Scientists Find a New Spot in the PEDV Genome for Inserting Foreign Genes

Porcine epidemic diarrhea virus, an enteric coronavirus that causes acute watery diarrhea, vomiting and mortality that can approach one hundred percent in suckling piglets, has now been repurposed in the laboratory as a delivery vehicle for a foreign antigen. In a short communication published in Animal Diseases, researchers in China report that a region of the PEDV genome lying upstream of the M gene can accept inserted foreign genes and keep them stably expressed through serial passaging in cell culture and through replication in experimentally infected piglets. As a demonstration, the team inserted the gene for VP1, the major immunodominant antigen of foot-and-mouth disease virus, and recovered a recombinant virus that grew like the parental strain while carrying the heterologous cargo intact.

The work began with the construction of a reverse genetics platform for PEDV, the technical foundation that makes any such engineering possible. The researchers assembled a full-length infectious cDNA clone of the viral genome using transformation-associated recombination, or TAR, a yeast-based method in which overlapping DNA fragments and a linearized vector are cotransformed into Saccharomyces cerevisiae and stitched together by the cell’s own homologous recombination machinery. The PEDV genome, roughly 28 kilobases of positive-sense single-stranded RNA, was divided into eight overlapping fragments for this assembly. The vector cassette carried a CMV promoter, a hepatitis delta virus ribozyme and a bovine growth hormone polyadenylation signal, the standard elements needed to launch and terminate correct genome transcription after transfection.

To verify that any recovered virus truly came from the clone rather than from contamination with the parental laboratory strain, the team introduced synonymous mutations into the N gene, deliberately disrupting a SacII restriction site. These silent changes served as a genetic marker. When the recombinant plasmid was transfected into Vero cells engineered to stably express the PEDV N protein, typical coronavirus cytopathic effects, including cell fusion and syncytium formation, appeared within twelve hours. Indirect immunofluorescence and Western blotting confirmed N protein expression, and sequencing of the marker region showed the designed C-to-A change at the SacII site, confirming the clonal origin of the rescued virus, designated rPEDV.

With the rescue system in hand, the central question could be addressed: where in the PEDV genome can foreign genes be inserted without wrecking the virus? The choice of insertion site is a known bottleneck for coronavirus vectors. An ideal locus must not impair replication, must drive stable expression, and must preserve genetic integrity over many passages. Previous work in other nidoviruses, including infectious bronchitis virus and porcine deltacoronavirus, showed that insertion site choice can markedly influence recombinant virus stability, and two permissive loci had already been described in PEDV, the ORF3 site and the intergenic region between N and the three prime untranslated region. But no systematic characterization of the M upstream region had been reported.

To screen candidate sites, the researchers built reporter viruses carrying an enhanced green fluorescent protein expression cassette at multiple positions. Among the sites tested, the region upstream of M supported efficient EGFP expression and was selected for deeper analysis. The EGFP cassette was linked to the M protein coding sequence through a T2A peptide, an 18 to 22 amino acid ribosomal skipping sequence derived from Thosea asigna virus that allows equimolar, independent expression of the proteins flanking it from a single open reading frame. The recombinant virus induced typical PEDV cytopathic effects in Vero cells, and RT-PCR consistently amplified the EGFP-containing fragment from the viral genome across serial passages, indicating the insertion was not being lost or excised.

Protein-level evidence backed up the genetic data. Immunofluorescence microscopy showed EGFP signal colocalizing with the PEDV N protein inside infected cells, and Western blotting detected EGFP-specific bands in lysates of cells infected with the recombinant virus but not in parental-virus-infected or mock-infected controls. Taken together, these experiments established upstream of M as a novel, previously uncharacterized permissive insertion site that supports stable foreign gene expression without appreciably affecting viral replication.

The team then swapped the reporter for a payload of practical interest: the VP1 coding sequence of foot-and-mouth disease virus, replacing EGFP at the same genomic location. VP1 carries critical neutralizing epitopes and is the primary target of subunit and peptide vaccine development against FMD, a devastating disease of swine and other cloven-hoofed animals. The resulting recombinant, rPEDV-VP1, produced typical cytopathic effects in Vero cells, and RT-PCR, immunofluorescence and Western blotting each confirmed the presence and expression of the VP1 insert. Because both PEDV and FMDV are major swine pathogens with limited vaccine cross-protection, the authors frame a recombinant PEDV expressing FMDV VP1 as a candidate single-shot bivalent vaccine strategy, though the study itself tested expression and stability, not protective efficacy.

Replication kinetics were compared among rPEDV, rPEDV-EGFP, rPEDV-VP1 and the parental virus in Vero cells infected at a multiplicity of 0.1, with titers measured by TCID50 assay at intervals from 6 to 42 hours post infection. The replication curves of the four viruses were similar, and plaque assays revealed no obvious differences in plaque size or morphology among the recombinants and the parental strain. In other words, inserting either EGFP or VP1 upstream of M left the virus’s growth characteristics essentially unchanged, and the interchangeability of the inserted genes illustrates the flexibility of the platform for multivalent designs.

Genetic stability was examined in two settings. In vitro, rPEDV-VP1 was serially passaged for ten generations in Vero cells; RT-PCR amplified the VP1 fragment from every passage tested, and Western blotting and immunofluorescence confirmed that VP1 protein expression persisted through passage ten, with no spontaneous loss of the insert. In vivo, the tenth-passage virus was inoculated into six one-day-old colostrum-deprived piglets confirmed negative for PEDV antibodies and viral nucleic acids, split between oral and intramuscular routes at a dose of 10 to the 6.5 TCID50 per animal. Seventy-two hours after infection, the VP1 gene was amplified by RT-PCR from stomach, ileum, colon, blood, oral swab and feces samples, showing that the recombinant virus replicated in the animals. Viral loads were significantly higher in the orally inoculated group than in the intramuscular group, and the ileum proved to be the optimal site of viral proliferation, consistent with the natural enteric tropism of PEDV.

To test whether the insert survived replication in a living host, intestinal tissue homogenates from the infected piglets were clarified, inoculated onto Vero cells, and subjected to three freeze-thaw cycles and three blind passages. The recombinant rPEDV-VP1 was successfully recovered, and PCR analysis confirmed that the VP1 fragment remained intact. The authors conclude that the M upstream locus maintains genetic stability after in vivo replication while preserving the virus’s enteric tropism, and they position it as a candidate docking site for heterologous antigen expression in PEDV-based live viral vector platforms. The study is a proof of principle built on a single strain, XJ2022-P120, in a small number of animals, and it does not report immunogenicity or protection data; whether an rPEDV-VP1 vaccine can actually protect piglets against either pathogen remains a question for future work. What the results do establish is a technical foundation: a reliable yeast-based assembly route for a 28-kilobase coronavirus genome, a validated permissive insertion site, and a demonstration that a foreign antigen gene can ride along, intact and expressed, through cell culture passaging and through infection of its natural host.

Subject of Research: Identification of a genomic insertion site upstream of the M gene for stable foreign antigen expression in a porcine epidemic diarrhea virus vector platform

Article Title: Identification of a novel insertion site for efficient expression of FMDV VP1 in the PEDV genome

Article References: Lu, X., Yang, X., Qin, W., Liu, Y., Wang, J., Tong, G., Kong, N., & Shan, T. (2026). Identification of a novel insertion site for efficient expression of FMDV VP1 in the PEDV genome. Animal Diseases, 6(1), Article 57. https://doi.org/10.1186/s44149-026-00279-x

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00279-x

Keywords: PEDV, FMDV, VP1, viral vector, reverse genetics, yeast TAR, EGFP reporter, genetic stability, piglets, vaccine platform, coronavirus, swine