2.3. Viral isolation
For viral isolation, we tested two different cell lines known to support coronavirus replication. We used PK-15 and Vero cells which were plated in 24-well plates at 90% of confluence. PK-15 cells were kindly provided by Dr. Jhon Pasick from the Canadian Food Inspection Agency (Ontario, Canada) and Vero cells by the Naval Medical Research Unit Six (NAMRU-6). Briefly, 100 µl of intestinal content was filtered and treated with TPCK-trypsin (10 µg/ml) to allow ligand cleavage for further virus attachment to cell receptors. Treated samples were inoculated and incubated for 1 h, followed by replacement of maintenance media with trypsin. Cells were incubated for 5 days at 37 °C with 5% CO2, with a second blind passage. Cytopathic effect was evaluated daily, and pictures recorded under light microscopy (Leica Microsystems) using a Leica MC170 HD camera (Leica Microsystems). Following isolation, we performed an immunofluorescence test using a PDCoV anti-nucleoprotein monoclonal antibody (SD55-197), kindly provided by Drs. Eric Nelson and Steven Lawson from the South Dakota State University (South Dakota, US), to confirm the presence of the PDCoV.