A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences

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-CSs) of PEDV can make the recombination impossible between the engineered vaccine virus and field strains or wild-type viruses. We used an infectious clone-derived reporter PEDV, dORF3-enhanced green fluorescent protein (EGFP), as the backbone to generate a remodeled TRS (RMT) mutant that carries the recoded leader and body TRS-CSs (except for the EGFP). The RMT and dORF3-EGFP showed comparable replication efficiency in Vero cells. However, the incompatibility between the rewired and wild-type 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 heterogeneous 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 wild-type 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.