Postnatal Persistent Infection with Classical Swine Fever Virus and Its Immunological Implications

Muñoz-González, Sara; Ruggli, Nicolas; Rosell, Rosa; Pérez, Lester Josué; Frías-Leuporeau, Maria Teresa; Fraile, Lorenzo; Montoya, Maria; Cordoba, Lorena; Domingo, Mariano; Ehrensperger, Felix; Summerfield, Artur; Ganges, Llilianne

PBMCs and ELISPOT assay for CSFV-specific IFN-γ-producing cells Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood collected at 4 and 6 weeks post-infection, and these cells were separated by density-gradient centrifugation with Histopaque 1077 (Sigma). The number and viability of the PBMCs were determined by staining with Trypan Blue [42] . ELISPOT assay to detect CSFV-specific IFN-γ cells was performed as previously described [41] . Briefly, plates (Costar 3590, Corning) were coated overnight with 5 μg/ml of capture antibody (P2G10, Pharmigen). Detection was performed using a biotinylated antibody (P2C11, Pharmigin). A total of 5x10 5 PBMCs/well were plated in triplicate at 0.1 multiplicity of infection (MOI) of the Cat01 and PR CSFV strains. Moreover, the same samples were incubated in the presence of Thiverval strain at 0.01 MOI and phytohaemagglutinin (PHA) (10 μg/ml). The controls were incubated in the presence of mock-stimulated wells. The numbers of spots in the media for mock-stimulated wells were considered to be the baseline for the calculation of antigen-specific frequencies of IFN-γ-producing cells.