Equine herpesvirus 1 (EHV1) replicates in the respiratory tract of horses, after which infected leukocytes transport virus throughout the body, resulting in abortion or nervous system disorders. Two EHV1 strains circulate in the field: neurovirulent and non-neurovirulent. To investigate differences in replication in the upper respiratory tract (URT), an experimental inoculation study in ponies was performed with both strains. Two groups of six ponies, were inoculated intranasally with 10 6.5 TCID 50 of either strain. Clinical signs, nasal shedding and viremia were evaluated. At early time points post inoculation (pi), one pony of each group was euthanized. Tissues were collected for titration and immunostainings. Number and size of EHV1-induced plaques were calculated, and individual EHV1-infected cells were quantified and characterized. Inoculation with either strain resulted in nasal shedding and replication in several tissues of the URT. Both strains replicated in a plaquewise manner in epithelium of the nasal mucosa, but replication in epithelium of the nasopharynx was largely limited to nonneurovirulent EHV1. Plaques were never able to cross the basement membrane, but individual infected cells were noticed in the connective tissue of all examined tissues for both strains. The total number of these cells however, was 3-7 times lower with non-neurovirulent EHV1 compared to neurovirulent EHV1. CD172a + cells and CD5+ lymphocytes were important target cells for both strains. Interestingly, in lymph nodes, B-lymphocytes were also important target cells for EHV1, irrespective of the strain. Viremia was detected very early pi and infected cells were mainly CD172a + for both strains. In summary, these results are valuable for understanding EHV1 pathogenesis at the port of entry, the URT.

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 (NAD) 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.

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BackgroundHerpes simplex virus infections are highly prevalent in humans. However, the current therapeutics suffer important drawbacks such as limited results in neonates, increasing occurrence of resistance and impeded treatment of stromal infections. Remarkably, interactions of herpesviruses with human mucosa, the locus of infection, remain poorly understood and the underlying mechanisms in stromal infection remain controversial.Methodology/Principal FindingsA human model consisting of nasal respiratory mucosa explants was characterised. Viability and integrity were examined during 96 h of cultivation. HSV1-mucosa interactions were analysed. In particular, we investigated whether HSV1 is able to reach the stroma.Explant viability and integrity remained preserved. HSV1 induced rounding up and loosening of epithelial cells with very few apoptotic and necrotic cells observed. Following 16–24 h of infection, HSV1 penetrated the basement membrane and replicated in the underlying lamina propria.Conclusions/SignificanceThis human explant model can be used to study virus-mucosa interactions and viral mucosal invasion mechanisms. Using this model, our results provide a novel insight into the HSV1 stromal invasion mechanism and for the first time directly demonstrate that HSV1 can penetrate the basement membrane.

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