Porcine Kidney Cells and CSFV Infection Protocols

The porcine kidney cell line PK-15 was obtained from ATCC (CCL-33, Middlesex, England) and were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% foetal bovine serum (FBS), Pestivirus-free, at 37 °C in 5% CO₂.
The CSFV moderately virulent Catalonia 01 (Cat01) strain, which belongs to the 2.3 subgenotype and has been proven to induce postnatal persistent infection in newborn piglets, was used [15 (link),26 (link)]. The Alfort 187 strain, genogroup 1.1, was used in the neutralisation peroxidase-linked assay (NPLA). Viruses were grown in the PK-15 cell line that were infected with 0.1 TCID₅₀/cell in 2% FBS during 72 h of incubation. Peroxidase-linked assay (PLA) [27 (link)] was used for viral isolation and titration following the statistical methods previously described [28 (link)]. The attenuated vaccine (C-strain) belongs to the CSFV 1.1 genogroup and was used in Spain in the 1980s for CSF control. This vaccine has 100% homology with the Z46258 strain. This vaccine strain was employed to immunize animals in order to obtain IFN-γ producing PBMCs for the co-culture assays [29 (link),30 (link)].