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

To serve as a novel MLV vaccine strain, we generated an Aram-derived attenuated PEDV strain using a cold adaptation attenuation method. Our approach for viral attenuation was to obtain a cold-adapted strain with impaired growth at physiological temperature (37°C), which can sufficiently induce mucosal immunity in the natural host. The cold-adapted Aram-P29-CA strain was derived by 24 serial passages of the Aram isolate at a low temperature (32°C). The Aram-P29-CA strain developed unsuccessful in vitro infection at the physiological temperature, as a significant decrease in infectious units was observed. However, this virus showed significantly high immunogenicity in orally inoculated pigs. Furthermore, when investigating the phenotype of Aram-P29-CA in highly susceptible 5 day-old piglets, none of the inoculated animals became clinically sick and grossly abnormal. Our experimental data indicate that the cold-adapted Aram-P29-CA virus is attenuated virologically in vitro and clinically in vivo. Subsequently, we sequenced the entire genome of the Aram-P29-CA strain to decode the genetic mutations that emerged during sequential cell passages at 32°C. Upon cold adaptation, a total of 12 non-silent mutations arose in ORFs 1 through 5, except for ORF3, without extra INDELs throughout the genome, whereas the two S and ORF3 DEL signatures of the Aram strain were entirely maintained. Intriguingly, 7-aa substitutions were accumulated in the S protein, among which 4-and 3-aa substitutions were distributed in the N-terminus of S1 and S2 domains, respectively, suggesting that some of these changes are associated with viral attenuation. Since the PEDV S protein is heavily glycosylated, altered glycan motifs may be involved in viral pathogenesis by modifying the protein conformation and function [37] . However, no genetic changes were found in the putative N-glycosylation sites between the parental and cold-adapted strains. As the major antigenic determinant, the S glycoprotein possesses at least four neutralizing domains of PEDV at aa positions 19-220 (NTD/S0), 499-600, 744-774, and 1371-1377 [38] . The aa residues that comprise these domains remained nearly unchanged throughout the cold adaptation, except for one N209Y mutation in the NTD/S0 region. Furthermore, no aa changes were found in additional discrete domains, including a C-terminal domain (residues 477-629) of S1 that can interact with cellular receptor(s) and a S2 fusion domain comprising of a hydrophobic fusion peptide (residues 891-908) and two heptad repeat regions (residues 978-1117 and 1274-1313). Although the polygenic traits are likely associated with PEDV virulence, it is of utmost importance that future work using reverse genetics technology should aim to address whether genetic drift, especially in the S protein, influences the pathogenicity of PEDV. In addition, attenuated viruses often have the propensity to revert to virulent form under certain circumstances, which is a main drawback for their use as an MLV vaccine. Indeed, the Aram-P29-CA strain had an ability to replicate at 37°C since it denoted impaired, but not entirely halted, growth at 37°C, as the viral titer was determined to be 10 2.5 TCID 50 /mL. This incompletely abolished growth of Aram-P29-CA at the physiological temperature implies a residual risk of reversion to virulence in vivo. Thus, further safety studies will be necessary to evaluate the phenotypic stability of the attenuated Aram-P29-CA virus in neonatal