Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation

A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped approximately using fragments from within EcoR1-I, and the precise location of the variable sites determined from the DNA sequence of this fragment. Oligonucleotide primers flanking the variable sites were synthesized, and used in PCR assays to detect variable fragments. The AluI variable fragment was found to result from the presence or absence of a single AluI site. In contrast, the variable bands seen with HaeIII and RsaI, resulted from variation in the copy number of two tandemly repeated sequences, one of which had not previously been recognized. In addition, HaeIII digests of EHV-1 isolates probed with the glycoprotein B (gB) gene of EHV-1 also separated isolates into two groups. The variable HaeIII site was mapped towards the 5′-end of the gB gene and a PCR assay established. The distribution of the variable AluI site within the EcoR1-I fragment and the HaeIII site within the gB gene were estimated on a large number of clinical isolates using PCR on unpurified viral tissue culture medium. Both sites had a good distribution and together with additional variable sites should provide the basis for the rapid DNA fingerprinting of EHV-1 isolates.