Fig. 2. Functional profile of SILV, SAEILV, and CSILV HAs.
(A) Hemagglutination assays were performed using recombinant HAs from SILV, SAEILV, and CSILV with turkey and chicken RBCs. HA proteins were added at 10 μg/ml and serially diluted twofold. (B to E) Glycan microarray analysis was used to assess binding of recombinant SILV, SAEILV, and CSILV HAs to synthetic glycans with α2,3- or α2,6-linked sialic acids (Neu5Ac), including linear, biantennary, triantennary, and SLex structures. B/Netherlands/2914/2015 HA was used as a positive control. Glycans A to M and O to W represent linear and biantennary structures, whereas glycans 1 to 22 represent triantennary N-glycans with LacNAc extensions. Glycans are color coded by terminal motif: yellow (no NeuAc), pink (α2,6-NeuAc), white (α2,3-NeuAc), blue (Lex), and black (SLex). Bars with dual colors indicate distinct epitopes on different arms. Data are shown as mean RFUs ± SD. (F) HA cleavage was assessed in HEK293T cells cotransfected with plasmids expressing full-length HAs and human airway proteases. After 48 hours, cleavage of HA0 to HA1 was evaluated by Western blot. Cells transfected with pCAGGS-HA, treated or untreated with TPCK-treated trypsin, served as controls. (G) Cell-cell fusion was assessed in HeLa cells expressing the respective HAs. Following trypsin treatment and low-pH exposure, polykaryon formation was evaluated. Representative images show fusion induced by SILV, SAEILV, CSILV, and B/Malaysia/2506/2004 HAs. White arrows indicate polykaryons, with insets showing magnified views of the fused cells. UT, untransfected.