Molecular evolution of H3N8 equine influenza virus in Argentina

Abstract

Since 1963, Equine influenza virus (EIV) H3N8 subtype has been responsible for numerous outbreaks of respiratory disease in horses worldwide. The aim of this work was to study the molecular characteristics of H3N8 EIV detected in Argentina, estimate their diversification and infer the potential source of infections. Nucleotide and deduced amino acid (aa) sequences for the complete HA gene of the EIV detected in Argentina between 1985 and 2012 were analyzed along with sequences obtained from the Influenza Research database. Maximum Likelihood (ML) and Bayesian phylogenetic trees were built up. Bayesian coalescent analyses were carried out to estimate the time of the most recent common ancestors (tMRCA) and the dynamic population growth. The phylogenetic trees demonstrate that H3N8 EIV detected in Argentina grouped in 4 well-supported monophyletic clades: Group VIII (G.VIII), South American clade 1 (SA1) and 2 (SA2), and Florida clade 1 (FC1). The viruses circulating in Argentina in 1985 (n1⁄42), together with a Chilean strain, formed G.VIII (tMRCA~1984). The EIVs detected during the period 19931996 (n1⁄45) grouped in the SA1 (tMRCA~1992), composed only by Argentinian strains, and their most probable origin was a Kentucky 1992 strain. The strains circulating in the period 1997-2005 (n1⁄48) grouped in the SA2 (tMRCA~1997), formed a monophyletic group together with a 2006 Chilean strain, and were closely related to California 1997 and Kentucky 1994 strains, feasible ancestors of this group. The viruses detected in 2012 (n1⁄46) grouped within the FC1 (tMRCA~2011) and formed a cluster with the viruses detected in Florida in 2011. The demographic reconstruction showed a remarkable increase in viral diversity in 2006, a slight decrease between 2007 and 2009, and an abrupt decrease since 2009. Compared with A/eq/Fontainebleau/79 (pre-divergent prototype strain), Argentinean strains belonging to G.VIII have 9 aa substitutions: T46I, A93T, R140K, T182S, G222W, V223I, I267V, I187B and L199S, these last two situated at the antigenic site B. In comparison with A/eq/Newmarket/1/93 (American lineage reference strain), the SA 1 Argentinean viruses carried 4 aa substitutions: N132K, Q189N, Q190E and E193K, the last 3 at the antigenic site B, and the SA2 strains showed only one aa substitution at the antigenic site D (I214V). The possible ancestors for SA1 and SA2 also possess the same aa substitutions at the antigenic sites B and D, respectively. EIV Argentinean strains detected in 2012 belonging to FC1 contained 5 aa substitutions in comparison with A/eq/Ohio/01/03 (FC1 reference strain): G7D, R62K, D104N, A138S and V223I. In addition, two different subpopulations can be recognized among them, one with a substitution in K-14A and the other one in K-14T and M70V, this last (M70V) being present only in Argentinean strains. Except for K14A and M70V, the strain considered to be the origin of the group (A/eq/Florida/146609/11) has the same aa substitutions. The obtained results allow us to hypothesize that the outbreaks of equine influenza in Argentina were due to 4 introductions of virus, presumably from North America. The rapid increase in the relative genetic diversity in 2006 could be due to the co-circulation of different lineages, while the abrupt decline observed after 2009 could be related to a subsequent reduction in transmission of EIV H3N8 connected to the incorporation of Florida Clade 2 strains in vaccines.