Generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus

Won, Hokeun; Lee, Dong-Uk; Jang, Guehwan; Noh, Yun-Hee; Lee, Seung-Chul; Choi, Hwan-Won; Yoon, In-Joong; Yoo, Han Sang; Lee, Changhee

The PEDV strain, Aram-P5, was plaque-purified twice in Vero cells, and the purified virus was serially passaged in Vero cells by gradually reducing the incubation temperature from 37°C to 32°C as described previously with some modifications [24] . Confluent Vero cells grown in 100-mm diameter tissue culture dishes were washed with PBS and inoculated with PEDV Aram-P5 along with trypsin. After incubation at 37°C for 1 h, virus growth medium [BMpro-V medium (Cell Science & Technology Institute, Inc., Japan) supplemented with 5 μg/mL of trypsin] was added. The inoculated cells were maintained at 37°C under 5% CO 2 and monitored daily for CPE. When > 70% of the cells showed CPE, the inoculated cells were subjected to three rounds of freezing and thawing. The culture supernatants were then centrifuged for 10 min at 400 × g (Hanil Centrifuge FLETA5, Korea) and filtered through a 0.45-μm pore-size filter (Millipore, USA). The clarified supernatants were aliquoted and stored at −80°C as a viral stock (Aram-P1-CA) for the next passage. In the same manner, Aram-P1-CA was passaged 4 times with step-wise descending temperatures of 1°C per passage in Vero cells up to 32°C. Subsequently, Aram-P5-CA was serially cultivated at 32°C for an additional 45 passages for cold adaptation, and virus stock were produced at 24, 28, 35, and 45 passages that were designated Aram-P29-CA, P33-CA, P40-CA, and P50-CA.