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Background: Equine influenza is a major health problem of equines worldwide. The polymerase genes of influenza virus have key roles in virus replication, transcription, transmission between hosts and pathogenesis. Hence, the comprehensive genetic and codon usage bias of polymerase genes of equine influenza virus (EIV) were analyzed to elucidate the genetic and evolutionary relationships in a novel perspective. Results: The group - specific consensus amino acid substitutions were identified in all polymerase genes of EIVs that led to divergence of EIVs into various clades. The consistent amino acid changes were also detected in the Florida clade 2 EIVs circulating in Europe and Asia since 2007. To study the codon usage patterns, a total of 281,324 codons of polymerase genes of EIV H3N8 isolates from 1963 to 2015 were systemically analyzed. The polymerase genes of EIVs exhibit a weak codon usage bias. The ENc-GC3s and Neutrality plots indicated that natural selection is the major influencing factor of codon usage bias, and that the impact of mutation pressure is comparatively minor. The methods for estimating host imposed translation pressure suggested that the polymerase acidic (PA) gene seems to be under less translational pressure compared to polymerase basic 1 (PB1) and polymerase basic 2 (PB2) genes. The multivariate statistical analysis of polymerase genes divided EIVs into four evolutionary diverged clusters - Pre-divergent, Eurasian, Florida sub-lineage 1 and 2. Conclusions: Various lineage specific amino acid substitutions observed in all polymerase genes of EIVs and especially, clade 2 EIVs underwent major variations which led to the emergence of a phylogenetically distinct group of EIVs originating from Richmond/1/07. The codon usage bias was low in all the polymerase genes of EIVs that was influenced by the multiple factors such as the nucleotide compositions, mutation pressure, aromaticity and hydropathicity. However, natural selection was the major influencing factor in defining the codon usage patterns and evolution of polymerase genes of EIVs. [ABSTRACT FROM AUTHOR]

Equine influenza viruses (EIV) are a major cause of acute respiratory disease in horses worldwide and occasionally also affect vaccinated animals. Like other influenza A viruses, they undergo antigenic drift, highlighting the importance of both surveillance and virus characterisation in order for vaccine strains to be kept up to date. The aim of the work reported here was to monitor the genetic and antigenic changes occurring in EIV circulating in the UK from 2013 to 2015 and to identify any evidence of vaccine breakdown in the field. Virus isolation, reverse transcription polymerase chain reaction (RT-PCR) and sequencing were performed on EIV-positive nasopharyngeal swab samples submitted to the Diagnostic Laboratory Services at the Animal Health Trust (AHT). Phylogenetic analyses were completed for the haemagglutinin-1 (HA1) and neuraminidase (NA) genes using PhyML and amino acid sequences compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 2 vaccine strain. Substitutions between the new isolates and the vaccine strain were mapped onto the three-dimensional structure protein structures using PyMol. Antigenic analyses were carried out by haemagglutination inhibition assay using a panel of post-infection ferret antisera. Sixty-nine outbreaks of equine influenza in the UK were reported by the AHT between January 2013 and December 2015. Forty-seven viruses were successfully isolated in eggs from 41 of the outbreaks. Only three cases of vaccine breakdown were identified and in each case the vaccine used contained a virus antigen not currently recommended for equine influenza vaccines. Nucleotide sequencing of the HA and NA genes revealed that all of the viruses belonged to the Florida clade 2 sub-lineage of H3N8 EIV. Phylogenetic and sequence analyses showed that the two sub-populations, previously identified within clade 2, continued to circulate and had accrued further amino acid substitutions. Antigenic characterisation using post-infection ferret antisera in haemagglutination inhibition assays however, failed to detect any marked antigenic differences between the isolates. These findings show that Florida clade 2 EIV continue to circulate in the UK and support the current OIE recommendation to include an example of Florida clade 2 in vaccines. [ABSTRACT FROM AUTHOR]

Background: An 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. Methods: Equine 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. Results: Nucleotide 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. Conclusions: An 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. [ABSTRACT FROM AUTHOR]

Background: H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA), one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and determine the level of reassortment occurring between sub-lineages. Methods: To facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the design of novel primers for an N8 neuraminidase segment. Results: Two field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07, representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length from 400-600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates. Conclusions: These methods can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR products than previously reported methods for influenza A viruses, the techniques used are affordable and the equipment required is available in most research laboratories. The adoption of these methods will hopefully allow for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better understanding of EIV evolution. [ABSTRACT FROM AUTHOR]

Abstract: 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 inter-relationships 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. [Copyright &y& Elsevier]

Abstract: Like other influenza A viruses, equine influenza virus undergoes antigenic drift. It is therefore essential that surveillance is carried out to ensure that recommended strains for inclusion in vaccines are kept up to date. Here we report antigenic and genetic characterisation carried out on equine influenza virus strains isolated in North America and Europe over a 2-year period from 2008 to 2009. Nasopharyngeal swabs were taken from equines showing acute clinical signs and submitted to diagnostic laboratories for testing and virus isolation in eggs. The sequence of the HA1 portion of the viral haemagglutinin was determined for each strain. Where possible, sequence was determined directly from swab material as well as from virus isolated in eggs. In Europe, 20 viruses were isolated from 15 sporadic outbreaks and 5 viruses were isolated from North America. All of the European and North American viruses were characterised as members of the Florida sublineage, with similarity to A/eq/Lincolnshire/1/07 (clade 1) or A/eq/Richmond/1/07 (clade 2). Antigenic characterisation by haemagglutination inhibition assay indicated that the two clades could be readily distinguished and there were also at least seven amino acid differences between them. The selection of vaccine strains for 2010 by the expert surveillance panel have taken these differences into account and it is now recommended that representatives of both Florida clade 1 and clade 2 are included in vaccines. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]