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.