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.