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

Abstract Porcine epidemic diarrhea virus (PEDV) is an important enteric pathogen that causes severe diarrhea and high mortality in suckling piglets, posing a major threat to the global swine industry. In recent years, viral vectors expressing heterologous protective antigens have emerged as a promising strategy for novel vaccine development. However, whether PEDV can serve as a viral vector for the efficient and stable expression of foreign proteins remains unclear. In this study, a novel insertion site upstream of M in the PEDV genome was identified as a permissive locus for foreign gene insertion using a PEDV reverse genetics platform via yeast-based transformation-associated recombination (TAR) technology. The EGFP reporter system confirmed that the site supported stable foreign gene expression without compromising viral replication. Furthermore, the VP1 of foot-and-mouth disease virus (FMDV), a major immunodominant antigen, was introduced into the site, generating the recombinant virus rPEDV-VP1, which stably maintained the foreign gene not only during serial passaging but also during subsequent in vivo infection in piglets. Collectively, these findings identify a candidate insertion site for efficient expression of foreign antigens in the PEDV genome and provide a technical basis for the development of PEDV-based viral vector platforms.