Pluronic acid solution

Porcine kidney LLC-PK cells were routinely grown in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a 5% CO₂ atmosphere. CHO cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO₂ atmosphere. Spodoptera frugiperda ovarian cells (Sf9 cells) purchased from Gibco were cultured at 27°C in SF-900 II SFM medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, a lipid medium supplement, and 0.1% pluronic acid solution (Sigma-Aldrich, St. Louis, MO, USA). PSaV strain Cowden was generated from a full-length infectious cDNA clone, pCV4A (a kind gift from K. O. Chang, Kansas State University, Manhattan, KS, USA), and cultured in LLC-PK cells supplemented with 200 μM GCDCA (Sigma-Aldrich), 2.5% FBS, and 1% penicillin-streptomycin (33 (link), 62 (link)). To obtain a high virus titer and to remove cellular factors, PSaVs were purified by cesium chloride (CsCl) gradient centrifugation as described below (57 (link)). The viral titer was determined by 50% tissue culture infective dose (TCID₅₀) assay as described previously (51 (link)).

Porcine kidney epithelial cells (LLC-PK), obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), were grown in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Spodoptera frugiperda ovarian cells (Sf9 cells), purchased from Gibco (Fort Worth, TX, USA), were cultured at 27°C in SF-900 II SFM medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, lipid medium supplementation, and 0.1% pluronic acid solution (Sigma-Aldrich, St. Louis, MO, USA).
The tissue culture-adapted PSaV Cowden strain was recovered from the full-length infectious clone pCV4A and propagated in LLC-PK cells with supplementation with 200 μM GCDCA (Sigma-Aldrich) (31 (link)). PSaV was concentrated by ultracentrifugation, and the viral titer was calculated by the method of Reed and Muench (52 (link)) and expressed as the median tissue culture infectious dose (TCID₅₀) per milliliter.