title: An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein.
abstract: 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.
authors.affiliation: Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK. adam.rash@aht.org.uk.
authors.forename: Adam
authors.initials: A
authors.lastname: Rash
authors.forename: Alana
authors.initials: A
authors.lastname: Woodward
authors.forename: Neil
authors.initials: N
authors.lastname: Bryant
authors.forename: John
authors.initials: J
authors.lastname: McCauley
authors.forename: Debra
authors.initials: D
authors.lastname: Elton
mesh_terms: D000818:Animals; D018076:DNA, Complementary; D016679:Genome, Viral; D006734:Horse Diseases / Q000821:virology*; D006736:Horses; D053123:Influenza A Virus, H3N8 Subtype / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000662:veterinary* / Q000821:virology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D022661:RNA, Untranslated / Q000235:genetics; D012367:RNA, Viral
chemical_list: D018076:DNA, Complementary; D022661:RNA, Untranslated; D012367:RNA, Viral