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

Abstract

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 10(3) times more sensitive than virus isolation by cell culture for EHV2 and 10(6) 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.

Prevalence of equine herpesvirus types 2 and 5 in horse populations by using type-specific PCR assays

“…Further samples have to be tested in order to make a complete study on this aspect. A difference in the prevalence of EHV-2 and EHV-5 was also reported in Australia where 31% of the tested horses were positive to EHV-2 while only 16% reacted in EHV-5 PCR [17]. Other explanations on the low number of EHV-5 positive cases could be that PBLs are probably not the main reservoirs of this virus.…”

Association of Two Newly Recognized Herpesviruses with Interstitial Pneumonia in Donkeys (Equus Asinus)

“…1,6,41 Like EHV2, EHV5 also appears to be widespread, although somewhat less prevalent. 33 For the 2 recently identified gammaherpesviruses of equids ZHV and WAHV, no disease symptoms were reported in the animals from which these viruses were detected. 12 For animals from which ZHV was identified, blood from a total of 20 free-ranging zebras from Namibia was initially tested and found positive for anti-bodies to EHV2.…”

“…4,9,19,20,28,[39][40][41] However, infection with EHV2 appears to be widespread, and several studies have also found EHV2 in the respiratory tract and peripheral blood leukocytes of horses without clinical disease, with isolation and/ or PCR detection rates ranging from 31 to 89%. 4,19,33,35,39 Following experimental inoculation of EHV2 into both juvenile and adult horses, chronic pharyngitis was observed in the young horses while the adult horses remained clinically normal. 3 As with other gammaherpesviruses, latency in lymphocytes has been demonstrated for EHV2.…”

Multiplex PCR: Optimization and Application in Diagnostic Virology

“…In addition, the methodology has proved to be a powerful tool for characterization of nonhuman viruses (50,70,85,104,114,116).…”