New Real-Time PCR Assay Using Allelic Discrimination for Detection and Differentiation of Equine Herpesvirus-1 Strains with A 2254 and G 2254 Polymorphisms

Abstract

ABSTRACT A single-nucleotide polymorphism (A 2254 or G 2254 ) 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 (E 2 ) 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 (E 1 ) was developed by redesigning primers and probes specific to ORF30. The E 1 and E 2 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 (A 2254 or G 2254 ) in all archived isolates plus 168 of the clinical samples. The E 1 assay was 10 times more sensitive than E 2 , with a lower detection limit of 10 infectious virus particles. Furthermore, all A 2254 and G 2254 genotypes along with samples from three cases of dual infection (A 2254 +G 2254 ) were correctly identified by E 1 , whereas E 2 produced 20 false dual positive results with only one actual mixed A 2254 +G 2254 genotype confirmed. Based on these findings, E 1 offers greater sensitivity and accuracy for the detection and A/G 2254 genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.