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Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.

Summary Reasons for performing study: Neurological disease in horses caused by infection with certain ‘paralytic’ strains of equine herpesvirus‐1 (EHV‐1) is a potentially devastating condition the pathogenesis of which is poorly understood. Preliminary observations in both experimentally induced and naturally occurring cases of the central nervous system disease have revealed a more robust cell‐associated viraemia in horses infected with paralytic isolates of EHV‐1, relative to horses infected with abortigenic isolates. To investigate further this pathogenesis ‐ rdevant question, the present study was performed using a greater number of horses and a more precise method for quantification of EHV‐1 DNA present in viraemic leucocytes. Objective: To compare the magnitude and duration of leucocyte‐associated viraemia in seronegative, age‐matched foals following infection with paralytic vs. abortigenic isolates of EHV‐1. Methods: Peripheral blood mononuclear cells (PBMC) were collected from 20 weanling foals at 2, 4, 7, 9, 11, 14 and 21 days after intranasal inoculation with either paralytic or abortigenic isolates of EHV‐1. The amount of EHV‐1 DNA present in each PBMC sample was measured by real‐time quantitative PCR. Results: Foals inoculated with paralytic strains of EHV‐1 developed both a greater magnitude and longer duration of PBMC‐associated viraemia than foals inoculated with abortigenic strains of the virus. Conclusions: Both the higher magnitude and longer duration of cell‐associated viraemia contribute to the risk for development of neurological signs in horses infected with paralytic strains of EHV‐1. Potential relevance: Our results provide empirically derived, scientific data that contributes to a better understanding of the pathogenetic basis for the differing abilities of paralytic and abortigenic strains of EHV‐1 to cause post infection central nervous system disease in the horse. The findings identify the importance of minimising the quantitative burden of viraemic leucocytes that follows exposure to the virus, by the use of effective therapeutic antiviral drugs and efficacious prophylactic vaccines that stimulate cytotoxic immune responses against EHV‐1 infected cells.

Summary Equine herpesvirus‐1 (EHV‐1) remains a frequent cause of upper respiratory tract infection and abortion in horses worldwide. However, little is known about the local antibody response elicited in the upper airways of horses following exposure to EHV‐1. This study analysed the mucosal humoral immune response of weanling foals following experimental infection with virulent EHV‐1, or vaccination with either of 2 commercial vaccines. Twenty weanlings were assigned to 5 groups and were inoculated with, or vaccinated against, EHV‐1 following different regimens. Finally, all weanlings were simultaneously challenged intranasally with virulent EHV‐1 Army 183 (A183). Nasal wash and serum samples were collected at regular intervals until 13 weeks after final challenge. Nasal washes were assayed for EHV‐1‐specific equine IgGa, IgGb, IgG(T), IgA, IgM and total virus‐specific antibody using an indirect, quantitative ELISA. Total serum antibody responses were also monitored, and clinical signs of EHV‐disease were recorded for each individual. Virus‐specific IgA dominated the mucosal antibody response elicited in weanlings inoculated with A183, being detectable at up to 3.1 μg/mg total IgA 13 weeks after challenge. Neither inactivated EHV‐1 administered i.m., nor attenuated EHV‐1 administered intranasally induced detectable mucosal antibodies. EHV‐1‐specific mucosal antibodies impeded EHV‐1 plaque formation in vitro. Such virus‐neutralising antibody probably contributes to a reduction of shedding of EHV‐1 from the respiratory tract of virus‐infected horses.

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