Emerging and re-emerging swine viral infectious diseases impose substantial economic burdens. Additionally, swine, which frequently interact with humans, may facilitate virus evolution, posing a risk to public health security. Consequently, there is a pressing need to develop safe, effective, and rapid vaccine platforms, with vector vaccine being a viable option. In this study, we utilized the enhanced green fluorescent protein (eGFP) as an exogenous reporter to investigate the intergenic regions of the PIV5-JS17 strain for expressing exogenous proteins. These regions included N-P, P-M, M-F, F-SH, SH-HN, and HN-L. Our findings revealed that exogenous gene expression varied at different positions, with the expression cassette containing the non-coding sequence within the P-M intergenic region achieving the highest eGFP fluorescence intensity. Then, we successfully established a porcine infection model through oral administration of the recombinant virus, identified the target organs infected, and verified the safety of the viral vector in swine. Furthermore, using porcine deltacoronavirus (PDCoV)-S protein as a model antigen, it was demonstrated that the recombinant virus triggered a robust humoral and cellular immune responses. In conclusion, we have developed a novel oral gene delivery system for swine, providing insights and guidance for the design of vector vaccines based on the newly discovered porcine PIV5, the selection of appropriate exogenous gene insertion sites, and vaccine delivery strategies.IMPORTANCEThe research presented in this paper hinges on the fortunate isolation of the PIV5-JS17 strain from the intestines of pigs. Given the pressing demand for oral vaccines, the emergence of this novel PIV5 strain capable of infecting both the respiratory and intestinal tracts has sparked our interest in developing it as a vector vaccine. Utilizing eGFP as a model exogenous gene, our findings reveal that the P/M intergenic region serves as the optimal site for the insertion of exogenous genes. Using the PDCoV-S protein as a model antigen, the study shows that this novel porcine-derived PIV5 virus vector presents innovative prevention methods and gene delivery strategies for addressing porcine infectious diseases.

Keywords: exogenous gene expression cassette; gene delivery; optimal insertion site; parainfluenza virus type 5; viral vector.