Porcine epidemic diarrhea virus (PEDV) is a deadly coronavirus for neonatal piglets and no effective vaccines are available. Transcriptional regulatory sequences (TRSs) are critical in regulating coronavirus discontinuous transcription. Also, TRSs contribute to a high recombination rate of coronaviruses, leading to difficulty in developing safe live vaccines. We hypothesize that recoding the TRS core sequences (TRS-CS) of PEDV can make the recombination impossible between the engineered vaccine virus and field strains or wildtype viruses. We used an infectious clone-derived reporter PEDV, dORF3-EGFP, as the backbone to generate a remodeled TRS (RMT) mutant that carries the recoded leader and body TRS-CSs. The RMT and dORF3-EGFP showed comparable replication efficiency in Vero cells. However, the incompatibility between the rewired and wildtype TRS-CSs led to few EGFP in RMT-infected cells. Furthermore, RMT and dORF3-EGFP had a similar attenuated phenotype, replication efficiency, and protective immunogenicity in neonatal pigs. RNA sequencing analysis indicated that EGFP transcription directed by the heterogenous TRS-CSs was significantly reduced to an extremely low level. Meanwhile, recombinant viruses were not detected in Vero cells and in pigs that were co-infected with RMT and a PEDV S-INDEL strain, Iowa106. In vitro and in vivo passaging of the RMT did not result in reversion mutations in the rewired TRS-CSs, introduced gaps, and disrupted wildtype TRSs. In summary, the RMT mutant was resistant to recombination and genetically stable and can be further optimized (e.g., deletion of the EGFP) to serve as a platform to develop safe PEDV live attenuated vaccines.
Overall design: The PEDV mutants were generated from the five-plasmid (A, B, C, D, and E) reverse genetic system for a highly virulent PEDV strain PC22A (28). For dORF3-EGFP, the accessory gene, ORF3, was removed and a reporter gene, EGFP, was inserted into plasmid E under the control of the original TRS for ORF3, designated as pE-dORF3. As for the RMT mutant, the wildtype leader TRS-CS (5’-CUAAAC-3’) in plasmid A was recoded (5’-GUGAAC-3’) using a NEB Q5® Site-Directed Mutagenesis Kit (NEB, Ipswich, MA). And the pE-dORF3 were modified accordingly to rewire all body TRS-CS to 5’-GUGAAC-3’, except the one for EGFP gene. Then the sequencing confirmed plasmids were digested with restriction enzymes for purification of DNA fragments that were assembled to form the full-length cDNA of PEDV mutants by in vitro ligation with T4 ligase (NEB, Ipswich, MA) at 4 °C overnight in an equal molar ratio. After purification with chloroform extraction, the cDNA was used as templates for in vitro transcription to generate the PEDV mutant mRNA genome using a mMessage mMachine T7 transcription kit (Ambion, Austin, CA). Meanwhile, a polyadenylated PEDV N gene transcript was generated to be co-electroporated with the mRNA genome into Vero cells at 450 V and 50 μF using a Gene Pulser II electroporator (Bio-Rad, Hercules, CA). At 6 hours post electroporation, cells were washed with PBS and cultured in the maintenance medium in the presence of 5 μg/mL of trypsin. Cells were monitored for CPE daily and harvested when CPE was observed for about 50% cells. This rescued virus was designed as P0 virus. The P0 virus was subjected to plaque purification and one clone was propagated. After the whole genome was confirmed by sanger sequencing, it was designated as the P1 stock of the mutants.
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