FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / BMC Vet Res / Development of one-step TaqMan® real-time reverse trans...
BMC veterinary research2012; 8; 120; doi: 10.1186/1746-6148-8-120

Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses.

Abstract: Equine rhinitis viruses A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. Sero-surveillance studies have shown that these two viral infections are prevalent in many countries. Currently, the diagnosis of ERAV and ERBV infections in horses is mainly based on virus isolation (VI). However, the sensitivity of VI testing varies between laboratories due to inefficient viral growth in cell culture and lack of cytopathic effect. Therefore, the objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) to detect and distinguish ERAV from ERBV without the inherent problems traditionally associated with laboratory diagnosis of these infections. Results: Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, with the two remaining assays specific for ERBV. Additionally, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were developed. Both rRT-PCR and cRT-PCR assays were evaluated using RNA extracted from 21 archived tissue culture fluid (TCF) samples previously confirmed to be positive for ERAV (n = 11) or ERBV (n = 10) with mono-specific rabbit antisera. The ERAV rRT-PCR and cRT-PCR assays could only detect ERAV isolates and not ERBV isolates. Similarly, the ERBV rRT-PCR and cRT-PCR assays could only detect ERBV isolates and not ERAV isolates. None of the rRT-PCR or cRT-PCR assays cross-reacted with any of the other common equine respiratory viruses. With the exception of one cRT-PCR assay, the detection limit of all of these assays was 1 plaque forming unit per ml (pfu/ml). Conclusions: The newly developed rRT-PCR and cRT-PCR assays provide improved diagnostic capability for the detection and differentiation of ERAV and ERBV. However, a larger number of clinical specimens will need to be tested before each assay is adequately validated for the detection of ERAV and/or ERBV in suspect cases of either viral infection.
Get Full Text
Publication Date: 2012-07-25 PubMed ID: 22830930PubMed Central: PMC3542198DOI: 10.1186/1746-6148-8-120Google 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 paper discusses the development of molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) aimed at detecting and distinguishing between equine rhinitis viruses A and B (ERAV and ERBV), overcoming the challenges of traditional laboratory diagnosis methods for these infections.

Background and Aim

  • The research sets out to enhance the diagnosis of ERAV and ERBV infections in horses, which are prevalent in many countries and currently diagnosed chiefly via virus isolation (VI). This method experiences varying success due to differences in lab practice, incomplete viral growth in cell cultures, and lack of cytopathic effect.
  • The intent of the study was to create molecular diagnostic assays for detecting and identifying ERAV from ERBV, thereby resolving the issues traditionally associated with these infections’ laboratory diagnosis.

Methods and Experimental Design

  • Three rRT-PCR assays aimed at the 5′-UTR of ERAV and ERBV were developed. One was specific for ERAV, and the remaining two were ERBV-specific. Similarly, six cRT-PCR assays were designed targeting the 5′-UTR and 3D polymerase areas of both ERAV and ERBV.
  • These rRT-PCR and cRT-PCR assays were evaluated using RNA derived from 21 archived tissue culture fluid (TCF) samples, previously confirmed as positive for ERAV or ERBV with mono-specific rabbit antisera.

Results

  • The results revealed that the ERAV-specific rRT-PCR and cRT-PCR assays could only detect ERAV isolates, not ERBV. Similarly, ERBV-specific rRT-PCR and cRT-PCR assays could exclusively detect ERBV isolates and not ERAV.
  • Crucially, none of these assays cross-reacted with other prevalent equine respiratory viruses, assuring their specificity.
  • The detection limit for almost all these assays was established at 1 plaque forming unit per milliliter (pfu/ml).

Conclusions

  • The new rRT-PCR and cRT-PCR assays offer an improved diagnostic capability for the detection and differentiation of ERAV and ERBV. They promise to provide a more reliable alternative to the traditional virus isolation method, ensuring dependable detection of ERAV and ERBV in horses.’s respiratory infections.
  • However, a larger number of clinical specimens must be tested before these assays can be fully validated for identifying ERAV and/or ERBV in suspected cases of either viral infection.

Cite This Article

APA
Lu Z, Timoney PJ, White J, Balasuriya UB. (2012). Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription-PCR assays for the detection of equine rhinitis A and B viruses. BMC Vet Res, 8, 120. https://doi.org/10.1186/1746-6148-8-120

Publication

BMC veterinary research
ISSN: 1746-6148
NlmUniqueID: 101249759
Country: England
Language: English
Volume: 8
Pages: 120

Researcher Affiliations

Lu, Zhengchun
  • Maxwell H, Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, 108 Maxwell H, Lexington, KY 40546, USA.
Timoney, Peter J
    White, Jena
      Balasuriya, Udeni Br

        MeSH Terms

        • Animals
        • Aphthovirus / genetics
        • Aphthovirus / isolation & purification
        • Cell Line
        • Erbovirus / genetics
        • Erbovirus / isolation & purification
        • Rabbits
        • Real-Time Polymerase Chain Reaction / methods
        • Real-Time Polymerase Chain Reaction / veterinary
        • Reverse Transcriptase Polymerase Chain Reaction / methods
        • Reverse Transcriptase Polymerase Chain Reaction / veterinary
        • Sensitivity and Specificity
        • Serotyping

        References

        This article includes 39 references
        1. Stanway G, Brown F, Christian P. Family picornaviridae. Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses 2005; pp. 757–778.
        2. Steil BP, Barton DJ. Cis-active RNA elements (CREs) and picornavirus RNA replication.. Virus Res 2009 Feb;139(2):240-52.
          doi: 10.1016/j.virusres.2008.07.027pmc: PMC2692539pubmed: 18773930google scholar: lookup
        3. Paul AV. Possible unifying mechanism of picornavirus genome replication. Molecular Biology of Picornaviruses 2002; pp. 227–246.
        4. Kok CC, McMinn PC. Picornavirus RNA-dependent RNA polymerase.. Int J Biochem Cell Biol 2009 Mar;41(3):498-502.
          doi: 10.1016/j.biocel.2008.03.019pubmed: 18487072google scholar: lookup
        5. PLUMMER G. An equine respiratory virus with enterovirus properties.. Nature 1962 Aug 4;195:519-20.
          doi: 10.1038/195519a0pubmed: 14487261google scholar: lookup
        6. Li F, Browning GF, Studdert MJ, Crabb BS. Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.. Proc Natl Acad Sci U S A 1996 Feb 6;93(3):990-5.
          doi: 10.1073/pnas.93.3.990pmc: PMC40017pubmed: 8577774google scholar: lookup
        7. Wutz G, Auer H, Nowotny N, Grosse B, Skern T, Kuechler E. Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses.. J Gen Virol 1996 Aug;77 ( Pt 8):1719-30.
          pubmed: 8760418doi: 10.1099/0022-1317-77-8-1719google scholar: lookup
        8. Steck F, Hofer B, Schaeren B, Nicolet J, Gerber H. Equine rhinoviruses : new serotypes. Veterinary Publication 1978; pp. 321–328.
        9. Black WD, Hartley CA, Ficorilli NP, Studdert MJ. Sequence variation divides Equine rhinitis B virus into three distinct phylogenetic groups that correlate with serotype and acid stability.. J Gen Virol 2005 Aug;86(Pt 8):2323-2332.
          pubmed: 16033980doi: 10.1099/vir.0.80778-0google scholar: lookup
        10. Horsington JJ, Gilkerson JR, Hartley CA. Identification of mixed equine rhinitis B virus infections leading to further insight on the relationship between genotype, serotype and acid stability phenotype.. Virus Res 2011 Feb;155(2):506-13.
          doi: 10.1016/j.virusres.2010.12.007pubmed: 21185890google scholar: lookup
        11. Black WD, Studdert MJ. Formerly unclassified, acid-stable equine picornaviruses are a third equine rhinitis B virus serotype in the genus Erbovirus.. J Gen Virol 2006 Oct;87(Pt 10):3023-3027.
          pubmed: 16963761doi: 10.1099/vir.0.81937-0google scholar: lookup
        12. Huang JA, Ficorilli N, Hartley CA, Wilcox RS, Weiss M, Studdert MJ. Equine rhinitis B virus: a new serotype.. J Gen Virol 2001 Nov;82(Pt 11):2641-2645.
          pubmed: 11602775doi: 10.1099/0022-1317-82-11-2641google scholar: lookup
        13. Burrell MH, Wood JL, Whitwell KE, Chanter N, Mackintosh ME, Mumford JA. Respiratory disease in thoroughbred horses in training: the relationships between disease and viruses, bacteria and environment.. Vet Rec 1996 Sep 28;139(13):308-13.
          doi: 10.1136/vr.139.13.308pubmed: 8893488google scholar: lookup
        14. Carman S, Rosendal S, Huber L, Gyles C, McKee S, Willoughby RA, Dubovi E, Thorsen J, Lein D. Infectious agents in acute respiratory disease in horses in Ontario.. J Vet Diagn Invest 1997 Jan;9(1):17-23.
          doi: 10.1177/104063879700900104pubmed: 9087920google scholar: lookup
        15. Black WD, Wilcox RS, Stevenson RA, Hartley CA, Ficorilli NP, Gilkerson JR, Studdert MJ. Prevalence of serum neutralising antibody to equine rhinitis A virus (ERAV), equine rhinitis B virus 1 (ERBV1) and ERBV2.. Vet Microbiol 2007 Jan 17;119(1):65-71.
          doi: 10.1016/j.vetmic.2006.08.031pubmed: 17046179google scholar: lookup
        16. McCollum WH, Timoney PJ. Studies on the seroprevalence and frequencey of equine rhinovirus-I and -II infection in normal horse urine. Equine Infectious Diseases VI 1992; pp. 83–87.
        17. Hartley CA, Ficorilli N, Dynon K, Drummer HE, Huang JA, Studdert MJ. Equine rhinitis A virus: structural proteins and immune response.. J Gen Virol 2001 Jul;82(Pt 7):1725-1728.
          pubmed: 11413384doi: 10.1099/0022-1317-82-7-1725google scholar: lookup
        18. Kriegshäuser G, Deutz A, Kuechler E, Skern T, Lussy H, Nowotny N. Prevalence of neutralizing antibodies to Equine rhinitis A and B virus in horses and man.. Vet Microbiol 2005 Apr 10;106(3-4):293-6.
          pubmed: 15778036doi: 10.1016/j.vetmic.2004.12.029google scholar: lookup
        19. Studdert MJ, Gleeson LJ. Isolation and characterisation of an equine rhinovirus.. Zentralbl Veterinarmed B 1978;25(3):225-37.
          pubmed: 207059doi: 10.1111/j.1439-0450.1978.tb01180.xgoogle scholar: lookup
        20. Diaz-Mendez A, Viel L, Hewson J, Doig P, Carman S, Chambers T, Tiwari A, Dewey C. Surveillance of equine respiratory viruses in Ontario.. Can J Vet Res 2010 Oct;74(4):271-8.
          pmc: PMC2949340pubmed: 21197227
        21. Fukunaga Y, Kumanomido T, Kamada M, Wada R. Equine picornavirus: isolation of virus from the oral cavity of healthy horses. Bull Equine Res Inst 1983;20:103–109.
        22. Johnson DJ, Ostlund EN, Palmer TJ, Fett KL, Schmitt BJ. Isolation of Equine rhinitis A virus from a horse semen sample.. J Vet Diagn Invest 2012 Jul;24(4):801-3.
          doi: 10.1177/1040638712447936pubmed: 22621949google scholar: lookup
        23. Powell DG, Timoney PJ, Murphy T, Allen G, Donahue JM, Wilson J, Tudor L, Ferris K, Kawaoka Y. The application of advanced molecular techniques to investigate epizootics of infectious disease in the equine population.. Acta Vet Scand Suppl 1988;84:337-9.
          pubmed: 3068979
        24. Powell DG. Viral respiratory disease of the horse.. Vet Clin North Am Equine Pract 1991 Apr;7(1):27-52.
          pubmed: 1647261doi: 10.1016/s0749-0739(17)30514-xgoogle scholar: lookup
        25. Plummer G, Kerry JB. Studies on an equine respiratory virus. Vet Rec 1962;74:967–970.
        26. Hoffer B, Steck F, Gerber H, Loher J, Nicolet J, Paccaud MF. An investigation of the etiology of viral respiratory disease in a remount depot. 3rd International Conference of Equine Infectious Disease 1972; pp. 527–545.
        27. Black WD, Hartley CA, Ficorilli NP, Studdert MJ. Reverse transcriptase-polymerase chain reaction for the detection equine rhinitis B viruses and cell culture isolation of the virus.. Arch Virol 2007 Jan;152(1):137-49.
          doi: 10.1007/s00705-006-0810-3pmc: PMC7086902pubmed: 16932985google scholar: lookup
        28. Li F, Drummer HE, Ficorilli N, Studdert MJ, Crabb BS. Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses.. J Clin Microbiol 1997 Apr;35(4):937-43.
          pmc: PMC229704pubmed: 9157156doi: 10.1128/jcm.35.4.937-943.1997google scholar: lookup
        29. Burrows R. Laboratory diagnosis of some virus infections of the upper respiratory tract of the horse. Equine Vet J 1968;1:32–36.
          doi: 10.1111/j.2042-3306.1968.tb03342.xgoogle scholar: lookup
        30. Klaey M, Sanchez-Higgins M, Leadon DP, Cullinane A, Straub R, Gerber H. Field case study of equine rhinovirus 1 infection: clinical signs and clinicopathology.. Equine Vet J 1998 May;30(3):267-9.
          doi: 10.1111/j.2042-3306.1998.tb04499.xpubmed: 9622330google scholar: lookup
        31. Dynon K, Varrasso A, Ficorilli N, Holloway S, Reubel G, Li F, Hartley C, Studdert M, Drummer H. Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction.. Aust Vet J 2001 Oct;79(10):695-702.
          doi: 10.1111/j.1751-0813.2001.tb10674.xpubmed: 11712710google scholar: lookup
        32. Mori A, De Benedictis P, Marciano S, Zecchin B, Zuin A, Zecchin B, Capua I, Cattoli G. Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens.. J Virol Methods 2009 Feb;155(2):175-81.
          doi: 10.1016/j.jviromet.2008.10.009pubmed: 19013197google scholar: lookup
        33. Quinlivan M, Maxwell G, Lyons P, Arkins S, Cullinane A. Real-time RT-PCR for the detection and quantitative analysis of equine rhinitis viruses.. Equine Vet J 2010 Mar;42(2):98-104.
          doi: 10.2746/042516409X479559pubmed: 20156243google scholar: lookup
        34. Balasuriya UB, Leutenegger CM, Topol JB, McCollum WH, Timoney PJ, MacLachlan NJ. Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay.. J Virol Methods 2002 Mar;101(1-2):21-8.
          pubmed: 11849680doi: 10.1016/s0166-0934(01)00416-5google scholar: lookup
        35. Willoughby R, Ecker G, McKee S, Riddolls L, Vernaillen C, Dubovi E, Lein D, Mahony JB, Chernesky M, Nagy E. The effects of equine rhinovirus, influenza virus and herpesvirus infection on tracheal clearance rate in horses.. Can J Vet Res 1992 Apr;56(2):115-21.
          pmc: PMC1263518pubmed: 1317244
        36. Powell DG, Burrows R, Spooner PR, Goodridge D, Thompson GR, Mumford J. A study of infectious respiratory disease among horses in Great Britain, 1971–1976. Veterinary Publications 1978; pp. 451–459.
        37. Bell SA, Balasuriya UB, Nordhausen RW, MacLachlan NJ. Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.. J Vet Diagn Invest 2006 Sep;18(5):472-5.
          doi: 10.1177/104063870601800509pubmed: 17037617google scholar: lookup
        38. Glaser AL, Renshaw RW, Trock SC, Brady RC, Dubovi EJ. Isolation of Salem virus, a novel equine paramyxovirus, and assessment of its etiologic role in a disease outbreak.. Vet Microbiol 2002 Jul 9;87(3):205-12.
          doi: 10.1016/S0378-1135(02)00067-6pubmed: 12052331google scholar: lookup
        39. Lu Z, Branscum AJ, Shuck KM, Zhang J, Dubovi EJ, Timoney PJ, Balasuriya UB. Comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of Equine arteritis virus nucleic acid in equine semen and tissue culture fluid.. J Vet Diagn Invest 2008 Mar;20(2):147-55.
          doi: 10.1177/104063870802000202pubmed: 18319426google scholar: lookup

        Citations

        This article has been cited 3 times.
        1. Uchida-Fujii E, Kinoshita Y, Niwa H, Maeda T, Nukada T, Ueno T. High prevalence of Mycoplasma equirhinis in Thoroughbred horses with respiratory symptoms in autumn 2018. J Vet Med Sci 2021 Dec 9;83(12):1907-1912.
          doi: 10.1292/jvms.21-0163pubmed: 34732605google scholar: lookup
        2. Ambagala A, Fisher M, Goolia M, Nfon C, Furukawa-Stoffer T, Ortega Polo R, Lung O. Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus. Transbound Emerg Dis 2017 Oct;64(5):1610-1623.
          doi: 10.1111/tbed.12554pubmed: 27589902google scholar: lookup
        3. Stasiak K, Dunowska M, Rola J. Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland. Viruses 2024 Jul 26;16(8).
          doi: 10.3390/v16081204pubmed: 39205178google scholar: lookup
        Subscribe to our newsletter
        Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.