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. 2014 May;88(9):4752-63.
doi: 10.1128/JVI.03805-13. Epub 2014 Feb 12.

Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Nicola S Lewis  1 , Tavis K Anderson, Pravina Kitikoon, Eugene Skepner, David F Burke, Amy L Vincent
Affiliations

Substitutions near the hemagglutinin receptor-binding site determine the antigenic evolution of influenza A H3N2 viruses in U.S. swine

Nicola S Lewis et al. J Virol. 2014 May.

Abstract

Swine influenza A virus is an endemic and economically important pathogen in pigs, with the potential to infect other host species. The hemagglutinin (HA) protein is the primary _target of protective immune responses and the major component in swine influenza A vaccines. However, as a result of antigenic drift, vaccine strains must be regularly updated to reflect currently circulating strains. Characterizing the cross-reactivity between strains in pigs and seasonal influenza virus strains in humans is also important in assessing the relative risk of interspecies transmission of viruses from one host population to the other. Hemagglutination inhibition (HI) assay data for swine and human H3N2 viruses were used with antigenic cartography to quantify the antigenic differences among H3N2 viruses isolated from pigs in the United States from 1998 to 2013 and the relative cross-reactivity between these viruses and current human seasonal influenza A virus strains. Two primary antigenic clusters were found circulating in the pig population, but with enough diversity within and between the clusters to suggest updates in vaccine strains are needed. We identified single amino acid substitutions that are likely responsible for antigenic differences between the two primary antigenic clusters and between each antigenic cluster and outliers. The antigenic distance between current seasonal influenza virus H3 strains in humans and those endemic in swine suggests that population immunity may not prevent the introduction of human viruses into pigs, and possibly vice versa, reinforcing the need to monitor and prepare for potential incursions.

Importance: Influenza A virus (IAV) is an important pathogen in pigs and humans. The hemagglutinin (HA) protein is the primary _target of protective immune responses and the major _target of vaccines. However, vaccine strains must be updated to reflect current strains. Characterizing the differences between seasonal IAV in humans and swine IAV is important in assessing the relative risk of interspecies transmission of viruses. We found two primary antigenic clusters of H3N2 in the U.S. pig population, with enough diversity to suggest updates in swine vaccine strains are needed. We identified changes in the HA protein that are likely responsible for these differences and that may be useful in predicting when vaccines need to be updated. The difference between human H3N2 viruses and those in swine is enough that population immunity is unlikely to prevent new introductions of human IAV into pigs or vice versa, reinforcing the need to monitor and prepare for potential introductions.

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Figures

FIG 1
FIG 1
3D antigenic maps of swine influenza A(H3N2) and human influenza A(H3N2) viruses from 1998 to 2013 and positions of key amino acids on the three-dimensional trimeric model of the hemagglutinin protein. (A) The relative positions of isolates (colored spheres) and antisera (open gray cubes) were computed so that the distances between isolates and antisera in the map correspond with the least error to measurements in the HI assay (26). The swine isolate color represents the antigenic cluster to which each isolate belongs, and the gray spheres represent recent human influenza A(H3N2) viruses. The large gray sphere is A/Victoria/361/2011. The scale bar represents 1 unit of antigenic distance, corresponding to a 2-fold dilution of antiserum in the HI assay. (B and C) Antigenic maps with only swine influenza A(H3N2) viruses showing the antigenic effects of the amino acid substitutions for each antigenic variant that was not located within the red (B) or the cyan (C) antigenic clusters. The arrows radiate from the consensus in each cluster to the outlying antigen, and the numeric values show the number of antigenic units (AU) separating the outlier from the antigens representing the consensus. (D) A trimeric structure of A/swine/Illinois/A01241469/2012 (red antigenic cluster) was generated to demonstrate the location of the antigenic cluster-differentiating amino acid positions. The receptor-binding site is colored in tan. An α2,6 glycan (LSTc) is shown as sticks docked in the binding site. The six amino acid positions associated with antigenic outliers are colored red. The images were produced using PyMOL (33).
FIG 2
FIG 2
Maximum-likelihood phylogenies (A and B) and genetic maps (C and D) of representative H3N2 swine influenza A virus isolates using HA1 domain amino acid sequences. The numbers above or below branches in the phylogenetic trees indicate bootstrap support (%) estimated from 1,000 resamplings of the sequence data; bootstrap values of ≤50% are not shown. H3N2 HA sublineages are indicated on the right (clusters I, II, II, and IV-A/B/C/D/E/F). Taxon names indicate viral isolates, followed by GenBank or GISAID EpiFlu accession identifiers in parentheses. The branches are colored by genetic cluster (A) and antigenic cluster (B); the branches in light gray were not part of the antigenic study. The scale bars in the phylogenies indicate the numbers of amino acid substitutions per site. The genetic maps were based on pairwise differences among strains, and the spheres representing virus strains are colored by genetic cluster (C) or antigenic cluster (D). The scale bars in the genetic maps correspond to 5 amino acid substitutions.
FIG 3
FIG 3
Maximum-likelihood phylogeny of NA gene amino acid sequences from viruses in the antigenic study and representative H3N2 swine influenza A isolates. The branches are colored by HA genetic cluster; the branches in light gray were not part of the study. The numbers above or below branches in the phylogenetic trees indicate bootstrap support (%) estimated from 1,000 resamplings of the sequence data; bootstrap values of ≤50% are not shown. H3N2 NA sublineages are indicated on the right (1998 versus 2002). Taxon names indicate viral isolates, followed by GenBank or GISAID EpiFlu accession identifiers in parentheses. The scale bar in the phylogeny indicates amino acid substitutions.
FIG 4
FIG 4
Patristic distance from A/Wuhan/359/95 in the maximum-likelihood phylogenetic tree presented in Fig. S1 in the supplemental material to each isolate in the cluster IV H3N2 swine influenza A virus clade plotted as a function of time. The solid line represents the regression for the 3 years prior to 2009, with a slope of 0.003 (x intercept = −7.84 ± 1.31 SE; adjusted R2 = 0.30: P < 0.0001), whereas the dashed line represents the regression for the isolates from 2010 to the present, with a slope of 0.005 (x intercept = −10.40 ± 0.68 SE; adjusted R2 = 0.31: P value < 0.0001).
FIG 5
FIG 5
Antigenic distances from putative cluster I (A) and cluster IV (B) swine vaccine sera and antigenic distances from the human seasonal vaccine strain A/Victoria/361/2011 swine sera (C) to circulating strains in pigs by year.
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