,Alby, & Hensley, 2015). Briefly, the chicken erythrocytes were pretreated with different amounts of α2-3,6,8 neuraminidase (New England Biolabs, Beverly, MA, USA) for 1 h at 37 °C. The chicken erythrocytes were washed with PBS and added (as 1% (v /v) solutions) to 4 HAU of each virus (as determined using nontreated chicken erythrocytes). Agglutination was measured after incubation for 1 hour. Virus with higher receptor binding avidity is able to bind to chicken erythrocytes that are treated with high amounts of α2-3,6,8 neuraminidases.