Figure 4
Number of antigen-positive cells/8 mm2in agarose embedded nasal (a) and nasopharyngeal (b) explants. Of each donor animal and at each collection time, sixty 8 μm-thick cryosections of nasal mucosa and sixty 8 μm-thick cryosections of nasopharyngeal mucosa were cut and fixed in methanol. EAV nucleocapsid (N) protein was visualized with mAb 17D3, directly labeled with Zenon® Alexa Fluor® 488 Mouse IgG1 Labeling Kit, according to the manufacturer’s instructions. The nuclei were counterstained with Hoechst 33342. The number of viral-antigen positive cells was determined in an area of 8 mm2 for each nasal and nasopharyngeal mucosae. The number of EAV-positive cells increased in time with a mean of 68 in 8 mm2 of nasal mucosa and 225 in 8 mm2 of nasopharyngeal mucosa at 72 hpi. In particular, 54, 74 and 76 antigen-positive cells/8 mm2 were counted in nasal explants and 201, 220 and 254 EAV-positive cells/8 mm2 were quantified in nasopharyngeal explants of horse 1, 2 and 3, respectively.