Intra- and interhost evolutionary dynamics of equine influenza virus

Abstract

Determining the evolutionary basis of cross-species transmission and immune evasion is key to understanding the mechanisms that control the emergence of either new viruses or novel antigenic variants with pandemic potential. The hemagglutinin glycoprotein of influenza A viruses is a critical host range determinant and a major target of neutralizing antibodies. Equine influenza virus (EIV) is a significant pathogen of the horse that causes periodical outbreaks of disease even in populations with high vaccination coverage. EIV has also jumped the species barrier and emerged as a novel respiratory pathogen in dogs, canine influenza virus. We studied the dynamics of equine influenza virus evolution in horses at the intrahost level and how this evolutionary process is affected by interhost transmission in a natural setting. To this end, we performed clonal sequencing of the hemagglutinin 1 gene derived from individual animals at different times postinfection. Our results show that despite the population consensus sequence remaining invariant, genetically distinct subpopulations persist during the course of infection and are also transmitted, with some variants likely to change antigenicity. We also detected a natural case of mixed infection in an animal infected during an outbreak of equine influenza, raising the possibility of reassortment between different strains of virus. In sum, our data suggest that transmission bottlenecks may not be as narrow as originally perceived and that the genetic diversity required to adapt to new host species may be partially present in the donor host and potentially transmitted to the recipient host.

FIG. 1.

Intrahost genetic variation of EIV in horses. (A) Layout of the transmission chain. Of the two horses experimentally inoculated with EIV, only one horse (7D36) was used to start the transmission chain (broken-line arrow). No virus was detected in nasal swabs from horse 282E (indicated with a stop sign). (B) Estimation of viral copy numbers from individual swabs by real-time PCR. (C) Number of clones with nucleotide (nt) and amino acid (aa) mutations. (D) Position and frequency of synonymous (red) and nonsynonymous (blue) mutations relative to the reference sequence (nucleotide level).

FIG. 2.

Intrahost EIV populations are constituted by a mixture of closely related genomes. Minimum spanning trees of horses 5D1A (A) and 6005 (B). Each tree was inferred by compiling sequences from multiple days. With the exception of singletons, mutations are indicated on respective branches. Synonymous mutations are underlined. The size of the circle is proportional to the number of sequences that exhibit each variant.

FIG. 3.

Unrooted maximum likelihood phylogenetic trees for HA1 segment clones from samples obtained from chain 1 (A) and chain 2 (B). Branch lengths are drawn to scale, with those displaying variants detected in more than one animal magnified. Colored circles represent different animals. Horizontal branch lengths are drawn to a scale of nucleotide substitutions per site. Bars = 0.001 substitution/site.

FIG. 4.

Maximum likelihood phylogenetic tree for HA1 segment clones from a natural case of EIV (sample OB151) compared with the global phylogeny of EIV H3N8. Colored boxes represent distinct EIV phylogenetic groups according to the World Organization for Animal Health (OIE) nomenclature of H3N8 strains. Clones from sample OB151 are represented in red boxes. Bootstrap values (>70%) are shown for key nodes relating to the phylogenetic position of those sequences sampled from horse OB151. Horizontal branches are drawn to a scale of nucleotide substitutions per site, and the tree is rooted on the EIV Miami isolate from 1963. Bar = 0.001 substitution/site.