Unravelling the first key steps in equine herpesvirus type 5 (EHV5) pathogenesis using ex vivo and in vitro equine...

Equine herpesvirus type 5 (EHV5) is a ubiquitous, yet obscure pathogen in the horse population and is commonly associated with fatal equine multinodular pulmonary fibrosis (EMPF). To date, little is known about the precise pathogenesis of EHV5. Here, we evaluated the dynamics of EHV5 infection in representative ex vivo and in vitro equine models, using immunofluorescence staining and virus titration. EHV5 was unable to infect epithelial cells lining the mucosa of nasal and tracheal explants. Similarly, primary equine respiratory epithelial cells (EREC) were not susceptible to EHV5 following inoculation at the apical or basolateral surfaces. Upon direct delivery of EHV5 particles to lung explants, few EHV5-positive cell clusters were observed at 72 hours post-inoculation (hpi). These EHV5-positive cells were identified as cytokeratin-positive alveolar cells. Next, we examined the potential of EHV5 to infect three distinct equine PBMC populations (CD172a⁺ monocytes, CD3⁺ T lymphocytes and Ig light chain⁺ B lymphocytes). Monocytes did not support EHV5 replication. In contrast, up to 10% of inoculated equine T and B lymphocytes synthetized intracellular viral antigens 24 hpi and 72 hpi, respectively. Still, the production of mature virus particles was hampered, as we did not observe an increase in extracellular virus titer. After reaching a peak, the percentage of infected T and B lymphocytes decayed, which was partly due to the onset of apoptosis, but not necrosis. Based on these findings, we propose a model for EHV5 pathogenesis in the horse. Uncovering EHV5 pathogenesis is the corner step to finally contain or even eradicate the virus.