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

As the pandemic of the G2b PEDV strain ravages the global swine industry, the PEDV field is moving towards the development of novel vaccines and vaccination strategies. To accomplish this task, we first isolated field G2b PEDV that can be efficiently propagated in vitro. The G2b isolate, Aram-P5, exhibited comparable growth characteristics with other G2b strains in regard to cytopathology, infectious titers, and replication kinetics in Vero cells as reported previously [23] . Genetic and phylogenetic analyses showed that the Aram isolates are most closely related to the recent G2b strains prevalent worldwide. However, sequence comparisons with other PEDV strains revealed outstanding genomic features in the Aram strain, which naturally included two independent DELs in S and ORF3, respectively. More interestingly, due to the 2-nt DEL in ORF3, Aram is predicted to encode an alternative ORF3 protein with a large 87-aa excised C-terminus. Although the exact function of PEDV ORF3 remains unknown, a number of attenuated PEDV strains contain their signature DELs in several parts of ORF3, suggesting its critical role in viral pathogenesis [24, 34, 35] . In particular, Wang et al. [11] reported an attenuated PEDV that encodes a C-terminal truncated ORF3 protein of 91-aa residues, which is form the most analogous to the ORF3 of the Aram strain. However, experimental oral inoculation of newborn pigs with Aram-P5 induced severe clinical presentations and macroscopic and microscopic intestinal lesions typical of acute PEDV infection as reported previously [23] . In general, death rates average 50% in suckling piglets up to one week of age, often approaching 100% in neonates less than 3 days of age, and decrease to 10% thereafter [1, 2] . Likewise, our study reproduced 100% mortality in 5-day-old conventional piglets inoculated orally with the 10 −2 diluted Aram virus (calculated dose of 10 3 TCID 50 per pig). The data demonstrated that the Aram isolate was highly enteropathogenic in neonatal piglets, implying that the ORF3 product would be irrelevant to PEDV virulence. Since a growing body of evidence proposes that a combination of multiple genetic mutations in S, ORF3, and functional nsps affects the virulence of PEDV, we cannot exclude that the C-terminal defect in ORF3 may be one of molecular and physiological factors that may simultaneously alter pathogenic mechanisms. However, consistent with previous works [24, 36] , growth kinetics of the Aram strain in cell culture supports the notion that the accessory ORF3 gene is dispensable for PEDV replication in vitro. Collectively, despite a redundant role in virus propagation in vitro, conservation of the complete ORF3 in PEDV field isolates still suggests its importance in causing natural infection in the animal host.