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

Pathogenicity tests were performed using a total of 12 suckling piglets of 3 days of age obtained from commercial crossbred sows (Great Yorkshire × Dutch Landrace). Pigs were randomly assigned to three experimental groups: the parental Aram-P5-inoculated (n = 4), cold-adapted P29-CA-inoculated (n = 4), and sham-inoculated control (n = 4) groups. Animals were fed commercial milk replacer frequently (4 times daily) and had ad libitum access to water for the duration of the study (9 days). After a 2-day acclimation period, piglets (5-day-old) in the virusinoculated groups received a 1 mL dose of 10 4.0 TCID 50 /mL (equivalent to 1000 LD 50 as determined in this study) of one of the viruses orally. The sham-inoculated pigs were administered cell culture medium as a placebo. Animals were monitored daily for clinical signs of vomiting, diarrhea, and mortality throughout the experiment. Rectal swabs were collected from all pigs and diluted with PBS to 10% (w/v) suspensions followed by centrifugation. The clarified supernatants were subjected to conventional reverse transcription polymerase chain reaction (RT-PCR) using an i-TGE/PED Detection Kit (iNtRON Biotechnology, Korea) and real-time RT-PCR to detect the presence of PEDV shedding. A clinical significance score (CSS) was determined with the following scoring criteria based on visual examination for 7 DPI used to measure diarrheal severity: 0 = normal and no diarrhea (mean cycle threshold [Ct] values of > 45); 1 = mild and fluidic feces; 2 = moderate watery diarrhea; 3 = severe watery and projectile diarrhea (mean Ct values of < 20); 4 = death. Piglets were necropsied upon death after challenge throughout the study, whereas all surviving pigs from the challenge and control groups were euthanized at 7 DPI for post-mortem examinations.