Receptor-Binding Assay for Influenza Viruses

Receptor-binding property is a key determinant of the mammalian adaption of the influenza virus (Gao et al., 2009 (link); Zhang et al., 2012 (link)). Hemagglutination assays using resialylated chicken red blood cells (cRBCs) and sheep red blood cells (sRBCs) were performed to analyze the receptor-binding property of the CIVs. The surface of cRBCs expresses both human-like α2,6 and avian-like α2,3 sialic acid receptors (Ito et al., 1997 (link)), while sRBCs only contain avian-like α2,3 sialic acid receptors (Medeiros et al., 2001 (link)). The α2,3 sialic acids were removed from the cRBCs' surface by incubating with α2,3-sialidase (Takara, Dalian, Liaoning, China). The desialylation cRBCs were generated by incubating with Vibrio cholerae neuraminidase (VCNA, Roche, San Francisco, CA, United States). The desialylation cRBCs were used as the negative control. The A/Sichuan/1/2009(H1N1) and A/chicken/Hebei/3/2013(H5N2), which bind to α2,6-cRBCs and α2,3-cRBCs, respectively (Shi et al., 2017 (link)), were used as controls.