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.