The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties

Abstract

In 1997, 18 confirmed cases of human influenza arising from multiple independent transmissions of H5N1 viruses from infected chickens were reported from Hong Kong. To identify possible phenotypic changes in the hemagglutinin (HA) and neuraminidase (NA) of the H5 viruses during interspecies transfer, we compared the receptor-binding properties and NA activities of the human and chicken H5N1 isolates from Hong Kong and of H5N3 and H5N1 viruses from wild aquatic birds. All H5N1 viruses, including the human isolate bound to Sia2-3Gal-containing receptors but not to Sia2-6Gal-containing receptors. This finding formally demonstrates for the first time that receptor specificity of avian influenza viruses may not restrict initial avian-to-human transmission. The H5N1 chicken viruses differed from H5 viruses of wild aquatic birds by a 19-amino-acid deletion in the stalk of the NA and the presence of a carbohydrate at the globular head of the HA. We found that a deletion in the NA decreased its ability to release the virus from cells, whereas carbohydrate at the HA head decreased the affinity of the virus for cell receptors. Comparison of amino acid sequences from GenBank of the HAs and NAs from different avian species revealed that additional glycosylation of the HA and a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses. This finding indicates that changes in both HA and NA may be required for the adaptation of influenza viruses from wild aquatic birds to domestic chickens and raises the possibility that chickens may be a possible intermediate host in zoonotic transmission.

FIG. 1

Analysis of glycosylation sites on the HA1 globular head of H5 and H7 influenza viruses. Columns: A, Phylogenetic relationships based on the HA1 nucleotide sequences of the virus strains; B, NA subtype; C, GenBank accession number; D, number of basic amino acids at the HA cleavage site, if there were more than three such amino acids at this site; E, positions of glycosylation sequons by H3 numbering (see Fig. 2 for the location of sequons on the three-dimensional model of the HA). The asterisks next to the GenBank accession number mark the H5 HA sequences determined in this study.

FIG. 1

Analysis of glycosylation sites on the HA1 globular head of H5 and H7 influenza viruses. Columns: A, Phylogenetic relationships based on the HA1 nucleotide sequences of the virus strains; B, NA subtype; C, GenBank accession number; D, number of basic amino acids at the HA cleavage site, if there were more than three such amino acids at this site; E, positions of glycosylation sequons by H3 numbering (see Fig. 2 for the location of sequons on the three-dimensional model of the HA). The asterisks next to the GenBank accession number mark the H5 HA sequences determined in this study.

FIG. 2

Positions of glycosylation sites on the upper part of the HA1 subunit (amino acid residues 90 to 260) of avian influenza viruses shown on a model of the complex between X31 virus HA and 3′-SL (1HGG structure; Brookhaven Protein Databank [37]). The HA monomers are depicted in shades of gray. Positions corresponding to the asparagine residues in the N-linked glycosylation triplets are shown in black and numbered. The sialyllactose molecules are shown as stick models. The figure was generated with WebLab ViewerPro 3.1 (Molecular Simulations, Inc., San Diego, Calif.).

FIG. 3

Amino acid sequences of the stalk region of N1 and N2 influenza virus NAs (top) and a phylogenetic tree for some of the N1 NAs (bottom). The positions of deletions are shown by dashes; potential glycosylation sites are shaded. Only a partial sequence was available for the NA stalk region of the three human H1N1 strains marked with an asterisk (∗). Numbers at the tree nodes show bootstrap values (100 replications); the horizontal lengths of the branches are proportional to these values.