Vibrio cholerae neuraminidase vcna

The receptor binding affinity of H13 AIVs was evaluated by its ability to bind to treated chicken erythrocytes or sheep erythrocytes by the HA assays. The sheep erythrocytes have only α-2,3 glycans on the surface. When the chicken erythrocytes were treated with α-2,3-N-sialidase (Takara, Dalian, China), only α-2,6 glycans remained in the treated chicken erythrocytes. The chicken erythrocytes were treated with Vibrio cholerae neuraminidase (VCNA, Roche, San Francisco, CA, USA) to destroy the receptors. The F076, S201, DZ137, and ZH385 strains were diluted in the hemagglutinin titre of 2⁷. We used these erythrocytes in a 0.5% RBCs suspension to evaluate the receptor binding specificity.

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.

The A549, HBE, and HEp-2 cells were treated with ST6GAL1 or control siRNAs and cultured for 72 h. Cells were washed with phosphate-buffered saline (PBS) and fixed in 3.7% formaldehyde in PBS for 30 min. For detection of sialic acid residues on the surface of cells, apical monolayers were blocked with 3% bovine serum albumin (BSA; Merck, Darmstadt, Germany) in PBS for 30 min and then incubated with 5 μg/mL fluorescein isothiocyanate (FITC)-conjugated Sambucus nigra lectin (SNA; Vector Laboratories, Burlingame, CA, USA) for 1 h. To confirm the specificity of lectin binding, monolayers were treated with 50 mU Vibrio cholerae neuraminidase (VCNA; Roche, Almere, Netherlands) for 1 h prior to fixation and then examined with a rapid-scanning confocal laser microscope (Nikon Corp, Tokyo, Japan).