ASFV Propagation in Porcine Alveolar Macrophages

Porcine alveolar macrophage (PAM) cells were freshly harvested from swine lungs according to the OIE Manual’s instructions [27 ]. PAM cells were used for the propagation of the highly virulent ASFV_HU_2018 isolate of the African swine fever virus (Accession Number: MN715134.1). PAM cells were grown in RPMI 1640-containing L-glutamine (Lonza, Basel, Switzerland) medium supplemented with 10% fetal bovine serum (Euro Clone, Pero, Italy), 1% Na-pyruvate (Lonza), 1% non-essential amino acid solution (Lonza), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO₂ in air gas phase. The infectious titer of serially diluted viral stock was calculated using an immunofluorescence (IF) assay as described earlier [22 (link)]. PAMs were cultivated in 6-well plates at a density of 3.3 × 10⁵ cells and infected at a multiplicity of infection (MOI) of 10 plaque-forming units per cell at 4 h after cell seeding. The supernatant was replaced with a fresh medium after 1 h post-infection (hpi). Infected PAM cells were harvested at 4, 8, 12, and 20 hpi, whereas mock-infected cells were harvested at 20 hpi IF assay was used for monitoring the efficiency of infection in an infected control well fixed at 20 hpi.