Virus isolation
Virus isolation was performed on PK-15 cells grown in 96-well tissue culture plates. Briefly, PK-15 cells were seeded at 500,000 cells per well in 96 well tissue culture plates using α-MEM supplemented with 2 mM L-glutamine, 5mg/mL gentamicin and 2% γ-irradiated Fetal Bovine Serum (FBS). Ten percent (W/V) homogenates were prepared from each tissue and 25 µl of each suspension was added to each well of the PK-15 cells. A positive control plate was inoculated with a PRV variant strain (JS-2012). The plates were incubated for 48 hours in a 5% CO2 humid air incubator at 37°C and were checked daily for cytopathic effect (CPE) and possible contamination using a light microscope. Two days post infection, the cells were fixed using 200 µl of fixation fluid (Acetone, PBS, and Bovine serum albumin fraction V) per well, and stained using porcine-origin PRV antiserum. Briefly, the plates were rehydrated for 30 minutes (using 150 µl of PBS at room temperature), incubated with PRV antibody positive polyclonal serum (diluted 1:100 in PBS-0.1%Tween 20) for 30 minutes at 37°C, followed by a 30-minute incubation with rabbit anti-swine IgG HRP conjugate at 37°C and finally for 15 minutes at 37°C with t3-Amino-9-ethylcarbazole (AEC) substrate solution. After the substrate incubation, the plates were washed, and positive staining recorded under a light microscope. PRV JS-2012 and Bristol strains with known titers were used as positive controls.