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 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).