2.3. Generation and Recovery of Recombinant iPEDVPT-P5, iPEDVPT-P96, iPEDVPT-P5-96S and iPEDVPT-P96-5S Viruses
The strategy used to recover iPEDVPT-P96 has been described previously [19]. The approach to constructing a cDNA clone of iPEDVPT-P5 was technically identical to that of the iPEDVPT-P96. However, we split the plasmid B into two fragments because the sequence remained toxic to the One Shot™ TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, USA) despite propagation in LB broth supplemented with 10% SOC medium and being incubated at 30 °C. To generate chimeric viruses carrying heterologous spike (S) genes, namely the iPEDVPT-P5-96S and iPEDVPT-P96-5S, cDNA clones of iPEDVPT-P5 and iPEDVPT-P96, respectively, were used as the backbones. The sequences covering the complete S gene of each virus were exchanged without disruption to the remaining genomic structure. Sequence differences in the S genes of iPEDVPT-P5 and iPEDVPT-P96 viruses are summarized in Table 1. Each plasmid was digested with corresponding type-IIS restriction enzymes as designated in Figure S1, gel-purified, assembled and phenol-chloroform extracted to generate the full-length cDNAs. The cDNAs were then in vitro transcribed to the full-length RNA transcripts using a mMessage mMachine T7 transcription kit (Ambion, Austin, CA, USA) and immediately electroporated into 800 μL of 107 cells/mL Vero cells in RNase-free phosphate buffered saline (PBS) along with 5 μg of PEDV nucleocapsid (N) transcripts. After electroporation, the cells were allowed to recover in growth medium for approximately 16 h and then maintained in PI-medium until cytopathic effects involved over 90% of cell monolayers. The whole flasks were subjected to one freeze-and-thaw cycle and the rescued viruses were passaged once to generate viral stocks (P1). The viral stocks were titrated on Vero cells in 96-well plates to determine the viral titer (see below).