Biological activities of the original HNs. The expression of each HN protein at the cell surface was determined by A IIFA and FCM B analysis at 24 h post-transfection of BHK-21 cells by using a primary HN monoclonal antibody and a fluorescein-labelled secondary antibody. Empty cells were used as mock cells, and empty pCAGGS was used as the negative control. C The HAd activity was determined based on the ability of HN expressed at the cell surface at 16 h post-transfection to adsorb chicken erythrocytes at 4 °C. D NA activity was determined as the ability of the cell surface HN proteins to catalyse the release of sialic acid at 16 h post-transfection. All marks indicate significance in comparison to HN-CE16 (100%), and the results are presented as the mean ± SD of the results of three independent experiments. *p < 0.05, **p < 0.01.