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An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein
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 th...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161859/ https://www.ncbi.nlm.nih.gov/pubmed/25183201 http://dx.doi.org/10.1186/1743-422X-11-159 |
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author | Rash, Adam Woodward, Alana Bryant, Neil McCauley, John Elton, Debra |
author_facet | Rash, Adam Woodward, Alana Bryant, Neil McCauley, John Elton, Debra |
author_sort | Rash, Adam |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1743-422X-11-159) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4161859 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-41618592014-09-13 An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein Rash, Adam Woodward, Alana Bryant, Neil McCauley, John Elton, Debra Virol J Methodology 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1743-422X-11-159) contains supplementary material, which is available to authorized users. BioMed Central 2014-09-02 /pmc/articles/PMC4161859/ /pubmed/25183201 http://dx.doi.org/10.1186/1743-422X-11-159 Text en © Rash et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Rash, Adam Woodward, Alana Bryant, Neil McCauley, John Elton, Debra An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title | An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title_full | An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title_fullStr | An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title_full_unstemmed | An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title_short | An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein |
title_sort | efficient genome sequencing method for equine influenza [h3n8] virus reveals a new polymorphism in the pa-x protein |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161859/ https://www.ncbi.nlm.nih.gov/pubmed/25183201 http://dx.doi.org/10.1186/1743-422X-11-159 |
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