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

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.

Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017

“…Since Nugent et al (2006) [21] put forward the hypothesis that variants with the G2254/D752 substitution in the DNA polymerase have increased likelihood of association with neuropathogenicity, there has been international focus on the characterisation of the genotypes of EHV-1 isolates and allelic discrimination assays have been widely used to distinguish between neuropathogenic and non-neuropathogenic strains [42,43]. Many studies concentrate on the retrospective investigation of archived viruses and the prevalence data generated must be interpreted with caution due to sampling and storage bias.…”

Equid herpesvirus 8: Complete genome sequence and association with abortion in mares

“…The number of outbreaks of neurological disease in horses attributed to the EHV-1 hypervirulent phenotype may be inflated, as may the increased prevalence of this phenotype in EHV-1 abortions [ 48 ], due to the inclusion of EHV-8 cases. The ORF30 variation (D752/N752) has been applied to the development of PCR tests to discriminate between EHV-1 hypervirulent and non-hypervirulent strains [ 49 – 51 ]. All sequenced EHV-8 strains have the D752 genotype, and the specificity of the EHV-1 allelic discrimination test has not been assessed for EHV-8 [ 49 ].…”

“…The ORF30 variation (D752/N752) has been applied to the development of PCR tests to discriminate between EHV-1 hypervirulent and non-hypervirulent strains [ 49 – 51 ]. All sequenced EHV-8 strains have the D752 genotype, and the specificity of the EHV-1 allelic discrimination test has not been assessed for EHV-8 [ 49 ]. Given the high level of sequence identity to EHV-8 at the primer-binding sites used in this assay [ 49 ], with one mismatch in the forward primer and none in the reverse primer or the probe (G2254), there is a strong likelihood of misidentified detections.…”

“…All sequenced EHV-8 strains have the D752 genotype, and the specificity of the EHV-1 allelic discrimination test has not been assessed for EHV-8 [ 49 ]. Given the high level of sequence identity to EHV-8 at the primer-binding sites used in this assay [ 49 ], with one mismatch in the forward primer and none in the reverse primer or the probe (G2254), there is a strong likelihood of misidentified detections. Furthermore, an assessment of the EHV-1 gC primers used initially in this study [ 12 ] showed that there were only two mismatches in the forward primer and one in the reverse primer.…”

Cryptic etiopathological conditions of equine nervous system with special emphasis on viral diseases

“…A real-time PCR assay using allelic discrimination (E2) to distinguish between neuro-pathogenic and non-neuropathogenic strains of EHV1 has been used [ 118 ]. Similarly, the development of ORF30 region specific, allelic discrimination (E1) EHV1 real-time PCR test has also been implicated [ 119 ]. The effective vaccination together with good management practices is effective tool for prevention of EHV-1 [ 120 ].…”