α2 3 n sialyltransferase

Affinity to avian α2,3 and human-like α2,6- sialic acid (SA) receptors was assessed using modified turkey erythrocytes (TRBCs) (Herfst et al., 2012 (link)). Briefly, SA was removed from TRBCs by incubation with Vibrio cholerae neuraminidase (Sigma-Aldrich, Germany) in the presence of calcium chloride (Herfst et al., 2012 (link)). After washing with PBS, desialylated TRBCs were suspended in PBS containing 1% BSA. Complete loss of hemagglutination of the TRBCs was confirmed by incubation with control viruses (i.e., PR8 and H4N2). Resialylation was done using α2,6-(N)-sialyltransferase (Takara, Germany) or α2,3-(N)-sialyltransferase (Sigma-Aldrich, Germany) in final concentrations of 1.5 mM Cytidine 5′-monophosphate (CMP)-sialic acid (Sigma-Aldrich, Germany). Modified TRBCs were suspended in PBS containing 1% bovine serum albumin to a final concentration of 0.5%. Resialylation was confirmed by hemagglutination of viruses using human PR8 with high affinity to α2,6-SA and H4N2 with high affinity to avian α2,3-SA receptors. HA test was done using the modified TRBCs, desialylated RBCs and original turkey RBCs (OIE, 2015 ). The assay was run in duplicates and repeated twice.

All sialic acids (SA) were removed from the surfaces of TRBCs by incubating 1% TRBCs in PBS with 50 mU of Vibrio cholerae NA (VCNA) (Roche) in 8 mM calcium chloride (CaCl₂) at 37°C for 1 h. After two washing steps with PBS, VCNA-treated TRBCs were resuspended in PBS containing 1% bovine serum albumin (BSA) to a final concentration of 0.5% TRBCs. Removal of SA was confirmed by observation of a complete loss of hemagglutination of the TRBCs by control influenza A viruses. Subsequently, resialylation was performed using 0.5 mU of α2,3-N-sialyltransferase (Sigma) or 25 mU of α2,6-N-sialyltransferase (Sigma) and 1.5 mM CMP-sialic acid (Merck) at 37°C for 2 h in a final volume of 75 μl to produce α2,3-TRBCs and α2,6-TRBCs, respectively. After two washing steps, the TRBCs were resuspended in PBS containing 1% BSA to a final concentration of 0.5% TRBCs. Resialylation was confirmed by hemagglutination of viruses with known receptor specificity: A/H5N1 IN/05 and A/H3N2 NL/13. The receptor specificity of A/H5N8 ck/NL/14 and A/H5N6 GZ/14 was tested by performing a standard hemagglutination assay with the modified TRBCs.

All sialic acid residues were enzymatically removed from cRBCs by incubation with the 50 mU Vibrio cholera neuraminidase (VCNA; Roche) in 8 mM calcium chloride at 37 °C for 1 h and then resialylated followed by using either α2,6-(N)-sialyltransferase or α2,3-(N)-sialyltransferase (Sigma) at 37 °C for 4 h³⁵. The receptor binding avidity of NC/02 or NC/02^HA149 virus were determined by performing standard HA assays using 0.5% modified cRBCs.