Alterations in hemagglutinin receptor-binding specificity accompany the emergence of highly pathogenic avian influenza viruses

Abstract

Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3'SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLe(c)) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLe(x)), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors.

Importance: Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.

FIG 1

Structure of sialyloligosaccharide moieties of biotinylated sialylglycopolymers and BODIPY-labeled neuraminidase substrates.

FIG 2

Avian influenza viruses used and evolutionary relationships of H5 and H7 subtype avian influenza viruses. A phylogenetic tree for amino acid sequences of the H5 and H7 HA1 protein was generated by the minimum-evolution method using MEGA software, version 5.2. The scale bar represents 0.01 or 0.005 units of amino acid per site for H5 or H7, respectively; black circles indicate LPAIVs. The tree is based on HA sequences presented in this study GenBank accession numbers are given (ns, not sequenced).

FIG 3

Receptor-binding specificity of avian influenza viruses. The data are presented as affinity constants of virus in complexes with sialyloligosaccharides (K_aff ± SD μM⁻¹ sialic acid). Viruses are grouped according to their subtypes and evolutionary relationships. The two circled LPAIVs were found to be exceptions as they revealed significantly higher binding affinities than other LPAIVs. Differences in the affinity constants (K_affs) between two groups of viruses, HPAIVs and LPAIVs, were statistically significant (P < 0.0001) and were determined using an unpaired two-tailed t test. See Fig. 2 for virus abbreviations.

FIG 4

Relative binding affinities of the viruses for 3′SLN versus SiaLe^x, SiaLe^c versus SiaLe^a, and 3′SLN versus SiaLe^c, determined K_aff(3′SLN)/K_aff(SiaLe^x), K_aff(SiaLe^c)/K_aff(SiaLe^a), and K_aff(3′SLN)/K_aff(SiaLe^c), respectively. Red indicates HPAIVs, and blue indicates LPAIVs. Differences in the ratio values of K_aff(3′SLN)/K_aff(SiaLe^x) between two groups of viruses, HPAIVs and LPAIVs, were statistically significant (P < 0.0001) and were determined using unpaired two-tailed t test. See Fig. 2 for virus abbreviations.

FIG 5

Partial amino acid sequences of H5 and H7 in viruses (H3 numbering). Positions described in the text are highlighted in yellow. The RBS is marked in gray. HPAIVs are marked with asterisks. Additional glycosylation sites are marked with circles. The figure was generated using BioEdit. See Fig. 2 for virus abbreviations.

FIG 6

Molecular models of the H5 HA of the influenza virus A/Vietnam/1194/2004 complexed with 3′SLN (PDB accession number 4BGY). Panels A and B show different views of the same model. Sialosides are shown in stick representation, with carbon in blue, oxygen in red, and nitrogen in violet. HA is shown in surface representation; amino acids in positions discussed in the text (H3 HA numbering) are shown in color.

FIG 7

NA substrate specificity of the avian influenza viruses was calculated for each sialoside as the slope of the linear region of the V₀-versus-S₀ kinetic curve (V₀, initial rate of the desialylation; S₀, initial substrate concentration). Substrate specificity of each virus NA was determined by comparison of V₀/S₀ values obtained for all substrates under the same conditions. Colors indicate the sialyloligosaccharide moiety of the SGP. Data are presented as indicated on the y axis, calculated for 1 HAU of virus. See Fig. 2 for virus abbreviations.

FIG 8

Profiles of NA substrate specificities of the avian influenza viruses as 3′SLN/SiaLe^x, 3′SLN/SiaLe^c, and SiaLe^c/SiaLe^a substrate specificity ratios. Red indicates HPAIVs, and blue indicates LPAIVs.