ASFV-specific PBMC Isolation and Analysis

Peripheral blood mononuclear cells (PBMCs) were prepared from animals immunized with ASFV Congo-a in the experiment described earlier (43 (link)). Briefly, pigs were infected intramuscularly with 10⁶ TCID50 of Congo-a virus. At 21 day post-infection (dpi), the animals were boosted with the same dose of the same virus. Three weeks later (42 dpi.), PBMCs were isolated from defibrinated blood using the Lymphocyte separation media (Gibco). The cells were resuspended in RPMI 1640 medium supplemented with 30% (v/v) plasma, 10% (v/v) fetal bovine serum (Gibco) and antimycotic-antibiotic (Gibco). The washed cells (1 × 10⁶cells/well) were seeded into 48 well plates and incubated for 2 h at 37°C with 5% CO₂. Then the cells were inoculated with two different virulent viruses with the multiplicity of infection of 1 (MOI = 1). Five hours after inoculation with the virus, PBMCs were washed once with sterile PBS and used to isolate total RNA.
To identify the genome of the ASFV, PCR of the B646L gene was performed in accordance with the protocol published by King et al. (44 (link)). PCR of the β-actin gene was used as endogenous control. PCR reactions were carried out on a CFX96TM thermal cycler (Bio-Rad, Hercules, CA, USA).