In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity

Abstract

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.

Fig. 1

Solid phase receptor binding assay of H3N2 and H5N1 variant viruses compared to wildtype. Binding of sialylglycopolymers 3′SLN-PAA (blue line) and 6′SLN-PAA (red lines) by influenza H3N2 and H5N1 viruses performed as described in Materials and methods section. The x axis denotes the concentration of biotinylated sialoglycan (μM) added to the wells, the y axis indicates the resulting absorbance reading. Data shown are representative from three independent binding experiments.

Fig. 2

Glycan microarray analysis of H5N1 variant viruses and including dual mutations from previous pandemic viruses. Different types of sialoglycans on the array (x-axis) are indicated by colors; the structure of each numbered glycan is provided in Table S1. Vertical bars denote mean binding signal (fluorescence intensity) and T bar extensions indicate the standard error. Glycan microarray analysis of H5N1 variant viruses and including dual mutations from previous pandemic viruses. Different types of sialoglycans on the array (x-axis) are indicated by colors; the structure of each numbered glycan is provided in Table S1. Vertical bars denote mean binding signal (fluorescence intensity) and T bar extensions indicate the standard error.

Fig. 3

Solid phase receptor binding assay of H5N1 variant with dual mutations close to the receptor binding site. Binding of sialylglycopolymers 3′SLN-PAA (blue line) and 6′SLN-PAA (red lines) by influenza H5N1 variants was described in Materials and methods section. The x and y axis are as in Fig. 1. Data shown are representative from three independent binding experiments.

Fig. 4

Glycan microarray analysis of clade 2 H5N1 viruses with dual mutations close to the receptor binding site. The color conventions and glycan key for the charts are described in Fig. 2.