Equine Influenza Virus in Asia: Phylogeographic Pattern and Molecular Features Reveal Circulation of an Autochthonous Lineage

Abstract

Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

Keywords: Asia; H3N8; H7N7; equine influenza; evolution; influenza; signature pattern; vaccine.

FIG 1

Time-scaled phylogeny of EIV H3N8 genes. The analyses were performed under the Bayesian skyline plot (BSP) demographic model and a relaxed clock model (UCLN) calibrated with terminal nodes (time of sampling, in years). FC1 and FC2 lineages are colored in blue and red, respectively. Strain A/equine/Urumqi/1/2015 sequenced in this study is indicated with a diamond.

FIG 2

Population dynamics of EIV H3N8 genes. The effective numbers of infections through time (Ne_τ) obtained from the analysis of EIV H3N8 individual genes, under the UCLN-BSP models. Median values are denoted by the solid lines, while dotted lines denote the 95% highest posterior density (HPD) values.

FIG 3

Phylodinamics of FC1 and FC2. (A) Time-scaled phylogeny from the discrete phylogeographic analysis. Branches are colored according to the most probable location of the parental node of each lineage (color codes are shown at bottom left). Circles on nodes are sized according to the location posterior probability. The major Asian lineages discussed in the text are shown with brackets. (B) Temporal fluctuation of the effective numbers of infections through time (Ne_τ,; y axis). Solid line, median Ne_τ values; dotted lines, 95% HPD.

FIG 4

Epidemiologically linked locations. The FC1 circulation is plotted in blue and that for FC2 in red. The figure shows all the nonzero rates with significant epidemiological links (Bayes factor [BF] ≥ 20). Asian countries with HA1 sequences available and included in the analysis are highlighted in blue and red. The map data were obtained from the National Geomatics Center of China (NGCC) and compiled by using QGIS 3.4 (https://www.qgis.org).

FIG 5

HA1 characteristic sites in the Asian FC1 and FC2 lineages. Representation of the modeled equine HA trimer representing the viral population described in both Asian FC1 and FC2. The amino acids labeled in green correspond to those characteristic of FC1 (G7, A78, and S159) or FC2 (N7, B78, and N159). (A) FC1a, the characteristic amino acid in light blue; (B) FC1b, the characteristic amino acid in medium blue and the latest amino acid acquisitions in blue; (C) Asian FC2a, the characteristic amino acid substitution in orange-red; (D) Asian-FC2b, the characteristic amino acid substitution in orange; (E) Asian FC2c, the characteristic amino acid substitution in red; (F) European FC2, the inner view of the HA trimer is also showed to highlight the characteristic amino acid substitution (in pink).

FIG 6

HA1 hydrophobicity and electrostatic potential. Representation of the modeled equine HA trimers (Asian FC1 and FC2) showing the electrostatic potential of the surfaces. Positive charges are indicated by blue, and negative by red. Scale, ±10 kcal/mol·e.