The mucosa of the upper respiratory tract (URT) represents the first line of defense against respiratory pathogens but often also serves as a portal of entry for several microorganisms, whether or not causing generalized infection. The principal viral agents responsible for respiratory disorders in horses are equine herpesvirus-1 (EHV-1) and equine herpesvirus-4 (EHV-4) , equine arteritis virus (EAV)  and equine influenza virus . A previous system used by Vandekerckhove et al. allowed cultivating four representative tissues of the equine URT up to 96 h without significant changes in morphology and viability . In that model, the explants were cultivated on gauzes allowing nutrients to penetrate through all sides of the mucosa. Because of the small size of the explants (0.5 cm2, few mm thick) and the open edges, nutrients have free access to the inner parts of the tissues. In this system, pathogens, just like nutrients, do not only enter the mucosa at the upper epithelium side, but also at the edges through the open spaces of the connective tissue. In fact, inoculation of such explant systems with PRV  or EAV 08P178 (our own studies, not published) resulted in an extensive spread of the virus, mainly at the bottom and lateral sides of the mucosal explants. Therefore, in order to better mimic the in vivo situation, we established a new model in which, through agarose embedded explants, a polarized system was obtained so that pathogens could only enter through the upper epithelium. The results of morphometric analysis of the epithelium, basement membrane and connective tissue and of TUNEL assays demonstrate that the agarose system allows cultivating explants up to 72 h without substantial alterations in the morphology and the viability of the tissues. Further, an assay based on streptavidin-labeled microspheres together with the absence of EAV-antigens in RK13 cells seeded underneath the agarose layer in which inoculated explants were embedded and the lack of viral antigen-positive cells at the cutting edges of the agarose embedded explants, demonstrate that the access of the pathogens in the system was restricted to the apical side of the epithelium.
We recently showed that, upon in vivo oro-nasal inoculation, EAV 08P178 starts its replication in nasal and nasopharyngeal regions and lungs . Therefore, to gain more insight into the early phase of EAV respiratory infection, kinetic studies were performed by inoculating the agarose embedded nasal and nasopharyngeal mucosal explants with EAV 08P178. Currently, little is known about the early phases of EAV pathogenesis such as (i) which strategy EAV uses to invade the host, (ii) what are the first cells to be infected at the entry site or (iii) how blood vessels are reached to disseminate the virus throughout the body. The current study showed that in the explant model, the majority of EAV-positive cells were CD172a+ myeloid cells followed by CD3+ T-lymphocytes, whereas only a small percentage were IgM+ B-lymphocytes. Previously, we speculated that, at the level of the URT, EAV hijacks mononuclear leukocytes to penetrate the BM and evade the immune response . In possible support of this, the present study shows a high percentage of EAV-infected CD172a+ cells. CD172a was expressed on equine monocytes, macrophages, dendritic cells (DC) and granulocytes. DC have specific characteristics: (i) they have a major role in activating naïve and resting antigen-specific T cells , (ii) they are migratory cells, unlike differentiated macrophages  and (iii) they are abundantly present within the airway epithelium  forming a network of antigen presenting cells in the respiratory mucosa [17, 18]. DC play a central role in initiating an immune response against infecting agents  but may also contribute to the pathogenesis and the spreading of several respiratory pathogens as shown for human  and simian  immunodeficiency viruses, cytomegalovirus  and Mycobacterium tuberculosis. Hence, it is tempting to speculate that DC could play an important role in the early phases of EAV pathogenesis. This theory could corroborate the findings of Del Piero that described EAV-antigens within stromal dendrite-like cells of lymph node sinuses and spleen . On the contrary, since the main function of myeloid cells is to capture antigens, CD172a+ cells may be positive for the EAV nucleocapsid protein due to phagocytosis rather than infection.
It is generally believed that EAV has a tropism mainly for the monocyte/macrophage population. However, Castillo-Olivares et al. showed that EAV-infected PBMC are negative for the human monocyte/granulocyte marker L1 (calprotectin) . In the present study, both T-lymphocytes and cells of the monocyte/macrophage lineage were found to be infected with EAV. Other studies have also demonstrated that EAV can infect CD3+ T lymphocytes in PBMC in vitro [25, 26].
In the present study, 37 to 64% of EAV-positive cells were identified. Since EAV has a wide tropism , the percentage of non-identified EAV-positive cells may be epithelial cells, endothelial cells, mesenchymal stromal cells, and natural killer cells. In fact, in vivo, EAV-antigens were demonstrated within the cytoplasm of endothelial cells, myometrial and cardiac myocytes, chorionic mesenchymal stromal cells and epithelial cells such as alveolar pneumocytes, enterocytes, adrenal cortical cells, trophoblast, thymus stroma, and renal tubular cells . Therefore, it will be interesting to further identify the remaining EAV-positive cells in future experiments using the agarose explant system.
Although polarized mucosal explants are much more physiological than non-polarized ones and in vitro cell cultures, they are only suitable to study the early stages of the pathogenesis of viruses that use the respiratory tract as a portal of entry. It must be kept in mind that the mucosal explants not fully represent the whole scope of virus-animal interactions since the inflammatory processes including invasion of blood elements are absent. However, preliminary results obtained with nasal mucosa from experimentally EAV-inoculated ponies were very similar to those of the present in vitro study, confirming the relevance of the polarized mucosal explant system. In conclusion, the present study demonstrates that the agarose embedded explant model represents a valid tool to study some aspects of the early steps in the pathogenesis of equine respiratory viruses such as EAV and that mucosal CD172a+ myeloid cells and CD3+ T lymphocytes represent important target cells for EAV.