Detection of EHV-1 neuropathogenic strains using real-time PCR in the neural tissue of horses with myeloencephalopathy.

EQUINE herpesvirus type 1 (EHV-1) infection is widespread in horse populations throughout the world and produces well-documented syndromes of respiratory disease, abortion, neonatal foal death and myeloencephalopathy (van Maanen 2002). The epidemiology of EHV-1 is characterised by lifelong latent infection after primary exposure. The reactivation of a latent stage in a subclinically affected animal often leads to viral shedding via nasal secretions and horizontal transmission (Edington and others 1985). PCR assays that are both more sensitive and rapid have largely replaced the time-consuming procedure of virus isolation (Wagner and others 1992, Kirisawa and others 1993, Lawrence and others 1994). Novel molecular platforms such as real-time PCR have allowed the study of EHV-1 viral kinetics by quantitating viral load (Allen and Breathnach 2006, Hussey and others 2006, Pusterla and others 2006). Molecular analysis of EHV-1 strains has recently shown a single-point mutation in the open reading frame 30 (ORF30) in 86 per cent of neuropathogenic EHV-1 strains (Nugent and others 2006). The sequence variation occurs in the DNA polymerase gene of the virus, which is involved in initial viral replication within infected cells and may also be involved in the establishment of latency and reactivation. The need to distinguish between neuropathogenic and non-neuropathogenic EHV-1 strains is crucial for implementation of management practices that decrease the risk of exposure of susceptible horses, as well as for the study of the pathogenesis of EHV-1. So far, strain differentiation has only been described by nucleotide sequence determination or sequence capture PCR coupled with restriction fragment length polymorphism (RFLP) (Allen 2006). Real-time TaqMan PCR is a unique and proven molecular diagnostic tool that allows for the differentiation of a single nucleotide poly morphism (SNP) as exemplified by the commercial availability of more than four million assays for human SNPs (Applied Biosystems). DNA TaqMan probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridisation-based assays. The goal of the study reported here was to design and validate real-time TaqMan PCR assays based on the MGB probe chemistry able to differentiate between non-neuropathogenic and neuropathogenic EHV-1 field strains based on the A2254G SNP in the ORF30 of the DNA polymerase gene. Formalin-fixed and paraffin-embedded brain tissues from eight horses with EHV-1 myeloencephalopathy confirmed by immunoperoxidase staining were used for this study. Formalinfixed and paraffin-embedded placentas from five mares with confirmed EHV-1 abortion by immuno peroxidase staining served as non-neurological EHV-1 controls. Additionally, 86 field samples (whole blood and nasopharyngeal secretions) collected from 52 adult horses known to have been exposed to an EHV-1 myeloencephalopathic strain during an outbreak were also analysed. DNA was extracted from the samples using the NucPrep chemistry on a semi-automated nucleic acid handling system (6100 Sample PrepStation; Applied Biosystems) according to the manufacturer’s guidelines. The genomic DNA (gDNA) was eluted in a volume of 200 μl. Each gDNA sample was run with a universal 18S rRNA (ssrRNA; Applied Biosystems) for quality control purposes. The following real-time TaqMan PCR assays were used: a glyco protein B-based EHV-1 real-time TaqMan PCR (ORF33; NC_001491) (Pusterla and others 2006); a DNA polymerase-based EHV-1 with a TaqMan MGB probe (5 ́ label 6FAM; Applied Biosystems) specific for the neuropathogenic EHV-1 strain (ORF30; reference strain Ab4, AY665713); and a DNA polymerase-based EHV-1 assay recognising the EHV-1 strain (same PCR primers, TaqMan MGB probe [5 ́ label VIC]) specific for the non-neuropathogenic reference strain V592 (AY464052; Table 1). Surrounding the real-time TaqMan PCR product, two sequencing primers were designed for the generation of a 141 base pair (bp) PCR product to re-sequence the TaqMan PCR target reaction. The PCR primers and TaqMan probes were tested in silico by BLAST analysis for the presence of the correct SNP at 2254 and absence of additional sequence variations within the primer region. Real-time TaqMan PCR reactions contained 400nM of each primer, 80nM of the TaqMan probe and mastermix (Universal TaqMan Mastermix with AmpErase UNG; Applied Biosystems) and 5 μl of the gDNA sample in a final volume of 12 μl. The samples were amplified in a combined thermocycler/fluorometer (7900 HTA; Applied Biosystems) with the standard thermal cycling protocol: two minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. The DNA polymerase EHV-1 real-time TaqMan PCR assays were validated for their discriminatory ability of the SNP at nucleotide position 2254 (A to G transition) using synthetic oligonucleotides (EHV-1 neuropathogenic control: