Primaria

The human bronchial epithelial cell line BEAS-2B
(CRL-9609) was purchased from ATCC (Manassas, VA). Cells were tested
regularly for mycoplasma contamination. BEAS-2B were cultured using
a BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza, Basel,
Switzerland) at 37 °C in a humidified atmosphere with 5% CO₂. In order to obtain a clonal parental BEAS-2B cell line,
cells were seeded at limited dilution (500 cells per 96-well plate).
Cells were left to recover until they were sufficiently viable to
transfer (4–8 days), after which wells were visually inspected
to identify those containing a single cell. These clones were expanded
and passaged to Primaria (Corning, Corning, NY) 6-well plates and
then to Primaria 10 cm dishes. The clonal parental population was
chosen on the criteria that the cells displayed the same morphology
as BEAS-2B cells and had the same division time. DNA was extracted
(see Section 2.6),
and clonality of the population was confirmed by whole-genome sequencing.
The resulting clonal parental BEAS-2B cell line was cultured in the
same way as the regular BEAS-2B cell line.

Titrations and virus isolations were performed on porcine peripheral blood mononuclear cell (PBMC) derived macrophages obtained from the EDTA-treated blood of a healthy donor pig. In brief, Pancoll animal, Density: 1.077 g/ml (PAN-biotech) was used for separation of the erythrocytes by mixture at 1:10 with the blood. After resting for 2 h, the clear supernatant was diluted 1:1 with PBS and cleared by centrifugation at 800 x g. The supernatant was discarded and the cell pellet resuspended with PBS; these washing steps were repeated three times. Finally, the cell pellet was resuspended in cell culture medium and counted using a TC20 automated cell counter (Bio-Rad) with trypan blue staining. For titrations, cells were seeded in 96-well culture plates (Primaria; Corning) with 100 μl/well at a density of 5 × 10⁶ cells/ml and cultivated for 48 h using GM-CSF as a growth factor (2 ng/ml). 100 μl of the respective samples diluted in cell culture medium at factors 10⁻¹ to 10⁻⁸ was added to each well for endpoint titration. 20 μl of a 1 % suspension of erythrocytes from the same donor pig in PBS was added after 24 h. For determination of titers, infected wells were read 48 and 72 h-post-infection using the hemadsorption as readout. The HAD₅₀ was calculated according to the method by REED and MUENCH^{20 (link)}.

The novel virus variant ΔMGFnV was isolated from the blood of pig #22 of the fifth passage and cultivated on macrophages in T25 cell culture flasks (Primaria; Corning) and titrated as described in the 2.2 Cells and titrations section. The absence of the MSV virus was assured by tailored qPCR. Growth kinetics were also conducted on macrophages obtained as described above. Cells were counted using a Bio-Rad TC 20 Automated Cell Counter with trypan blue staining, counting only living cells. T25 flasks were infected at a multiplicity of infection of 0.1 with either the ΔMGF MSV, the ΔMGFnV or both viruses simultaneously. After 2 h of incubation, medium supernatants were removed, cells were rinsed once with PBS- and flasks were resuspended with cell culture medium. 300 μl of supernatant were removed at -2 h postinfection (hpi, before adding the virus solution), 0 hpi (after incubation), and 4, 8, 12, 24, 48 and 72 hpi and immediately stored at −80 °C. Samples were analyzed by the commercial ASFV real-time PCR system virotype 2.0 (Indical Bioscience GmbH) and tailored qPCR for differentiation between ΔMGF MSV and ΔMGFnV replication, and by titration on macrophages using the methods described above.