Virus isolation on porcine macrophages and on WSL cells
Virus isolation and titration were completed with macrophages derived
from peripheral blood mononuclear cells (PBMCs). Blood was collected
from healthy domestic pigs in heparin tubes. The whole blood was diluted
1:1 in phosphate-buffered saline (PBS), 35 mL of diluted blood was
overlaid on 12 mL of Pancoll (PAN-Biotech, Aidenbach, Germany) and spun
at 730 x g for 40 minutes at 20°C with slow acceleration and no brake.
The PBMCs were collected and washed twice in PBS and passed over 70 µm
nylon strainers to remove any fatty debris. For blind passages, 5 x
106 PBMCs were seeded into each well of 24-well
Corning Primaria plates (Corning, Durham, USA). Titrations were
completed with 7.5 x 106 cells per mL in 96-well
Corning Primaria plates (100μL per well; Corning, Durham, USA).
PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS), 2% antibiotics, 75 µL mercaptoethanol (Merck,
Darmstadt, Germany) and 2.5 ng/mL granulocyte macrophage
colony-stimulating factor (GM-CSF; Biomol, Hamburg, Germany) for the
first day, then with 5 ng/mL GM-CSF from the second day after a media
change.
A volume of 300 µL soil supernatant was inoculated per well on a 24-well
plate with 700 µL of media. The following day, the cells were washed
with media once and the supernatant in each well was replaced with 1 mL
of fresh media. The cells were cultivated for 5 days prior to freezing
at -80°C for subsequent virus titration.
Wild boar lung cells (WSL) were cultivated in Iscove’s Modified
Dulbecco’s Medium with Ham’s F-12 Nutrient Mix (Thermo Fisher
Scientific), 10% FBS and 2% antibiotics.
For virus isolation, WSL cells were seeded the day before with
106 cells per well in a 24-well tissue culture plate
(Corning, Durham, USA). A volume of 300 µL soil supernatant was
inoculated on a 24-well plate with 700 µL of media, and subsequently
changed the next day and replaced with 1 mL of fresh media. The cells
were cultivated for 5 days prior to freezing at -80°C for subsequent
virus titration. Titrations were completed with 4 x
105 WSL cells per mL using 100 μL per well in a
96-well tissue culture plate (Corning, Durham, USA) seeded one day prior
to inoculation. Cells were inoculated with 100 µL of virus isolation
supernatant from one round of virus amplification in the 24-well plate
described above. Titrations were completed in ten-fold dilutions
starting with 10-1 to 10-8 and
calculated by the Spearman-Kärber method log10 50% end
point dilution with a limit of detection of 1.75
TCID50/mL.