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

PEDV isolation in cell culture is the first step toward the generation of a live attenuated vaccine. Thus, we initially attempted to isolate PEDV from clinical fecal suspensions and successfully propagated one Korean PEDV strain, designated as Aram, in Vero cells. The Aram strain produced obvious CPE typical of PEDV infection, such as cell fusion and multinucleated cell (syncytium) formation, in infected Vero cells, which was verified by detecting PEDV antigens by the IFA using a PEDV N protein-specific MAb (Fig. 1A) . Growth kinetics analysis indicated that Aram replicated efficiently in Vero cells, reaching a maximum viral titer of > 10 6 TCID 50 by 36 hpi (Fig. 1B) Virus titer (log TCID /mL) We subsequently determined the entire genomic sequences of the original fecal sample (Aram-feces) and the cell culture-passaged Aram-P5 virus using the Sanger technology. Both the identified genomes were identical in length [28,027-nucleotide (nt)], except for the 3′ poly(A) tails, and showed the typical genomic organization of alphacoronaviruses, consisting of the 292-nt 5′ UTR, the 20345-nt ORF1a/1b (nt 293 to 12601 for 1a and nt 12601 to 20637 for 1b), the 4152-nt S gene (nt 20634 to 24785), the 414-nt ORF3 (nt 24785 to 25198), the 231-nt E gene (nt 25438 to 25668), the 681-nt membrane (M) gene (nt 25676 to 26356), the 1326-nt N gene (nt 26368 to 27693), and the 334-nt 3′ UTR. Compared to the complete genome sequence of the Aram-feces, the Aram-P5 virus had no amino acid (aa) substitution throughout the entire genome, suggesting genetic stability during cell passages. Interestingly, the genome size of the Aram strain was 11-nt shorter than that of most G2b field viruses; this was due to the presence of the unique 9-nt and 2-nt deletions (DELs) at genomic positions 21,051-21,059 and 25,207-25,208, respectively. These DELs are completely absent in the genome sequences of the other global G1 and G2 strains available in the GenBank database. The former 9-nt DEL resulted in a 3-aa DEL at aa positions 140-142 in the N-terminal domain (NTD) of the S protein ( Fig. 2A) , while the latter 2-nt (thymine residues) DEL at nt positions 414-415 ( 412 TATTAT 417 ) in ORF3 caused premature termination 414-nt upstream from the authentic stop codon, thereby possibly producing a truncated ORF3 without C-terminal 87-aa residues (Fig. 2B) . (Fig. 2B) .