3.2. In Vitro Characterization of Recombinant PEDVs
The in vitro characteristics of the recombinant viruses were compared by evaluation of the size of syncytia, growth kinetics in Vero cells, and plaque morphologies. All recombinant viruses induced formation of syncytia by 18 h post-infection. Viruses carrying the identical S gene exhibited similar fusogenic ability as suggested by the comparable number of nuclei per syncytium; representative micrographs used to count the number of nuclei in syncytia are shown in Figure 1A. Remarkably, viruses carrying S gene derived from iPEDVPT-P96, induced 3.5 to 4 times larger syncytia than the other two viruses containing S gene derived from iPEDVPT-P5 (Figure 1B).
Replication of all recombinant viruses in Vero cells were examined by performing both one-step and multistep growth kinetics at a multiplicity of infection (MOI) of 1 and 0.001, respectively. Interestingly, although replacement of the S gene resulted in an alteration of the replication kinetics and efficiency, leading to similar growth curve patterns between viruses carrying the identical S gene sequence, it did not fully reverse the capability of virus yield, at least in Vero cells (Figure 1C).
We observed that plaque morphologies of the four recombinant viruses correlated to the results of syncytia size. At 72 h post-infection, both iPEDVPT-P5 and iPEDVPT-P96-5S induced barely visible viral plaques in Vero cells macroscopically, whereas iPEDVPT-P96 and iPEDVPT-5-96S generated distinct and comparable viral plaques with the plaque size of iPEDVPT-P5-96S being slightly smaller than that of the iPEDVPT-P96 (Figure 1D). These data suggested that the exchange of the S gene between iPEDVPT-P5 and iPEDVPT-P96 affected the fusogenicity and, to a lesser extent, replication kinetics in Vero cells.