Advertisement

Archives of Virology

, Volume 140, Issue 6, pp 1049–1060 | Cite as

Diagnosis of equine gammaherpesvirus 2 and 5 infections by polymerase chain reaction

  • G. H. Reubel
  • B. S. Crabb
  • M. J. Studdert
Original Papers

Summary

Nested polymerase chain reaction (PCR) assays were developed for the detection of equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5) using the nucleotide sequences from the glycoprotein B (gB) gene of EHV2 and the thymidine kinase (TK) gene of EHV5. The simultaneous use of EHV2 specific and EHV5 specific primers in one nested amplification assay (multiplex PCR) enabled a rapid, specific and sensitive diagnosis for each virus. PCR was found to be 103 times more sensitive than virus isolation by cell culture for EHV2 and 106 for EHV5. In separate PCR assays, the routine detection limit after ethidium bromide staining was 0.6 fg for EHV2 plasmid DNA and 2.3 fg for EHV5 plasmid DNA, equivalent for both viruses to approximately 100 genome copies. The detection limits in multiplex PCR were 6 pg for EHV2 and 2.3 fg for EHV5, respectively. PCR assays were applied to studies of the epidemiology of EHV2 and EHV5 infections of racehorses and breeding mares in Victoria and New South Wales, Australia. Peripheral blood leukocytes from 31% of horses were positive for EHV2, 16% positive for EHV5, 8% positive for both viruses and 63% negative for both viruses. EHV2 PCR was also successfully used to detect EHV2 DNA in nasal secretions from horses. The multiplex PCR assay proved to be a rapid and reliable method for the simultaneous detection and differentiation of 2 related equine gammaherpesviruses.

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Assay Thymidine Kinase Ethidium Bromide Staining Multiplex Polymerase Chain Reaction 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Agius CT, Nagesha HS, Studdert MJ (1992) Equine herpesvirus 5: comparisons with EHV2 (equine cytomegalovirus), cloning, and mapping of a new equine herpesvirus with a novel genome structure. Virology 191: 176–186Google Scholar
  2. 2.
    Belak S, Ballagi-Pordany A (1993) Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology. Vet Res Commun 17: 55–72Google Scholar
  3. 3.
    Browning GF, Studdert MJ (1987) Epidemiology of equine herpesvirus 2 (equine cytomegalovirus). J Clin Microbiol 25: 13–16Google Scholar
  4. 4.
    Browning GF, Studdert MJ (1987) Genomic heterogeneity of equine betaherpesviruses. J Gen Virol 68: 1441–1447Google Scholar
  5. 5.
    Browning GF, Studdert MJ (1988) Equine herpesvirus 2 (equine cytomegalovirus). Vet Bull 58: 775–790Google Scholar
  6. 6.
    Chamberlain JS, Gibbs RA, Rainier JE, Caskey CT (1992) Multiplex PCR for the diagnosis of Duchenne muscular dystrophy In Innis MA, Gelfand GH, White TJ (eds) PCR protocols: a guide methods and applications. Academic Press, San Diego, pp 272–281Google Scholar
  7. 7.
    Harden TJ, Bagust TJ, Pascoe RR, Spradbrow PB (1974) Studies on equine herpesviruses 5. Isolation and characterisation of slowly cytopathic equine herpesviruses in Queensland. Aust Vet J 50: 483–488Google Scholar
  8. 8.
    Kemeny L, Pearson JE (1970) Isolation of herpesvirus from equine leukocytes: comparison with equine rhinopneumonitis virus. Can J Comp Med 34: 59–65Google Scholar
  9. 9.
    Persing DH (1993) In vitro nucleic acid amplification techniques. In: Persing DH, Smith TF, Tenover FC, White TJ (eds) Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, pp 51–87Google Scholar
  10. 10.
    Persing DH, Smith TF, Tenover FC, White TJ (1993) Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, WashingtonGoogle Scholar
  11. 11.
    Reubel GH, Ramos RA, Rimstad E, Hickman A, Pedersen NC (1994) Detection of acute and latent feline herpesvirus 1 infections using the polymerase chain reaction. Arch Virol 132: 409–420Google Scholar
  12. 12.
    Roeder PL, Scott GR (1975) The prevalence of equid herpesvirus 2 infections. Vet Rec 95: 404–405Google Scholar
  13. 13.
    Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491Google Scholar
  14. 14.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  15. 15.
    Sherman J, Thorsen J, Barnum DA, Mitchell WR, Ingram DG (1977) Infectious causes of equine respiratory disease on Ontario Standardbred racetracks. J Clin Microbiol 5: 258–289Google Scholar
  16. 16.
    Telford EAR, Studdert MJ, Agius CT, Watson MS, Aird HC, Davison AJ (1993) Equine herpesviruses 2 and 5 are γ-herpesviruses. Virology 195: 492–499Google Scholar
  17. 17.
    Tenorio A, Echevarria JE, Casas I, Echevarria JM, Tabares E (1993) Detection and typing of human herpesviruses by multiplex polymerase chain reaction. J Virol Methods 44: 261–269Google Scholar
  18. 18.
    Turner AJ, Studdert MJ, Peterson JE (1970) Equine herpesviruses. 2. Persistence of equine herpesviruses in experimentally infected horses and the experimental induction of abortion. Aust Vet J 46: 90–98Google Scholar
  19. 19.
    Wilks CR, Studdert MJ (1974) Equine herpesviruses. 5. Epizootiology of slowly cytopathic viruses in foals. Aust Vet J 50: 438–442Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • G. H. Reubel
    • 1
  • B. S. Crabb
    • 1
  • M. J. Studdert
    • 1
  1. 1.Centre for Equine Virology, School of Veterinary ScienceThe University of MelbourneParkvilleAustralia

Personalised recommendations