Engineering of recombinant porcine deltacoronavirus stably expressing foreign gene

Porcine deltacoronavirus (PDCoV) is an emerging enteric virus characterized by high viral titers, strong immunogenicity, and broad host tropism, making it a promising candidate for developing vaccine vectors and foreign gene expression platforms. However, key aspects such as its transcription regulatory sequence (TRS) usage, reliable insertion sites, and the genetic stability of recombinant viruses remain incompletely understood. In this study, we established a reverse genetic system for the PDCoV strain DHeB1 and systematically evaluated the transcriptional activity of six TRS elements from the S, E, M, N, NS6, and NS7 genes. Each TRS was used to drive the expression of EGFP inserted between the N gene and 3' UTR of viral genome. The results showed that all TRSs could initiate EGFP expression, with the NS6-derived TRS exhibiting the strongest activity. However, all recombinant viruses carrying the EGFP cassette exhibited genetic instability, being passaged only up to seven times in LLC-PK1 cells, indicating that the region between the N gene and 3' UTR is unsuitable for stable foreign gene insertion. To improve genetic stability, while leveraging the strong promoter activity identified, we replaced the NS6 gene with the EGFP gene, as NS6 is an accessory protein whose deletion does not terminate viral replication. This recombinant virus was successfully rescued and stably propagated in LLC-PK1 cells for at least 15 passages, maintaining consistent EGFP expression. Collectively, this study provides an efficient reverse genetics platform for PDCoV, characterizes the transcriptional regulation of its TRS elements, and demonstrates the generation of a stable recombinant PDCoV expressing a foreign gene, thereby facilitating the development of PDCoV-based vector systems.