FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Scientific reports2025; 15(1); 38201; doi: 10.1038/s41598-025-22043-w

A multiplex real-time PCR assay for detection of equid herpesvirus 1 and 4.

Abstract: Equid herpesvirus (EHV) 1 and -4 are common viral pathogens of horses that can cause upper respiratory disease, neurological disease, abortion, and death. As characteristic alphaherpesviruses, both EHV-1 and EHV-4 can establish latency, resulting in a lifelong carrier state in infected animals. Here we describe the development and validation of a rapid and sensitive multiplex real-time PCR assay (EHV1-4MP) that simultaneously detects EHV-1 and EHV-4 and includes an endogenous internal control - melanocortin 1 receptor (MC1R) - targeting the equid genome. The EHV1-4MP assay analytical sensitivity was determined to be approximately two copies for EHV-1, four copies for EHV-4, and 10 copies for the equid MC1R gene per reaction. Analytical specificity was determined using a panel of 28 equine respiratory pathogens and commensal equine microorganisms. The EHV1-4MP assay detected reference and clinical isolates of EHV-1 and EHV-4, and did not detect other equid herpesviruses such as EHV-2, EHV-3, EHV-5, or several other viral and bacterial pathogens of horses. Importantly, the EHV1-4MP assay developed here has improved specificity compared to existing assays and is able to exclude the closely related EHV-3, EHV-8, and EHV-9 viruses. Diagnostic performance was evaluated using 60 clinical samples including upper respiratory swabs and washes, blood, placenta, lung, and brain. The EHV1-4MP assay results were in 100% concordance with singleplex EHV-1 and EHV-4 assays. Our results demonstrate that the EHV1-4MP real-time assay developed here offers rapid, sensitive, and simultaneous detection of EHV-1 and EHV-4.
© 2025. The Author(s).
Get Full Text
Publication Date: 2025-10-31 PubMed ID: 41173927PubMed Central: PMC12578806DOI: 10.1038/s41598-025-22043-wGoogle 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

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.

Overview

  • This study developed and validated a multiplex real-time PCR assay named EHV1-4MP that can quickly and accurately detect equid herpesvirus types 1 and 4 (EHV-1 and EHV-4) in horses.
  • The assay also includes a control for the host DNA to ensure test reliability and shows improved specificity compared to existing tests, avoiding false positives from closely related viruses.

Background

  • Equid herpesviruses 1 and 4 are common viral infections in horses causing respiratory illness, neurological disease, abortion, and sometimes death.
  • Both viruses are alphaherpesviruses capable of establishing latent infections, leading horses to become lifelong carriers capable of reactivation and transmission.
  • Rapid and accurate detection of these viruses is critical for disease control and management in equine populations.

Assay Development

  • The researchers created a multiplex real-time PCR assay (EHV1-4MP) that can simultaneously detect EHV-1 and EHV-4 in a single reaction.
  • They incorporated an endogenous internal control targeting the melanocortin 1 receptor (MC1R) gene, which is part of the horse’s genome, to confirm the presence of amplifiable host DNA and ensure test validity.

Sensitivity and Specificity

  • The assay’s analytical sensitivity, meaning the lowest number of DNA copies detectable, was approximately:
    • 2 copies per reaction for EHV-1
    • 4 copies per reaction for EHV-4
    • 10 copies per reaction for the equid MC1R gene
  • Specificity testing was conducted against 28 other equine respiratory pathogens and commensal microorganisms to ensure that the assay only detected EHV-1 and EHV-4.
  • The assay did not detect other related equid herpesviruses such as EHV-2, EHV-3, EHV-5, and importantly could discriminate against viruses closely related to EHV-1 and EHV-4, including EHV-3, EHV-8, and EHV-9.

Diagnostic Validation

  • The assay was tested on 60 clinical samples from various sources including upper respiratory swabs and washes, blood, placenta, lung, and brain tissues.
  • Results showed 100% agreement with established singleplex PCR assays that detect either EHV-1 or EHV-4 individually.
  • This confirmed the assay’s accuracy and reliability across different sample types encountered in clinical settings.

Advantages of the EHV1-4MP Assay

  • Rapid detection of both EHV-1 and EHV-4 simultaneously, saving time compared to running separate tests.
  • High sensitivity, enabling detection of very low viral loads.
  • Improved specificity preventing false positives from other equid herpesvirus types or unrelated pathogens.
  • Inclusion of an internal equid gene control enhances test robustness and helps identify invalid samples.
  • Applicable to a wide range of clinical samples relevant to different disease presentations.

Conclusion

  • The EHV1-4MP multiplex real-time PCR assay provides a valuable diagnostic tool for veterinary laboratories and equine health management.
  • Its high sensitivity, specificity, and ability to detect both important herpesviruses simultaneously make it well-suited for rapid and accurate diagnosis of EHV-related diseases in horses.
  • Such improved diagnostic capabilities can aid in disease surveillance, outbreak control, and better clinical decision-making to protect equine health.

Cite This Article

APA
Tallmadge RL, Laverack M, Lejeune M, Crossley B, Diel DG. (2025). A multiplex real-time PCR assay for detection of equid herpesvirus 1 and 4. Sci Rep, 15(1), 38201. https://doi.org/10.1038/s41598-025-22043-w

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 38201
PII: 38201

Researcher Affiliations

Tallmadge, Rebecca L
  • Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center (AHDC), College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Laverack, Melissa
  • Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center (AHDC), College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Lejeune, Manigandan
  • Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center (AHDC), College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Crossley, Beate
  • California Animal Health & Food Safety Laboratory System (CAHFS), University of California, Davis, CA, USA.
Diel, Diego G
  • Department of Population Medicine and Diagnostic Sciences, Animal Health Diagnostic Center (AHDC), College of Veterinary Medicine, Cornell University, Ithaca, NY, USA. dgdiel@cornell.edu.

MeSH Terms

  • Herpesvirus 1, Equid / genetics
  • Herpesvirus 1, Equid / isolation & purification
  • Animals
  • Horses / virology
  • Herpesviridae Infections / veterinary
  • Herpesviridae Infections / diagnosis
  • Herpesviridae Infections / virology
  • Herpesvirus 4, Equid / genetics
  • Herpesvirus 4, Equid / isolation & purification
  • Horse Diseases / virology
  • Horse Diseases / diagnosis
  • Real-Time Polymerase Chain Reaction / methods
  • Multiplex Polymerase Chain Reaction / methods
  • Sensitivity and Specificity

Conflict of Interest Statement

Declarations. Competing interests: The authors declare no competing interests.

References

This article includes 50 references
  1. Allen G, Kydd J, Slater J, Smith K. Equid herpesvirus 1 and equid herpesvirus 4 infections. 829–859 (2004).
  2. Oladunni FS, Horohov DW, Chambers TM. EHV-1: A constant threat to the horse industry. 2668 (2019).
    doi: 10.3389/fmicb.2019.02668pmc: PMC6901505pubmed: 31849857google scholar: lookup
  3. Pusterla N. Frequency of molecular detection of equine herpesvirus-4 in nasal secretions of 3028 horses with upper airway infection. 593 (2017).
    doi: 10.1136/vr.104240pubmed: 28386031google scholar: lookup
  4. Pusterla N. Frequency of detection and prevalence factors associated with common respiratory pathogens in equids with acute onset of fever and/or respiratory signs (2008–2021). 759 (2022).
    doi: 10.3390/pathogens11070759pmc: PMC9317490pubmed: 35890002google scholar: lookup
  5. Pusterla N. Surveillance programme for important equine infectious respiratory pathogens in the USA. 12 (2011).
    doi: 10.1136/vr.d2157pubmed: 21676986google scholar: lookup
  6. Cantón GJ. Equine abortion and stillbirth in california: a review of 1,774 cases received at a diagnostic laboratory, 1990–2022. 153–162 (2023).
    doi: 10.1177/10406387231152788pmc: PMC9999402pubmed: 36744759google scholar: lookup
  7. Akter R. Detection of Coxiella burnetii and equine herpesvirus 1, but not leptospira spp. Or Toxoplasma gondii, in cases of equine abortion in Australia - a 25 year retrospective study. e0233100 (2020).
    pmc: PMC7250447pubmed: 32453753
  8. Smith KC, Blunden AS, Whitwell KE, Dunn KA, Wales AD. A survey of equine abortion, stillbirth and neonatal death in the UK from 1988 to 1997. 496–501 (2003).
    doi: 10.2746/042516403775600578pubmed: 12875329google scholar: lookup
  9. Bazanów BA, Frącka AB, Jackulak NA, Staroniewicz ZM, Ploch SM. A 34-year retrospective study of equine viral abortion in Poland. 607–612 (2014).
    doi: 10.2478/pjvs-2014-0091pubmed: 25638974google scholar: lookup
  10. Couroucé A. Equine Herpesvirus-1 outbreak during a Show-Jumping competition: A clinical and epidemiological study. 104869 (2023).
    doi: 10.1016/j.jevs.2023.104869pubmed: 37339699google scholar: lookup
  11. Walter J, Seeh C, Fey K, Bleul U, Osterrieder N. Clinical observations and management of a severe equine herpesvirus type 1 outbreak with abortion and encephalomyelitis. 19 (2013).
    doi: 10.1186/1751-0147-55-19pmc: PMC3630004pubmed: 23497661google scholar: lookup
  12. Pronost S. Outbreak of equine herpesvirus myeloencephalopathy in france: a clinical and molecular investigation. 256–263 (2012).
    doi: 10.1111/j.1865-1682.2011.01263.xpubmed: 21975071google scholar: lookup
  13. Negussie H, Gizaw D, Tessema TS, Nauwynck HJ. Equine Herpesvirus-1 Myeloencephalopathy, an emerging threat of working equids in Ethiopia. 389–397 (2017).
    doi: 10.1111/tbed.12377pubmed: 26010868google scholar: lookup
  14. McFadden AMJ. The first reported outbreak of equine herpesvirus myeloencephalopathy in new Zealand. 125–134 (2016).
    doi: 10.1080/00480169.2015.1096853pubmed: 26414406google scholar: lookup
  15. Henninger RW. Outbreak of neurologic disease caused by equine Herpesvirus-1 at a university equestrian center. 157–165 (2007).
    pubmed: 17338164
  16. Gryspeerdt A. Description of an unusually large outbreak of nervous system disorders caused by equine herpesvirus 1 (EHV1) in 2009 in Belgium.. 147–153 (2011).
  17. Tong P. Outbreak of neuropathogenic equid herpesvirus 1 causing abortions in Yili horses of Zhaosu, North Xinjiang, China.. 83 (2022).
    pmc: PMC8886757pubmed: 35232435
  18. Mureşan A. An outbreak of equine HerpesVirus-4 in an ecological Donkey milk farm in Romania.. (3), 468 (2022).
    pmc: PMC8953855pubmed: 35335100
  19. Pavulraj S. Equine HerpesVirus Type 4 (EHV-4) Outbreak in Germany: Virological, Serological, and Molecular Investigations.. (2021).
    pmc: PMC8308676pubmed: 34202127
  20. Ryt-Hansen P, Johansen VK, Cuicani MM, Larsen LE, Hansen S. Outbreak of equine herpesvirus 4 (EHV-4) in denmark: tracing patient zero and viral characterization.. 1–12 (2024).
    doi: 10.1186/s12917-024-04149-xpmc: PMC11221098pubmed: 38961400google scholar: lookup
  21. Walker PJ. Recent changes to virus taxonomy ratified by the international committee on taxonomy of viruses (2022).. 2429–2440 (2022).
    doi: 10.1007/s00705-022-05516-5pmc: PMC10088433pubmed: 35999326google scholar: lookup
  22. Vissani MA, Damiani AM, Barrandeguy ME. Equine Coital Exanthema: New Insights on the Knowledge and Leading Perspectives for Treatment and Prevention.. (2021).
    pmc: PMC8398825pubmed: 34451519
  23. Roizman B. Herpesviridae.. 114–127 (Springer, 1995).
    doi: 10.1007/978-3-7091-6607-9google scholar: lookup
  24. Browning GF, Ficorilli N, Studdert MJ. Asinine herpesvirus genomes: comparison with those of the equine herpesviruses.. 183–190 (1988).
    doi: 10.1007/BF01310999pubmed: 2845891google scholar: lookup
  25. Wang T. Identification of equine herpesvirus 8 in Donkey abortion: a case report.. (1), 10 (2022).
    pmc: PMC8734136pubmed: 34991640
  26. Wang T. The emergence of viral encephalitis in donkeys by equid herpesvirus 8 in China.. 840754 (2022).
    pmc: PMC8930201pubmed: 35308333
  27. Liu C, Guo W, Lu G, Xiang W, Wang X. Complete genomic sequence of an equine herpesvirus type 8 Wh strain isolated from China.. 5407–5407 (2012).
    doi: 10.1128/JVI.00445-12pmc: PMC3347380pubmed: 22492929google scholar: lookup
  28. Garvey M. Equid herpesvirus 8: complete genome sequence and association with abortion in mares.. e0192301 (2018).
    doi: 10.1371/journal.pone.0192301pmc: PMC5802896pubmed: 29414990google scholar: lookup
  29. Fukushi H. Gazelle herpesvirus 1: a new neurotropic herpesvirus immunologically related to equine herpesvirus 1.. 34–44 (1997).
    doi: 10.1006/viro.1996.8296pubmed: 9015181google scholar: lookup
  30. Taniguchi A. Pathogenicity of a new neurotropic equine herpesvirus 9 (gazelle herpesvirus 1) in horses.. 215–218 (2000).
    doi: 10.1292/jvms.62.215pubmed: 10720196google scholar: lookup
  31. Wohlsein P. Fatal epizootic equine herpesvirus 1 infections in new and unnatural hosts.. 456–460 (2011).
    doi: 10.1016/j.vetmic.2010.11.024pubmed: 21167662google scholar: lookup
  32. Tau R. Comprehensive analysis of equid herpesvirus recombination: an insight into the repeat regions.. .
    pubmed: 37704182doi: 10.1016/j.jevs.2023.104916google scholar: lookup
  33. Greenwood AD. A potentially fatal mix of herpes in zoos.. 1727–1731 (2012).
    doi: 10.1016/j.cub.2012.07.035pubmed: 22902751google scholar: lookup
  34. Foote CE, Love DN, Gilkerson JR, Whalley JM. Detection of EHV-1 and EHV-4 DNA in unweaned thoroughbred foals from vaccinated mares on a large stud farm.. 341–345 (2004).
    doi: 10.2746/0425164044890634pubmed: 15163042google scholar: lookup
  35. Slater JD, Borchers K, Thackray AM, Field HJ. The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse.. 2007–2016 (1994).
    doi: 10.1099/0022-1317-75-8-2007pubmed: 8046404google scholar: lookup
  36. Giessler KS. Viral load and cell tropism during early latent equid herpesvirus 1 infection differ over time in lymphoid and neural tissue samples from experimentally infected horses.. 621 (2020).
    doi: 10.3389/fvets.2020.00621pmc: PMC7499125pubmed: 33102556google scholar: lookup
  37. Borchers K, Wolfinger U, Ludwig H. Latency-associated transcripts of equine herpesvirus type 4 in trigeminal ganglia of naturally infected horses.. 2165–2171 (1999).
    doi: 10.1099/0022-1317-80-8-2165pubmed: 10466816google scholar: lookup
  38. Pusterla N, Mapes S, Wilson D. Prevalence of latent alpha-herpesviruses in thoroughbred racing horses.. 579–582 (2012).
    doi: 10.1016/j.tvjl.2012.01.030pubmed: 22405721google scholar: lookup
  39. Carvelli A, Nielsen SS, Paillot R, Broglia A, Kohnle L. Clinical impact, diagnosis and control of equine HerpesVirus-1 infection in Europe.. 7230 (2022).
    pmc: PMC8985062pubmed: 35414834
  40. Lunn DP. Updated ACVIM consensus statement on equine herpesvirus-1.. 1290–1299 (2024).
    doi: 10.1111/jvim.17047pmc: PMC11099706pubmed: 38497217google scholar: lookup
  41. World Organisation for Animal Health. EQUINE RHINOPNEUMONITIS (INFECTION WITH VARICELLOVIRUS EQUIDALPHA1).. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (2024).
  42. Untergasser A. Primer3-new capabilities and interfaces.. e115 (2012).
    doi: 10.1093/nar/gks596pmc: PMC3424584pubmed: 22730293google scholar: lookup
  43. Dreier J, Störmer M, Kleesiek K. Use of bacteriophage MS2 as an internal control in viral reverse transcription-PCR assays.. 4551–4557 (2005).
    doi: 10.1128/JCM.43.9.4551-4557.2005pmc: PMC1234060pubmed: 16145106google scholar: lookup
  44. Elia G. Detection of equine herpesvirus type 1 by real time PCR.. 70–75 (2006).
    doi: 10.1016/j.jviromet.2005.10.024pubmed: 16309751google scholar: lookup
  45. Vandevanter DR. Detection and analysis of diverse herpesviral species by consensus primer PCR.. 1666–1671 (1996).
    doi: 10.1128/jcm.34.7.1666-1671.1996pmc: PMC229091pubmed: 8784566google scholar: lookup
  46. Cannon MV. In Silico assessment of primers for eDNA studies using primertree and application to characterize the biodiversity surrounding the Cuyahoga river.. 22908 (2016).
    doi: 10.1038/srep22908pmc: PMC4786790pubmed: 26965911google scholar: lookup
  47. Diallo IS. Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4).. 93–103 (2007).
    doi: 10.1016/j.vetmic.2007.02.004pubmed: 17346907google scholar: lookup
  48. Neubauer A, Braun B, Brandmüller C, Kaaden OR, Osterrieder N. Analysis of the contributions of the equine herpesvirus 1 glycoprotein gB homolog to virus entry and direct cell-to-cell spread.. 281–294 (1997).
    doi: 10.1006/viro.1996.8336pubmed: 9018127google scholar: lookup
  49. Spiesschaert B, Osterrieder N, Azab W. Comparative analysis of glycoprotein B (gB) of equine herpesvirus type 1 and type 4 (EHV-1 and EHV-4) in cellular tropism and cell-to-cell transmission.. 522–542 (2015).
    doi: 10.3390/v7020522pmc: PMC4353902pubmed: 25654240google scholar: lookup
  50. Smith FL. Frequency of shedding of respiratory pathogens in horses recently imported to the united States.. 1436–1441 (2018).
    doi: 10.1111/jvim.15145pmc: PMC6060314pubmed: 29761571google scholar: lookup

Citations

This article has been cited 0 times.
Subscribe to our newsletter
Join 99,928 horse owners like you who receive our email updates on horse health and nutrition.