The pathogenesis of EHV1 in horses : novel insights from experimental inoculations and field situations

Equine herpesvirus 1 (EHV1) is the most important cause of infectious abortion in horses worldwide and can also lead to severe nervous system disorders with frequent fatal outcome. Infection occurs via respiratory route and after local replication in tissues of the upper respiratory tract, the virus spreads via a cell-associated viremia to target internal organs such as the uterus or the nervous system. Secondary replication of EHV1 in endothelial cells of these organs can result in late term abortion or nervous system disorders. Vaccination with the currently available vaccines cannot prevent clinical symptoms such as abortion or nervous system disorders. Moreover, treatment of these symptoms is only supportive and specific medication is not available. Therefore, an improvement of existing vaccines and/or the development of therapies are necessary. Unfortunately, at the start of this thesis, several important steps in the pathogenesis of this disease were still not fully clarified, making it difficult to find new directions in the strategy to develop better vaccines and to create drugs that inhibit steps of the pathogenesis. In order to achieve this goal, studies in the natural host of EHV1, the horse, under both experimental and field conditions, are of paramount importance to obtain relevant and correct information about the pathogenesis of this disease. Firstly, a thorough knowledge of the complete pathogenesis from primary replication and invasion via cell-associated viremia to the target organs is urgent. Therefore, the first aim of this thesis was to gain a more detailed insight in the invasion of neurologic and non-neurologic strains of EHV1 from the upper respiratory tract to the carrier cells in the blood stream by means of experimental infection studies in horses (Chapter III). The second aim was to examine how individual EHV1-infected cells escape from immune effector mechanisms at the primary site of replication (Chapter IV). Secondly, to develop suitable vaccination and treatment strategies during natural occurring outbreaks of disease, field information about the epidemiology of the virus is needed. The third and last aim was therefore to gain more information about naturally occurring outbreaks in Belgium in terms of risk factors, protective properties of vaccination, the effect of experimental therapies and diagnosis (Chapter V)

Prevalence of respiratory pathogens in nasal swabs from horses with acute respiratory disease in Belgium

Contagious respiratory infections are an important cause of respiratory disease in horses, resulting in impaired pulmonary function, poor performance and sometimes severe illness. Although bacterial infections are often suspected to be involved, viruses are frequently overlooked and are an underestimated cause of respiratory disease outbreaks in horses. In this study, nasal swabs of 103 horses with acute symptoms of respiratory disease were analyzed for the presence of 13 different respiratory pathogens. Gamma herpesviruses were the most commonly detected, with 60% of the samples being positive, followed by streptococcus equi subsp. zooepidemicus infection (30%). Rhinovirus B, streptococcus equi subsp. equi, adenovirus 1 and EHV-4 were more rarely detected. Further research is necessary to correctly interpret the importance of gamma herpesviruses in horses, for example by screening a healthy control population. National surveillance of respiratory viruses in horses by PCR analysis on nasal swabs might be a useful, early warning system for viral epidemics

Neonatal foal death due to infection with equine arteritis virus in Belgium

This case report describes a small outbreak of neonatal foal death in Belgium due to infection with equine arteritis virus (EAV). The outbreak started with one foal suffering from acute dyspnea four days after birth. Despite intensive treatment, this foal died within a few hours. Three weeks later, another foal was born on the same farm in a healthy condition, although placental edema was found. At the age of 10 days, the foal showed acute respiratory distress and severe dyspnea and died shortly after. Post-mortem examination of both foals revealed consolidated diaphragmatic lung lobes with compensatory emphysema. Histologic lesions consisted of a mild acute interstitial pneumonia. Microscopic examination of the allantochorion from the second foal showed a focal necrotizing vasculitis, and immunolabeling demonstrated the presence of EAV antigens in chorionic vascular endothelial cells and macrophages. Upon inoculation of rabbit kidney (RK13) cell culture with a suspension of lung tissue, a cytopathic effect was observed. The agent was identified as equine arteritis virus by means of immunostainings

Description of an unusually large outbreak of nervous system disorders caused by equine herpesvirus 1 (EHV1) in 2009 in Belgium

Neurological diseases caused by neuropathogenic strains of EHV1 are being reported with increasing frequency. Consequently, concern is being voiced within the US horse industry that the neurologic form of EHV1 may be intensifying in prevalence and/or morbidity and mortality. In Belgium, outbreaks of EHV1-induced abortions are an annually recurrent phenomenon, but outbreaks of equine herpes myelitis (EHM) are rare. This report describes an unusually large outbreak of EHV1-induced EHM that involved at least 13 different premises. Seven of them were characterized in more detail and were included in this study. A morbidity of 26% was seen, with an EHM incidence of 43% in the affected horses. The outbreak was characterized by rapid occurrence of ataxia and paralysis immediately after disappearance of the fever. EHV1 was diagnosed by means of virus isolation and/or seroconversion. The isolated virus was classified either as neuropathogenic or as belonging to group 4 after sequencing in the ORF30 and ORF68 regions, respectively. The extent of this outbreak and the high percentage of neurological disease, along with the fact that EHM is only sporadically seen in Belgium, might indicate that the neurological form of EHV1 is possibly also emerging in Belgium

The protective properties of vaccination against equine herpesvirus 1-induced viremia, abortion and nervous system disorders

Equine herpesvirus (EHV) 1 is an important pathogen of horses. Upon infection, the virus replicates in the upper respiratory tract. Then it spreads to internal organs via a cell-associated viremia. Local replication in internal organs may result in abortion and nervous system disorders. The currently designed vaccines are not able to induce significant protection against EHV1-induced viremia. However, several vaccines are able to induce a significant level of protection against either abortion or nervous system disorders. Which immune responses correlate with this protection is so far unknown. Besides vaccination, management will remain a crucial factor in the prevention of EHV1-induced clinical signs. Management measures include the vaccination of all horses on the premises to reduce infection pressure; the separate housing of young horses, adult horses and pregnant mares; strict hygiene measures and strict control of contact with horses from outside the premises. If, despite vaccination and thorough management, an outbreak of EHV1-induced disease occurs, treatment with antiviral agents may be useful

The pathogenesis and immune evasive mechanisms of equine herpesvirus type 1

Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1

Replication kinetics of neurovirulent versus non-neurovirulent equine herpesvirus type 1 strains in equine nasal mucosal explants

Equine herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N -> D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D-752 or N-752, to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N-752 isolates and at 24 h p.i. for all D-752 isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D-752 isolates compared with N-752 isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals

Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies

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