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The objective of this study was to detect and characterize latent equine herpes virus (EHV)-1 and -4 from the submandibular (SMLN) and bronchial lymph (BLN) nodes, as well as from the trigeminal ganglia (TG) of 70 racing Thoroughbred horses submitted for necropsy following sustaining serious musculoskeletal injuries while racing. A combination of nucleic acid precipitation and pre-amplification steps was used to increase analytical sensitivity. Tissues were deemed positive for latent EHV-1 and/or -4 infection when found PCR positive for the corresponding glycoprotein B (gB) gene in the absence of detectable late structural protein gene (gB gene) mRNA. The EHV-1 genotype was also determined using a discriminatory real-time PCR assay targeting the DNA polymerase gene (ORF 30). Eighteen (25.7%) and 58 (82.8%) horses were PCR positive for the gB gene of EHV-1 and -4, respectively, in at least one of the three tissues sampled. Twelve horses were dually infected with EHV-1 and -4, two carried a latent neurotropic strain of EHV-1, six carried a non-neurotropic genotype of EHV-1 and 10 were dually infected with neurotropic and non-neurotropic EHV-1. The distribution of latent EHV-1 and -4 infection varied in the samples, with the TG found to be most commonly infected. Overall, non-neurotropic strains were more frequently detected than neurotropic strains, supporting the general consensus that non-neurotropic strains are more prevalent in horse populations, and hence the uncommon occurrence of equine herpes myeloencephalopathy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Recrudescence of latent equine herpesvirus 1 (EHV-1) with subsequent viral shedding via nasal secretions is a potential source of infection for susceptible horses and has been implicated in outbreaks occurring in closed populations., Objectives: To describe the viral kinetics of reactivated EHV-1 in blood and nasal secretions from latently infected horses after administration of corticosteroids, and to study the infectious nature of reactivated EHV-1 to sentinel horses., Animals: Eight healthy horses., Methods: Four horses infected 4 months previously with EHV-1 received dexamethasone on 5 consecutive days. Four seronegative horses served as sentinels and had direct contact with the latently infected horses. All horses were monitored daily for development of clinical signs. Whole blood and nasal secretions were collected daily for molecular detection and cell culture of EHV-1. Serum was collected weekly for the detection of antibodies against EHV-1., Results: All horses in the latently infected group showed transient molecular detection of EHV-1 in blood and nasal secretions, but only 1 horse developed fever. Three latently infected horses developed an increase in antibody concentrations against EHV-l. Viral cultures remained negative for all latently infected horses after corticosteroid administration. None of the sentinel horses developed clinical signs, viremia, viral shedding, or seroconversion., Conclusions and Clinical Importance: EHV-1 was successfully reactivated after corticosteroid administration in latently infected horses. However, transmission of reactivated virus to sentinel horses was unsuccessful. Failure to effectively transmit EHV-1 to susceptible horses may have resulted from the low level and short period of viral shedding in latently infected horses., (Copyright © 2010 by the American College of Veterinary Internal Medicine.)

This short review considers how the human herpesviruses were among the first viruses to be effectively treated by means of antiviral therapy although the ability of alphaherpsviruses to establish neuronal latency with reactivation remains the major obstacle to achieving a cure. Laboratory animals played an essential role in the development of herpes antivirals including our understanding of the complexity of the neurological infection in relation to chemotherapy. The existence of natural herpesvirus infections in domestic species also contributes to our understanding of latency and reactivation relevant to antiviral therapy although the use of antivirals to treat or prevent virus infections in veterinary species has been minimal, to date. The review briefly focuses on herpes infections in the horse and cat where some progress has already been achieved in the veterinary antiviral field.

Equid herpesvirus 1 (EHV-1) is the most common cause of virus-induced abortion in horses. After primary infection the virus becomes latent predominantly in the respiratory tract lymph nodes and the genome can also be detected in the peripheral nervous system. The role of mouse as a feasible model for the establishment of latency and reactivation of EHV-1 was investigated. Intracerebral and intranasal infections of 3- and 17-day-old mice were made and virus replication was confirmed by virus isolation and detected by indirect immunofluorescence (IIF) in brain. For reactivation studies, the mice were killed 8 weeks post infection and tissues were collected for cocultivation. In mice from both age groups, infectious virus was not detected by cocultivation. Following attempts to reactivate virus in vivo with corticosteroids, the viral antigen was detected at low levels by IIF and the expression of the gB gene by reverse transcription polymerase chain reaction (RT-PCR) in brain, trigeminal ganglia, olfactory lobe, lung and spleen. Virus was also detected by IIF following incubation of tissue explants in the growth medium containing pokeweed mitogen (PWM). These results show the limitations of the mouse model for investigating EHV-1 latency and highlights the issue of 'ineffective reactivation' of virus.

In this study, an improved polymerase chain reaction (PCR) was used for detection of DNA of latent EHV-1 strains from several sources. Three pairs of oligonucleotide primers spanning fragments of 333 bp, 226 bp and 268 bp of the thymidine kinase (tk) gene, and one primer pair spanning 225 bp of the glycoprotein C (gC) gene were used in specific amplifications. Primers for EHV-4 PCR were also designed. Restriction digests with TaqI confirmed the identity of tk PCR fragments from EHV-1. The sensitivity to detect PCR products was further improved by visualisation in silver-stained acrylamide gels. PCR assays were applied to 267 samples including pools of tissue, peripheral blood leukocytes (PBL) and nasal swabs of archived, farms and abattoir specimens from a total of 116 animals. The EHV-1 DNA was found in 88% of the analysed samples. The prevalence of the EHV-1 latent or persistent form in adult horses was similar to others reports but found higher than previously described in foetuses and young foals. EHV-4 latency was not detected in the Brazilian studied specimens.

IL-2 and equine chorionic gonadotrophin (eCG) initiated reactivation of equid herpesvirus-1 (EHV-1) from venous lymphocytes at a frequency of 1/10(-5). Indirect immunofluorescence showed that > 80% of virus-positive leukocytes were CD5+/CD8+ with the remaining 20% being CD5+/CD8-/CD4-. Cocultivation demonstrated that the reactivated virus was infectious. In addition, virus was reactivated in vitro from leukocytes of > 70% of horses by the mitogens phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Transfer of supernatants showed that IL-2 and eCG acted indirectly by causing the release of other mediators from adherent cells; these mediators then reactivated EHV-1 from T cells. Blocking experiments with anti-IL-2 showed that PWM and PHA acted via IL-2 but that eCG did not. This is the first clear definition of the lymphoid cells that harbour latent EHV-1 in vivo and correlates with current RT-PCR and in situ hybridization of latency-associated transcripts in lymphocytes. This method of reactivation in vitro can be used to detect horses carrying latent EHV-1 in vivo and also has the potential to dissect the sequence of events involved in reactivation in vitro.

Results from Southern hybridization and PCR amplification experiments using a randomly synthesized reverse transcription-PCR product showed that peripheral blood leukocytes from horses showing no clinical signs of disease expressed a putative latency-associated transcript antisense to and overlapping the 3' end of the equid herpesvirus 1 (EHV-1) immediate-early gene (gene 64). A PCR product derived from this transcript has > or =96% identity with the published EHV-1 sequence. In situ hybridization studies of equine bronchial lymph nodes corroborated these findings and are consistent with reactivation data (D. A. Smith, A. Hamblin, and N. Edington, unpublished data), indicating that EHV-1 latency is established predominantly in CD5+/CD8+ leukocytes.

Equine herpesvirus-1 (EHV-1) latency in murine olfactory bulbs was demonstrated by a novel combined in situ PCR and in vitro protein synthesis method (in situ PS-PCR). The Escherichia coli lacZ gene replacing a deletion in EHV-1 gene 71 (EUS4) was thus amplified and transcribed/translated in situ followed by enzymatic detection using X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside). beta-Galactosidase was found to be concentrated over mitral/tufted neurons indicating those to be the sites of latency. Our results suggest that, in common with other alpha-herpesviruses, EHV-1 can establish latency in central nervous system neurons and that the unique membrane glycoprotein encoded by EHV-1 gene 71 is nonessential for infection of neural tissues.

Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares a similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.

Equid herpesviruses 1 or 4 (EHV-1 or -4) were isolated by cocultivation from 60% of 40 horses examined at slaughter. The lymph nodes draining the respiratory tract were the most common source of virus. EHV-1 or EHV-4 was never isolated from the trigeminal ganglia (SLG). The polymerase chain reaction (PCR) detected virus in 87.5% of bronchial lymph nodes and a similar level in the trigeminal ganglia that were examined. By both assays approximately one third of the positive animals harboured both viruses. Equid herpesvirus 2 (EHV-2) was isolated from all but one of the horses and from > 75% of the lymph nodes draining the respiratory tract; alpha viruses were isolated only in the presence of EHV-2. The results indicate that latent EHV-1 and EHV-4 are widespread in the equine population and that the primary site of latency is the lymph nodes of the respiratory tract.