Equine influenza virus (EIV) surveillance is important in the management of equine influenza. It provides data on circulating and newly emerging strains for vaccine strain selection. To this end, antigenic characterisation by haemaggluttination inhibition (HI) assay and phylogenetic analysis was carried out on 28 EIV strains isolated in North America and Europe during 2006 and 2007. In the UK, 20 viruses were isolated from 28 nasopharyngeal swabs that tested positive by enzyme-linked immunosorbent assay. All except two of the UK viruses were characterised as members of the Florida sublineage with similarity to A/eq/Newmarket/5/03 (clade 2). One isolate, A/eq/Cheshire/1/06, was characterised as an American lineage strain similar to viruses isolated up to 10 years earlier. A second isolate, A/eq/Lincolnshire/1/07 was characterised as a member of the Florida sublineage (clade 1) with similarity to A/eq/Wisconsin/03. Furthermore, A/eq/Lincolnshire/1/06 was a member of the Florida sublineage (clade 2) by haemagglutinin (HA) gene sequence, but appeared to be a member of the Eurasian lineage by the non-structural gene (NS) sequence suggesting that reassortment had occurred. A/eq/Switzerland/P112/07 was characterised as a member of the Eurasian lineage, the first time since 2005 that isolation of a virus from this lineage has been reported. Seven viruses from North America were classified as members of the Florida sublineage (clade 1), similar to A/eq/Wisconsin/03. In conclusion, a variety of antigenically distinct EIVs continue to circulate worldwide. Florida sublineage clade 1 viruses appear to predominate in North America, clade 2 viruses in Europe.

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic interrelationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida Clade 2, all those from the USA belonged to Florida Clade 1. Two subpopulations of Clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida Clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida Clade 1 and Clade 2. Revision note VETMIC-D-13-8580Authors' response to reviewer comments: We agree with the reviewer that there are two topics here. However, we also believe that they are closely linked and that the increase in diagnostic sample submission should be sufficient justification to include the strategies used to encourage practitioners to submit nasal swabs. In response, we have therefore deleted surveillance data from the results & discussion, but retained the approaches used in the method section, for reference purposes.The scheme only relates to equine influenza as the financial support we have from the HBLB only covers 'flu. This is considered the major threat to horse racing and breeding in the UK, due to its rapid spread in an unprotected population.The 'free' surveillance scheme allows us to characterise samples sent from other countries as well as the UK. We believe that for countries where funding could potentially be sought, this scheme provides examples of low-cost methods that can be used to improve surveillance & sample submission, such as Twitter. We fully accept that this isn't possible in all countries. The isolate characterization data is certainly of value in monitoring the changes in EIV. What is disappointing is that all of the work done so far does not seem able to predict when a new vaccine will be needed. The antigenic mapping and serology tests tell us that changes are occurring and where in the HA and NA, but little about the efficacy of the current vaccines. Field data still seems most reliable. On...

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Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

BackgroundAn extensive outbreak of equine influenza occurred across multiple countries in South America during 2012. The epidemic was first reported in Chile then spread to Brazil, Uruguay and Argentina, where both vaccinated and unvaccinated animals were affected. In Brazil, infections were widespread within 3months of the first reported cases. Affected horses included animals vaccinated with outdated vaccine antigens, but also with the OIE-recommended Florida clade 1 strain South Africa/4/03.MethodsEquine influenza virus strains from infected horses were isolated in eggs, then a representative strain was subjected to full genome sequencing using segment-specific primers with M13 tags. Phylogenetic analyses of nucleotide sequences were completed using PhyML. Amino acid sequences of haemagglutinin and neuraminidase were compared against those of vaccine strains and recent isolates from America and Uruguay, substitutions were mapped onto 3D protein structures using PyMol. Antigenic analyses were completed by haemagglutination-inhibition assay using post-infection ferret sera.ResultsNucleotide sequences of the haemaglutinin (HA) and neuraminidase (NA) genes of Brazilian isolate A/equine/Rio Grande do Sul/2012 were very similar to those of viruses belonging to Florida clade 1 and clustered with contemporary isolates from the USA. Comparison of their amino acid sequences against the OIE-recommended Florida clade 1 vaccine strain A/equine/South Africa/4/03 revealed five amino acid substitutions in HA and seven in NA. Changes in HA included one within antigenic site A and one within the 220-loop of the sialic acid receptor binding site. However, antigenic analysis by haemagglutination inhibition (HI) assay with ferret antisera raised against representatives of European, Kentucky and Florida sublineages failed to indicate any obvious differences in antigenicity.ConclusionsAn extensive outbreak of equine influenza in South America during 2012 was caused by a virus belonging to Florida clade 1, closely related to strains circulating in the USA in 2011. Despite reports of vaccine breakdown with products containing the recommended strain South Africa/03, no evidence was found of significant antigenic drift. Other factors may have contributed to the rapid spread of this virus, including poor control of horse movement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0503-9) contains supplementary material, which is available to authorized users.