Functional analysis of HA variants. (A) Hemadsorption assay. Protein expressing HF cells were incubated with a suspension of hen red blood cells and hemadsorption was observed under light microscope. Non-expressing cells and dilution buffer were used as controls. (B) Hemagglutination. Lysates of cells expressing proteins were serially diluted and incubated with erythrocytes as described in Materials and Methods. Hemagglutination is shown by a uniform pink suspension of erythrocytes, while the lack of hemagglutination is accompanied by appearance of a red dot in the middle of the well, corresponding to sedimented erythrocytes. Non-expressing HF cells and dillution buffer were used as controls. Arrows indicate dilutions where specific hemagglutination was observed.