Trypsin solution from porcine pancreas

An isolate of the PEDV inoculum strain (Calaf-1; GenBank accession number MT602520) was used in viral neutralization test (VNT) and ELISPOT analyses. Briefly, intestinal contents of the inoculated piglets were centrifuged to 15,000 g for 15 min, diluted 1:10 in DMEM-high w/glutamax (Thermo Fisher Scientific), with 300 UI/mL penicillin and 300 µg/mL streptomycin (Thermo Fisher Scientific), 50 µg/mL nystatin (Merck, Madrid, Spain), 0.02% yeast extract (Thermo Fisher Scientific) and, 0.3% tryptone phosphate broth (Merck). The suspension was filtered through a 0.22 µm filter (Merck Millipore, Madrid, Spain) and trypsin was added to a final concentration of 10 µg/mL (Trypsin solution from porcine pancreas, Sigma). Then, 0.5 mL of the trypsintreated suspension was inoculated onto VERO cells (ATCC CCL-81) on 25cm 2 flasks. After 2h of adsorption at 37 ºC, 6 mL of the dilution medium was added, and cultures were incubated at 37 °C. After being cultured for 5 days a cytopathic effect (CPE) was observed and cultures were frozen and thawed to recover the virus. A single virus stock was used for the immunological analysis (10 5.3 TCID 50 /mL, passage 22).