Sialic acid species as a determinant of the host range of influenza A viruses

Abstract

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

FIG. 1

Structure of N-acetyl and N-glycolylneuraminic acids. These SA differ at position 5 of the pyranose ring. N-Acetylneuraminic acid is the precursor of N-glycolyneuraminic acid; enzymatic hydroxylation of the former results in the latter.

FIG. 2

Predominance of the SAα2,3Gal linkage as detected by lectin staining in horse trachea. The MAA lectin specific for SAα2,3Gal (α2-3; detected with fluorescein isothiocyanate (FITC)-labeled anti-DIG antibody) bound to horse and pig tracheal epithelium, whereas SNA lectin specific for SAα2,6Gal (α2-6; detected with rhodamine-labeled anti-DIG antibody) bound only to the latter. Blue staining is a nonspecific reaction.

FIG. 3

Chromatograms of 1,2-diamino-4,5-methylenedioxybenzene derivatives of NeuAc and NeuGc obtained from the epithelial cells of horse trachea. The standard mixture of NeuAc and NeuGc and epithelial cells from horse trachea was treated as described in Materials and Methods. The fluorescence of SA derivatives was detected at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.

FIG. 4

Detection of NeuGcα2,3Gal moieties in epithelial cells lining horse trachea. Antiserum specific for NeuGcα2,3Gal but not for NeuAcα2,3Gal or NeuAcα2,6Gal (14, 15) detected this moiety in tracheal samples from horses (FITC staining) but not chickens, which lack NeuGc (8). Blue staining is a nonspecific reaction.

FIG. 5

Binding reactivity of influenza A viruses with gangliosides of human and equine influenza viruses. Gangliosides (1 μmol), represented by II⁶(NeuAc)LacCer (NeuAcα2,6Gal), II³(NeuAc)LacCer (NeuAcα2,3Gal), and II³(NeuGc)LacCer (NeuGcα2,3Gal), were developed in thin-layer chromatography plates. The plates were incubated with virus (2⁸ hemagglutination units at 4°C for 9 h and then processed as described in Materials and Methods. Relative binding reactivity is shown, with the highest activity set at 100%.