Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus

Abstract

Equine influenza viruses (EIVs) of the H3N8 and H7N7 subtypes are the causative agents of an important disease of horses. While EIV H7N7 apparently is extinct, H3N8 viruses have circulated for more than 50 years. Like human influenza viruses, EIV H3N8 caused a transcontinental pandemic followed by further outbreaks and epidemics, even in populations with high vaccination coverage. Recently, EIV H3N8 jumped the species barrier to infect dogs. Despite its importance as an agent of infectious disease, the mechanisms that underpin the evolutionary and epidemiological dynamics of EIV are poorly understood, particularly at a genomic scale. To determine the evolutionary history and phylodynamics of EIV H3N8, we conducted an extensive analysis of 82 complete viral genomes sampled during a 45-year span. We show that both intra- and intersubtype reassortment have played a major role in the evolution of EIV, and we suggest that intrasubtype reassortment resulted in enhanced virulence while heterosubtypic reassortment contributed to the extinction of EIV H7N7. We also show that EIV evolves at a slower rate than other influenza viruses, even though it seems to be subject to similar immune selection pressures. However, a relatively high rate of amino acid replacement is observed in the polymerase acidic (PA) segment, with some evidence for adaptive evolution. Most notably, an analysis of viral population dynamics provided evidence for a major population bottleneck of EIV H3N8 during the 1980s, which we suggest resulted from changes in herd immunity due to an increase in vaccination coverage.

Fig. 1.

(A) Maximum-likelihood (ML) phylogenetic tree for the HA segment of EIV H3N8. Colored boxes represent distinct clades of EIV that are numbered I to X in chronological fashion, apart from Florida clades 1 and 2 (FC1 and FC2). Bootstrap (>70%) values are shown in black. Numbers in black boxes along the main trunk branch indicate the number of amino acid changes. Individual isolates involved in reassortment events are indicated with colored asterisks (see the text). Internal branches with d_N > d_S are shown in red. Horizontal branches are drawn to a scale of nucleotide substitutions per site, and the tree is rooted on the EIV Uruguay isolate from 1963. The same scale, color scheme, and rooting are used for each panel. (B) ML phylogeny of the NA gene of EIV H3N8. (C) ML phylogeny of the PA gene of EIV H3N8.

Fig. 1.

(A) Maximum-likelihood (ML) phylogenetic tree for the HA segment of EIV H3N8. Colored boxes represent distinct clades of EIV that are numbered I to X in chronological fashion, apart from Florida clades 1 and 2 (FC1 and FC2). Bootstrap (>70%) values are shown in black. Numbers in black boxes along the main trunk branch indicate the number of amino acid changes. Individual isolates involved in reassortment events are indicated with colored asterisks (see the text). Internal branches with d_N > d_S are shown in red. Horizontal branches are drawn to a scale of nucleotide substitutions per site, and the tree is rooted on the EIV Uruguay isolate from 1963. The same scale, color scheme, and rooting are used for each panel. (B) ML phylogeny of the NA gene of EIV H3N8. (C) ML phylogeny of the PA gene of EIV H3N8.

Fig. 1.

(A) Maximum-likelihood (ML) phylogenetic tree for the HA segment of EIV H3N8. Colored boxes represent distinct clades of EIV that are numbered I to X in chronological fashion, apart from Florida clades 1 and 2 (FC1 and FC2). Bootstrap (>70%) values are shown in black. Numbers in black boxes along the main trunk branch indicate the number of amino acid changes. Individual isolates involved in reassortment events are indicated with colored asterisks (see the text). Internal branches with d_N > d_S are shown in red. Horizontal branches are drawn to a scale of nucleotide substitutions per site, and the tree is rooted on the EIV Uruguay isolate from 1963. The same scale, color scheme, and rooting are used for each panel. (B) ML phylogeny of the NA gene of EIV H3N8. (C) ML phylogeny of the PA gene of EIV H3N8.

Fig. 2.

Maximum clade credibility (MCC) trees showing the contrast in phylogenetic relationships between EIV H3N8 and H7N7 among gene segments. H3N8 and H7N7 are shown as orange and blue circles, respectively. Posterior probability values are shown for the key nodes relating to the position of EIV H7N7 reassortant sequences. Branches exhibiting reassortant viruses are highlighted. The trees are automatically rooted through the assumption of a relaxed molecular clock, and tip times represent times (years) of sampling.

Fig. 3.

Population dynamics of EIV. Bayesian skyline plots showing the changing levels of relative genetic diversity through time (N_et) for all gene segments of EIV H3N8. Mean values of N_et are given by the boldface line, while the gray lines denote the 95% HPD values. Note the genome-wide drop in genetic diversity during the 1980s. The approximate dates on which vaccination was made compulsory for competition horses in some countries are indicated with a vertical arrow (see the text).