FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Virology journal2014; 11; 159; doi: 10.1186/1743-422X-11-159

An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein.

Abstract: 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.
Get Full Text
Publication Date: 2014-09-02 PubMed ID: 25183201PubMed Central: PMC4161859DOI: 10.1186/1743-422X-11-159Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research presents an efficient and cost-effective method for genome sequencing of Equine Influenza Virus (EIV), and indicates the discovery of a new polymorphism in the PA-X protein of some virus isolates.

Introduction and Methodology

This study was designed to unravel the full genome sequence of the equine influenza virus (EIV), a rampant disease among horses globally. The researchers note a lack of extensive sequence data for this virus compared to its counterparts in humans, swine, and birds. The newly developed method involves the use of M13-tagged primers to amplify relatively short and overlapping RT-PCR products, which are then analysed using Sanger dideoxynucleotide sequencing technology.

  • The researchers modified an existing method, previously used for human H3N2 and avian H5N1 influenza viruses, to sequence the non-coding parts of the EIV genome; an essential feature in determining the overall action and reproduction patterns of the virus in the host.
  • New primers were created for the N8 neuraminidase segment to expedite genome sequencing.

Results

Two virus samples were successfully sequenced with this method: A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07. These samples represented clades 1 and 2 of the Florida sub-lineage respectively.

  • By sequencing 26 PCR products varying in length from 400 to 600 nucleotides, the researchers covered the full coding sequences of the eight segments of the virus genome with sufficient overlap for sequence assembly without primer-derived sequences.
  • Sequences of the non-coding regions revealed a cytosine at nucleotide 4 in the polymerase segments.
  • In some isolates, the researchers discovered a novel polymorphism in the PA-X protein, an important find which could potentially inform better strategies for disease control and management.

Conclusion

This research methodology proves a salient tool for genome sequencing of EIV and could potentially be replicated on other virus genomes. The method is both cost-effective and efficient, requiring fewer PCR products than previously reported techniques. Embracing this approach could lead to an increase in the number of full EIV genomes available for study, which in turn might result in an improved understanding of the virus evolution and inform better strategies for disease surveillance and management.

Cite This Article

APA
Rash A, Woodward A, Bryant N, McCauley J, Elton D. (2014). An efficient genome sequencing method for equine influenza [H3N8] virus reveals a new polymorphism in the PA-X protein. Virol J, 11, 159. https://doi.org/10.1186/1743-422X-11-159

Publication

Virology journal
ISSN: 1743-422X
NlmUniqueID: 101231645
Country: England
Language: English
Volume: 11
Pages: 159
PII: 159

Researcher Affiliations

Rash, Adam
  • Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK. adam.rash@aht.org.uk.
Woodward, Alana
    Bryant, Neil
      McCauley, John
        Elton, Debra

          MeSH Terms

          • Animals
          • DNA, Complementary
          • Genome, Viral
          • Horse Diseases / virology
          • Horses
          • Influenza A Virus, H3N8 Subtype / genetics
          • Orthomyxoviridae Infections / veterinary
          • Orthomyxoviridae Infections / virology
          • Polymerase Chain Reaction
          • Polymorphism, Genetic
          • RNA, Untranslated / genetics
          • RNA, Viral

          Grant Funding

          • MC_U117512723 / Medical Research Council
          • MC_U117585868 / Medical Research Council

          References

          This article includes 28 references
          1. McGeoch D, Fellner P, Newton C. Influenza virus genome consists of eight distinct RNA species.. Proc Natl Acad Sci U S A 1976 Sep;73(9):3045-9.
            doi: 10.1073/pnas.73.9.3045pmc: PMC430922pubmed: 1067600google scholar: lookup
          2. WADDELL GH, TEIGLAND MB, SIGEL MM. A NEW INFLUENZA VIRUS ASSOCIATED WITH EQUINE RESPIRATORY DISEASE.. J Am Vet Med Assoc 1963 Sep 15;143:587-90.
            pubmed: 14077956
          3. Daly JM, Lai AC, Binns MM, Chambers TM, Barrandeguy M, Mumford JA. Antigenic and genetic evolution of equine H3N8 influenza A viruses.. J Gen Virol 1996 Apr;77 ( Pt 4):661-71.
            doi: 10.1099/0022-1317-77-4-661pubmed: 8627254google scholar: lookup
          4. Bryant NA, Rash AS, Russell CA, Ross J, Cooke A, Bowman S, MacRae S, Lewis NS, Paillot R, Zanoni R, Meier H, Griffiths LA, Daly JM, Tiwari A, Chambers TM, Newton JR, Elton DM. Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007.. Vet Microbiol 2009 Jul 2;138(1-2):41-52.
            doi: 10.1016/j.vetmic.2009.03.004pubmed: 19346084google scholar: lookup
          5. Murcia PR, Wood JL, Holmes EC. Genome-scale evolution and phylodynamics of equine H3N8 influenza A virus.. J Virol 2011 Jun;85(11):5312-22.
            doi: 10.1128/JVI.02619-10pmc: PMC3094979pubmed: 21430049google scholar: lookup
          6. Gorman OT, Donis RO, Kawaoka Y, Webster RG. Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus.. J Virol 1990 Oct;64(10):4893-902.
            pmc: PMC247979pubmed: 2398532doi: 10.1128/jvi.64.10.4893-4902.1990google scholar: lookup
          7. Ito T, Gorman OT, Kawaoka Y, Bean WJ, Webster RG. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins.. J Virol 1991 Oct;65(10):5491-8.
            pmc: PMC249043pubmed: 1895397doi: 10.1128/jvi.65.10.5491-5498.1991google scholar: lookup
          8. Gorman OT, Bean WJ, Kawaoka Y, Webster RG. Evolution of the nucleoprotein gene of influenza A virus.. J Virol 1990 Apr;64(4):1487-97.
            pmc: PMC249282pubmed: 2319644doi: 10.1128/jvi.64.4.1487-1497.1990google scholar: lookup
          9. Kawaoka Y, Krauss S, Webster RG. Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.. J Virol 1989 Nov;63(11):4603-8.
            pmc: PMC251093pubmed: 2795713doi: 10.1128/jvi.63.11.4603-4608.1989google scholar: lookup
          10. Okazaki K, Kawaoka Y, Webster RG. Evolutionary pathways of the PA genes of influenza A viruses.. Virology 1989 Oct;172(2):601-8.
            doi: 10.1016/0042-6822(89)90202-Xpubmed: 2800339google scholar: lookup
          11. Nakajima K, Nobusawa E, Ogawa T, Nakajima S. Evolution of the NS genes of the influenza A viruses. I. The genetic relatedness of the NS genes of animal influenza viruses.. Virus Genes 1990 Jun;4(1):5-13.
            doi: 10.1007/BF00308561pubmed: 2144066google scholar: lookup
          12. Worobey M, Han GZ, Rambaut A. A synchronized global sweep of the internal genes of modern avian influenza virus.. Nature 2014 Apr 10;508(7495):254-7.
            doi: 10.1038/nature13016pmc: PMC4098125pubmed: 24531761google scholar: lookup
          13. Livesay GJ, O'Neill T, Hannant D, Yadav MP, Mumford JA. The outbreak of equine influenza (H3N8) in the United Kingdom in 1989: diagnostic use of an antigen capture ELISA.. Vet Rec 1993 Nov 20;133(21):515-9.
            doi: 10.1136/vr.133.21.515pubmed: 8310627google scholar: lookup
          14. Binns MM, Daly JM, Chirnside ED, Mumford JA, Wood JM, Richards CM, Daniels RS. Genetic and antigenic analysis of an equine influenza H 3 isolate from the 1989 epidemic.. Arch Virol 1993;130(1-2):33-43.
            doi: 10.1007/BF01318994pubmed: 8503788google scholar: lookup
          15. Adeyefa CA, Quayle K, McCauley JW. A rapid method for the analysis of influenza virus genes: application to the reassortment of equine influenza virus genes.. Virus Res 1994 Jun;32(3):391-9.
            doi: 10.1016/0168-1702(94)90087-6pubmed: 7521550google scholar: lookup
          16. Bean WJ. Correlation of influenza A virus nucleoprotein genes with host species.. Virology 1984 Mar;133(2):438-42.
            doi: 10.1016/0042-6822(84)90410-0pubmed: 6710867google scholar: lookup
          17. Dong BB, Xu CL, Dong LB, Cheng HJ, Yang L, Zou SM, Chen M, Bai T, Zhang Y, Gao RB, Li XD, Shi JH, Yuan H, Yang J, Chen T, Zhu Y, Xiong Y, Yang S, Shu YL. A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic duck farm in Poyang Lake area.. Biomed Environ Sci 2013 Jul;26(7):546-51.
            pubmed: 23895699doi: 10.3967/0895-3988.2013.07.005google scholar: lookup
          18. Skehel JJ, Hay AJ. Nucleotide sequences at the 5' termini of influenza virus RNAs and their transcripts.. Nucleic Acids Res 1978 Apr;5(4):1207-19.
            doi: 10.1093/nar/5.4.1207pmc: PMC342071pubmed: 652519google scholar: lookup
          19. de Wit E, Spronken MI, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments.. Virus Res 2004 Jul;103(1-2):155-61.
            doi: 10.1016/j.virusres.2004.02.028pubmed: 15163504google scholar: lookup
          20. de Wit E, Bestebroer TM, Spronken MI, Rimmelzwaan GF, Osterhaus AD, Fouchier RA. Rapid sequencing of the non-coding regions of influenza A virus.. J Virol Methods 2007 Jan;139(1):85-9.
            doi: 10.1016/j.jviromet.2006.09.015pubmed: 17059848google scholar: lookup
          21. . Sequencing primers and protocol. .
          22. GISAID EpiFlu database [http://platform.gisaid.org]
          23. Lee KH, Seong BL. The position 4 nucleotide at the 3' end of the influenza virus neuraminidase vRNA is involved in temporal regulation of transcription and replication of neuraminidase RNAs and affects the repertoire of influenza virus surface antigens.. J Gen Virol 1998 Aug;79 ( Pt 8):1923-34.
            pubmed: 9714240doi: 10.1099/0022-1317-79-8-1923google scholar: lookup
          24. Jagger BW, Wise HM, Kash JC, Walters KA, Wills NM, Xiao YL, Dunfee RL, Schwartzman LM, Ozinsky A, Bell GL, Dalton RM, Lo A, Efstathiou S, Atkins JF, Firth AE, Taubenberger JK, Digard P. An overlapping protein-coding region in influenza A virus segment 3 modulates the host response.. Science 2012 Jul 13;337(6091):199-204.
            doi: 10.1126/science.1222213pmc: PMC3552242pubmed: 22745253google scholar: lookup
          25. Shi M, Jagger BW, Wise HM, Digard P, Holmes EC, Taubenberger JK. Evolutionary conservation of the PA-X open reading frame in segment 3 of influenza A virus.. J Virol 2012 Nov;86(22):12411-3.
            doi: 10.1128/JVI.01677-12pmc: PMC3486490pubmed: 22951836google scholar: lookup
          26. Robertson JS. 5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus.. Nucleic Acids Res 1979 Aug 24;6(12):3745-57.
            doi: 10.1093/nar/6.12.3745pmc: PMC327975pubmed: 493121google scholar: lookup
          27. Lee HK, Tang JW, Kong DH, Koay ES. Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus.. PLoS One 2013;8(5):e64785.
            doi: 10.1371/journal.pone.0064785pmc: PMC3669369pubmed: 23741393google scholar: lookup
          28. Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR. Universal primer set for the full-length amplification of all influenza A viruses.. Arch Virol 2001 Dec;146(12):2275-89.
            doi: 10.1007/s007050170002pubmed: 11811679google scholar: lookup

          Citations

          This article has been cited 16 times.
          1. Rozario C, Martínez-Sobrido L, McSorley HJ, Chauché C. Could Interleukin-33 (IL-33) Govern the Outcome of an Equine Influenza Virus Infection? Learning from Other Species. Viruses 2021 Dec 15;13(12).
            doi: 10.3390/v13122519pubmed: 34960788google scholar: lookup
          2. Alnaeem A, Shawaf T, Ali AM, Hemida MG. Clinical observations and molecular detection of Type-A influenza virus in some of the family Equidae in eastern Saudi Arabia winter-2019. Vet Res Commun 2021 Dec;45(4):423-430.
            doi: 10.1007/s11259-021-09822-2pubmed: 34435308google scholar: lookup
          3. Fougerolle S, Fortier C, Legrand L, Jourdan M, Marcillaud-Pitel C, Pronost S, Paillot R. Success and Limitation of Equine Influenza Vaccination: The First Incursion in a Decade of a Florida Clade 1 Equine Influenza Virus that Shakes Protection Despite High Vaccine Coverage. Vaccines (Basel) 2019 Nov 2;7(4).
            doi: 10.3390/vaccines7040174pubmed: 31684097google scholar: lookup
          4. Gahan J, Garvey M, Asmah Abd Samad R, Cullinane A. Whole Genome Sequencing of the First H3N8 Equine Influenza Virus Identified in Malaysia. Pathogens 2019 May 10;8(2).
            doi: 10.3390/pathogens8020062pubmed: 31083430google scholar: lookup
          5. Toh X, Soh ML, Ng MK, Yap SC, Harith N, Fernandez CJ, Huangfu T. Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015. Transbound Emerg Dis 2019 Sep;66(5):1884-1893.
            doi: 10.1111/tbed.13218pubmed: 31059176google scholar: lookup
          6. Gildea S, Garvey M, Lyons P, Lyons R, Gahan J, Walsh C, Cullinane A. Multifocal Equine Influenza Outbreak with Vaccination Breakdown in Thoroughbred Racehorses. Pathogens 2018 Apr 17;7(2).
            doi: 10.3390/pathogens7020043pubmed: 29673169google scholar: lookup
          7. Gahan J, Garvey M, Gildea S, Gür E, Kagankaya A, Cullinane A. Whole-genome sequencing and antigenic analysis of the first equine influenza virus identified in Turkey. Influenza Other Respir Viruses 2018 May;12(3):374-382.
            doi: 10.1111/irv.12485pubmed: 28940727google scholar: lookup
          8. Bera BC, Virmani N, Kumar N, Anand T, Pavulraj S, Rash A, Elton D, Rash N, Bhatia S, Sood R, Singh RK, Tripathi BN. Genetic and codon usage bias analyses of polymerase genes of equine influenza virus and its relation to evolution. BMC Genomics 2017 Aug 23;18(1):652.
            doi: 10.1186/s12864-017-4063-1pubmed: 28830350google scholar: lookup
          9. Rash A, Morton R, Woodward A, Maes O, McCauley J, Bryant N, Elton D. Evolution and Divergence of H3N8 Equine Influenza Viruses Circulating in the United Kingdom from 2013 to 2015. Pathogens 2017 Feb 8;6(1).
            doi: 10.3390/pathogens6010006pubmed: 28208721google scholar: lookup
          10. Lee J, Yu H, Li Y, Ma J, Lang Y, Duff M, Henningson J, Liu Q, Li Y, Nagy A, Bawa B, Li Z, Tong G, Richt JA, Ma W. Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses. Virology 2017 Apr;504:25-35.
            doi: 10.1016/j.virol.2017.01.015pubmed: 28142079google scholar: lookup
          11. Alves Beuttemmüller E, Woodward A, Rash A, Dos Santos Ferraz LE, Fernandes Alfieri A, Alfieri AA, Elton D. Characterisation of the epidemic strain of H3N8 equine influenza virus responsible for outbreaks in South America in 2012. Virol J 2016 Mar 19;13:45.
            doi: 10.1186/s12985-016-0503-9pubmed: 26993620google scholar: lookup
          12. Peacock T, Reddy K, James J, Adamiak B, Barclay W, Shelton H, Iqbal M. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape. Sci Rep 2016 Jan 7;6:18745.
            doi: 10.1038/srep18745pubmed: 26738561google scholar: lookup
          13. Bavagnoli L, Cucuzza S, Campanini G, Rovida F, Paolucci S, Baldanti F, Maga G. The novel influenza A virus protein PA-X and its naturally deleted variant show different enzymatic properties in comparison to the viral endonuclease PA. Nucleic Acids Res 2015 Oct 30;43(19):9405-17.
            doi: 10.1093/nar/gkv926pubmed: 26384413google scholar: lookup
          14. Kleij L, Bruder E, Raoux-Barbot D, Lejal N, Nevers Q, Deloizy C, Da Costa B, Legrand L, Barrey E, Chenal A, Pronost S, Delmas B, Dhorne-Pollet S. Genomic characterization of equine influenza A subtype H3N8 viruses by long read sequencing and functional analyses of the PB1-F2 virulence factor of A/equine/Paris/1/2018. Vet Res 2024 Mar 22;55(1):36.
            doi: 10.1186/s13567-024-01289-8pubmed: 38520035google scholar: lookup
          15. Pinto RM, Lycett S, Gaunt E, Digard P. Accessory Gene Products of Influenza A Virus. Cold Spring Harb Perspect Med 2021 Dec 1;11(12).
            doi: 10.1101/cshperspect.a038380pubmed: 32988983google scholar: lookup
          16. Chambers TM. Equine Influenza. Cold Spring Harb Perspect Med 2022 Jan 4;12(1).
            doi: 10.1101/cshperspect.a038331pubmed: 32152243google scholar: lookup
          Subscribe to our newsletter
          Join 99,780 horse owners like you who receive our email updates on horse health and nutrition.