Cells were washed in ice-cold elution buffer (PBS?+?2?mM EDTA?+?2% FCS) and re-suspended in elution buffer, containing 100 L rat anti-mouse IgG micro-beads (MACS Miltenyi Biotec, Cologne, Germany) for 1?h at 4?C

Cells were washed in ice-cold elution buffer (PBS?+?2?mM EDTA?+?2% FCS) and re-suspended in elution buffer, containing 100 L rat anti-mouse IgG micro-beads (MACS Miltenyi Biotec, Cologne, Germany) for 1?h at 4?C. 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 2′-Deoxycytidine hydrochloride 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. Electronic supplementary material The online version of this article (10.1186/s13567-019-0630-6) contains supplementary material, which is available to authorized users. Introduction As a member of the subfamily, equine herpesvirus type 5 (EHV5) is optimally adapted to its natural host, meaning that infected horses are mainly asymptomatic [1]. EHV5 is endemic in the horse population and plenty of horses shed the virus in nasal secretions and/or carry the virus in peripheral blood mononuclear cells (PBMC) or lymphoid organs. Nonetheless, only a small fraction 2′-Deoxycytidine hydrochloride of them develop severe clinical symptoms [2C10]. The virus typically causes upper respiratory tract disease (e.g. pharyngitis) or keratoconjunctivitis accompanied with clinical signs such as nasal and ocular discharge, tachypnea, coughing, fever, enlarged lymph nodes, anorexia, poor body condition and depression [2, 3, 11C13]. Single case reports linked EHV5 to B cell lymphomas, T 2′-Deoxycytidine hydrochloride FA-H cell leukemia and dermatitis [14C16]. However, the most dreadful complication of an EHV5 infection is the development of fatal equine multinodular pulmonary fibrosis (EMPF) [17]. EMPF is characterized by the presence of multiple fibrotic nodules throughout the lungs. Histologically, marked interstitial fibrosis with an alveolar-like architecture, lined by cuboidal epithelial cells and thickening of the alveolar walls is visible [2, 17, 18]. The high correlation between the presence of EMPF and EHV5 DNA suggests that the virus is involved in the development of lung fibrosis. This is corroborated by the findings of a study on a closely-related gammaherpesvirus murine herpesvirus type 4 (MuHV4). MuHV4 induces lung fibrosis in mice with a progressive deposition of interstitial collagen, increased transforming growth factor and T helper 2 cytokine expression and hyperplasia of type II pneumocytes [19]. Similarly in humans, the development of idiopathic pulmonary fibrosis has been linked to the gammaherpesvirus Epstein-Barr virus (EBV) [20, 21]. In addition, Williams et al. [22] were able to experimentally induce lung fibrosis in horses upon direct delivery of virulent EHV5.