Identification of equine herpesviruses 1 and 4 by polymerase chain reaction

Objective To develop and validate specific, sensitive and rapid (< 8 hour) diagnostic tests using polymerase chain reaction (PCR) for the diagnosis of abortion and respiratory disease caused by equine herpesvirus 1 (EHV1; equ ine abortion virus) and EHV4 (equine rhinopneumonitis virus). Design Primer sets based on nucleotide sequences encoding glycoprotein H (g H) of EHV1 and gB of EHV4 were designed and used in single round and second round (seminested) PCRs, and in a multiplex PCR for the diagnosis of EHV1 and EHV4 infections. Methods Oligonucleotide primers were designed for each virus, PCR condition s were defined and the specificity and sensitivity of the assays were deter mined. The tests were applied to tissue samples from aborted equine foetuse s and to nasopharyngeal swabs from horses with acute febrile respiratory di sease. Results Individual single round and a second round (seminested) EHV1 and EH V4 PCRs were specific in that EHV1 primers amplified all (n = 30) EHV1 isol ates and did not amplify EHV4. Similarly EHV4 primers amplified all (n = 6) EHV4 isolates and did not amplify EHV1. Both PCRs were sensitive in that t he first round EHV1 PCR detected 1220 molecules of EHV1 plasmid DNA and the first round EHV4 PCR detected 7280 molecules of EHV4 plasmid DNA. The EHV1 second round PCR was 100 times more sensitive in that it detected 12 molec ules of EHV1 DNA and the EHV4 second round PCR was 1000 times more sensitiv e in that it detected 8 molecules of EHV4 DNA. There was a high correlation between detection of EHV1 by virus isolation and PCR when tissue samples f rom 71 aborted foetuses were examined; all samples positive by virus isolat ion were positive by PCR. Similarly the EHV4 PCR was at least as sensitive as virus isolation when applied to nasaopharyngeal swabs from horses with r espiratory disease in that all samples positive by virus isolation were als o positive by PCR. Conclusion Individual single round and second round (seminested) PCRs and a seminested multiplex PCR were developed that enabled reliable, rapid detec tion of EHV1 and EHV4 in aborted foetal tissues and nasopharyngeal swab sam ples.