Abstract
BackgroundH3N8 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.MethodsTo 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.ResultsTwo 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1743-422X-11-159) contains supplementary material, which is available to authorized users.
Highlights
H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963
These methods can be used to determine the genome sequences of EIV, including the non-coding regions (NCRs), from both clade 1 and clade 2 of the Florida sublineage
Genome sequencing of equine influenza viruses At the time of writing, only 81 full genome sets were available from the NCBI Influenza Virus Resource for EIV and only one or two gene segment sequences had been published for the majority of strains
Summary
H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. Equine influenza virus (EIV) is an influenza A virus belonging to the Orthomyxoviridae family These viruses have a negative sense, single-stranded RNA genome consisting of eight viral gene segments [1]. During the 1980s the virus diverged into two antigenically distinct lineages [3], American and Eurasian, and since the American lineage has further evolved into the Florida sublineage clades 1 and 2, which continue to cocirculate today [4]. These lineages have historically been based on antigenic and genetic data for HA. Less than 100 complete viral genomes covering the 46 years from 1963 to 2008 were available at the time of the study
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