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.