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

All animals in group 1 were positive for PEDV, as determined by RT-PCR, by 1 DPI with a mean Ct value of 28.55 (equivalent to 10 2.59 TCID 50 /mL) and shed significantly higher amounts of PEDV in feces with Ct ranging from 6.80-12.35 (10 4.97 -10 6.14 TCID 50 /mL) until death (Fig. 6B) . Likewise, fecal shedding of PEDV was detected in all the piglets in group 2 by 1 DPI with the mean Ct values of 24.59 (10 3.11 TCID 50 /mL), but thereafter decreased to the lowest levels thereafter followed by a slight increase at 5 DPI. Overall, the quantities of viruses in the feces of animals of group 2 significantly declined compared to those in group 1, with wide Ct ranges of 34.46-24.34 ( Efficacy of cold-adapted attenuated PEDV vaccine All animals in the parental Aram-P5-infected group were necropsied upon death at 4 or 5 DPI, while piglets in the remaining groups were euthanized at the end of the study for postmortem examinations (Fig. 7) . Neither macroscopic nor microscopic intestinal lesions were evident in the negative control piglets (right panels C, F, and I). The virulent Aram-P5-inoculated pigs macroscopically showed archetypal PED-like gross lesions. Their small intestines were dilated with accumulated yellowish fecal fluid and had thin transparent walls as a result of villous atrophy (panel A), whereas the other internal organs appeared normal. In contrast, all animals infected with cold-adapted Aram-P29-CA in group 2 displayed no remarkable visible pathological lesions in their gastrointestinal tracts with normal bowel wall thickness, comparable to those in the negative control group (panel B). Microscopic assessment revealed that the small intestines from all dead piglets in group 1 were characterized by acute viral enteritis, with villous shortening and fusion, involving vacuolation of superficial epithelial cells, in the jejunum (panel D). Furthermore, IHC staining showed intense antigen labeling in the cytoplasm of epithelial cells in atrophied intestinal villi with a immunosignal score of 2.3 ± 0.6 (mean ± SDM) in the jejunum (panel G). However, two out of four cold-adapted virus-inoculated pigs in group 2 exhibited mild villous atrophy in the small intestines, while the remaining pigs showed normal intestinal histopathologies (panel E), analogous to those of the negative control group (panel F). PEDV antigen was rarely present in the small intestines in animals of group 2 with a significantly lower mean jejunal viral antigen score (0.3 ± 0.6) (panel H) than that in group 1 (panel G). Altogether, our results indicate that cold-adapted Aram-P29-CA showed noticeably weakened virulence with an attenuated phenotype in the highly susceptible piglets under experimental conditions.