Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding

Abstract

The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.

FIG. 1.

Weight loss and virus shedding in ferrets and mice inoculated with NL602-WT, NL602-D222E, or NL602-D222G. Data are shown for ferrets (A to C) and mice (D to F) inoculated with NL602-WT (▪), NL602-D222E (⧫), or NL602-D222G (▴). Body weights are depicted as the percentage of body weight at time of inoculation. Body weight is shown as average for six ferrets until 3 dpi and three ferrets from 4 to 7 dpi (A) or for five to six mice (D). Virus detection in throat swabs (B) and nose swabs (C) in ferrets are indicated for NL602-WT (▪), NL602-D222E (□), and NL602-D222G (░⃞). Geometric mean titers for positive samples are displayed, and error bars indicate the standard deviations. The lower limit of detection is indicated by the dotted line. The proportion of surviving mice is shown for each group (E). Virus titers in the lungs of inoculated mice (n = 3) were determined at 3 and 5 dpi, with the geometric mean titers and standard deviations indicated (F). Symbols and colors are consistent in panels A to F.

FIG. 2.

Attachment of NL602-WT and NL602-D222G viruses to avian and human tissues. The attachment patterns to cells in mallard duck colon (A), human upper respiratory tract (B), and human lower respiratory tract (C to E) are shown. The following tissues were analyzed: colon (A1 and A2), surface epithelium (B1 and B2) and submucosal glands (B3 and B4) of nasal turbinates, trachea (C1 and C2); bronchus (C3 and C4), bronchiole (C5 and C6), alveolus (C7 and C8), tracheal submucosal glands (D1 and D2), and alveolar macrophages (E1 and E2). Virus attachment is shown in red. Odd and even numbers show staining with NL602-WT and NL602-D222G, respectively. The panels were chosen to represent attachment patterns in the whole tissue section as much as possible, but small differences between the single panels and overall view may exist.

FIG. 3.

Representative structures for binding mode 1 (cyan), mode 2 (orange), and intermediate mode (magenta) for 1918 (A) and NL602 (B). All of the structures from the molecular dynamics simulations were classified as mode 1, mode 2, or intermediate based upon the distance between the backbone atoms of residue 224 and the O2 and O3 oxygen atoms of the galactose sugar. These atoms are shown as spheres. Interactions between α2,6-SA and the side chain of D222 are shown as dashes. Normalized frequency distributions of these distances are shown for the 1918-WT (C) and NL602-WT (D) strains as solid lines, with the D222G mutants shown as dashed lines. For these plots, the frequency was sampled every 0.1 Å. The positions of the representative structures shown in panels A and B are highlighted in these distributions by squares of the corresponding color.

FIG. 4.

Set of hydrogen bond interactions within the HA receptor binding site for 1918 (A), 1918-D222G (B), NL602 (C), and NL602-D222G (D). The frequency with which each hydrogen bond is observed in all structures from the molecular dynamics simulations is shown as a percentage and by the depth of color of the connecting lines.