Amino acid 226 in the hemagglutinin of H4N6 influenza virus determines binding affinity for alpha2,6-linked sialic acid and infectivity levels in primary swine and human respiratory epithelial cells

Abstract

Avian lineage H4N6 influenza viruses previously isolated from pigs differ at hemagglutinin amino acids 226 and 228 from H4 subtype viruses isolated from birds. Using a parental H4N6 swine isolate and hemagglutinin mutant viruses (at residues 226 and/or 228), we determined that viruses which contain L226 had a higher affinity for sialic acid alpha2,6 galactose (SAalpha2,6Gal) and a higher infectivity level for primary swine and human respiratory epithelial cells, whereas viruses which contain Q226 had lower SAalpha2,6Gal affinity and lower infectivity levels for both types of cells. Using specific neuraminidases, we found that irrespective of their relative binding preferences, all of the influenza viruses examined utilized SAalpha2,6Gal to infect swine and human cells.

FIG. 1.

Infectivity levels of the H4N6 influenza viruses in SRECs and HRECs. Cells were infected with three TCID₅₀s/cell for each virus, and infected cells were identified by immunocytochemistry (ICC) using the anti-influenza A NP antibody 68D2 (kindly provided by Y. Kawaoka, University of Wisconsin-Madison). Following ICC staining, the brightness and contrast of HREC micrographs were adjusted with ACDSee Photo Editor (ACD Systems) and PowerPoint (Microsoft) software to match that of the SREC micrographs. Similar patterns were observed in repeated experiments and with cells from different pig and human donors. Magnification, ×60.

FIG. 2.

Lectin staining and H4N6 influenza virus infectivity after NA depletion of cell surface sialic acid are shown. Removal of sialic acids from the surfaces of SRECs (A) and HRECs (B). Cells were treated with either an α2,3-specific NA or an α2,3/α2,6 NA, followed by cell surface lectin staining with MAA (SAα2,3Gal) or SNA (SAα2,6Gal). Values shown are the geometric means of fluorescent intensity (± standard errors of the means [SEM]) normalized such that the fluorescence intensity of untreated cells is 100% and the fluorescence intensity of mock lectin-treated cells is 0%. Data are the results of four separate experiments performed in duplicate. Virus infectivity levels after NA treatment of SRECs (C) and HRECs (D) are shown. Data are the results of four separate experiments with each virus assayed in triplicate for each experiment. Results are the means ± SEM. Statistically significant differences were observed between the infectivity levels of untreated and α2,3/α2,6 NA-treated cells (analysis of variance-protected Student's t tests; *, P < 0.05; **, P < 0.01).