Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction.

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.

Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV-1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.