Development and use of a polarized equine upper respiratory tract mucosal explant system to study the early phase of pathogenesis of a European strain of equine arteritis virus

The upper respiratory tract mucosa represents the first line of defense, which has to be overcome by pathogens before invading the host. Considering the economic and ethical aspects involved in using experimental animals for pathogenesis studies, respiratory mucosal explants, in which the tissue's three-dimensional architecture is preserved, may be ideal alternatives. Different respiratory mucosal explant cultures have been developed. However, none of them could be inoculated with pathogens solely at the epithelium side. In the present study, equine nasal and nasopharyngeal explants were embedded in agarose (3%), leaving the epithelium side exposed to allow apical inoculation. Morphometric analysis did not show degenerative changes during 72 h of cultivation. The number of apoptotic cells in the mucosa slightly increased over time. After validation, the system was used for apical infection with a European strain (08P178) of equine arteritis virus (EAV) (107.6TCID50/mL per explant). Impermeability of agarose to virus particles was demonstrated by the absence of labeled microspheres (40 nm) and a lack of EAV-antigens in RK13 cells seeded underneath the agarose layer in which inoculated explants were embedded. At 72 hpi, 27% of the EAV-positive cells were CD172a+ and 19% were CD3+ in nasal explants and 45% of the EAV-positive cells were CD172a+ and 15% were CD3+ in nasopharyngeal explants. Only a small percentage of EAV-positive cells were IgM+. This study validates the usefulness of a polarized mucosal explant system and shows that CD172a+ myeloid cells and CD3+ T lymphocytes represent important EAV-target cells in the respiratory mucosa.