Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Barnard, Karen N.; Wasik, Brian R.; LaClair, Justin R.; Buchholz, David W.; Weichert, Wendy S.; Alford-Lawrence, Brynn K.; Aguilar, Hector C.; Parrish, Colin R.

Mouse erythrocyte hemagglutination and lectin staining. Mouse erythrocytes were collected from C57B/6 mice by euthanizing the mice and immediately collecting blood by heart puncture into Alsever's solution. Erythrocytes were washed in PBS three times and diluted to 5% (vol/vol) in PBS. Then, 5% of the mouse erythrocytes were left untreated or treated with 30 g/ml BCoV HE-Fc for 18 h at 37°C, followed by dilution to 0.75% (vol/vol) in PBS for the HA assays. Briefly, HA assays were performed by 2-fold serially diluting virus in duplicate per treatment in a V-bottom 96-well plate. Treated or untreated mouse erythrocytes were added to virus and allowed to hemagglutinate at 4°C to prevent NA cleavage. To determine Sia linkage type, 5% washed erythrocytes were blocked for 1 h using 1ϫ Carbo Free blocking buffer (Vector Laboratories) and then stained for 1 h with fluoroscein-labeled plant lectins SNA, which binds ␣2,6-linked Sia, and MAA I, which binds ␣2,3-linked Sia (Vector Laboratories). Stained erythrocytes were then analyzed using flow cytometry as described above.