Isolation of macrophages, blind passage and haemadsorption test (HAT)
For blind passage and haemadsorption test (HAT), isolation of PBMC derived macrophages was performed from EDTA-anticoagulated blood from healthy domestic pigs as previously described (Fischer et al., 2020). PBMCs were seeded into 6-well tissue culture plates (Primeria; Corning, Durham, USA) with a density of 1x 107 cells/ml in Iscove′s Modified Dulbecco’s Medium with Ham’s F-12 Nutrient Mix (Thermo Fisher Scientific, Schwerte, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Antibiotic-Antimycotic mix (Thermo Fisher Scientific, Schwerte, Germany). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours. Subsequently, medium was changed to remove non-adherent cells, adherent macrophages were rinsed with 1 ml phosphate buffered saline (PBS) and afterwards wells were replenished with medium supplied with 2 ng/ml GM-CSF (granulocyte macrophage colony-stimulating factor; Biomol, Hamburg, Germany) to facilitate maturation of macrophages. PBMCs were incubated for 24 hours as described above. For blind passaging, medium was removed and replaced by 1 ml of reconstituted plasma (inoculum). After a 2 h adsorption period at 37°C the inoculum was discarded and cultures were rinsed with 1 ml PBS. Afterwards fresh medium without GM-CSF was added. Plates were incubated for 72 h and then stored at -80°C until real-time PCR analysis and processing in HAT.
For proof of infectious virus, PBMCs were seeded into 96-well microplates (Primeria; Corning, Durham, USA) with a density of 5x 106 cells/ml (100 µl per well) and cultured as described above. Subsequently, 100 µl of each blind passage sample was used to inoculate four wells of a 96-well plate respectively (quadruplicate values). Furthermore, virus suspension samples were prepared for endpoint titration. To this means, 10-fold dilution series of these samples were inoculated in quadruplicate. After an 24 h incubation at 37°C, 20 µl of a 1% homologous erythrocyte suspension in PBS were added to each well. For read-out, cultures were analyzed for haemadsorption reactions (formation of rosettes) over a period of 4 days. In a second-round HAT, virus positive blind passage samples and virus suspension samples were titrated in quadruplicate to obtain endpoint titers of biological replicates. Because of virus dilution and the number of replicate wells, the limit of detection is 1.75 log10 50% haemadsorbing doses per ml (HAD50/ml). Titers were calculated using the Reed and Muench method (Reed & Muench, 1938), and were expressed as log10 HAD50/ml. Graphical representation and calculations were performed using GraphPad Prism 8 (GraphPad Software Inc, San Diego, USA) and Excel version 2013 (Microsoft GmbH, Unterschleißheim, Germany), respectively.