New Real-Time PCR Assay Using Allelic Discrimination for Detection and Differentiation of Equine Herpesvirus-1 Strains with A2254 and G2254 Polymorphisms

A single-nucleotide polymorphism (A2254 or G2254) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E2) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69–72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E1) was developed by redesigning primers and probes specific to ORF30. The E1 and E2 rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A2254 or G2254) in all archived isolates plus 168 of the clinical samples. The E1 assay was 10 times more sensitive than E2, with a lower detection limit of 10 infectious virus particles. Furthermore, all A2254 and G2254 genotypes along with samples from three cases of dual infection (A2254+G2254) were correctly identified by E1, whereas E2 produced 20 false dual positive results with only one actual mixed A2254+G2254 genotype confirmed. Based on these findings, E1 offers greater sensitivity and accuracy for the detection and A/G2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.