Construction of Recombinant PRV Expressing PRRSV Antigens and Evaluation of Their Protective Efficacy in Pigs Against Homologous Challenge

Background/Objectives: Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to the global swine industry, while the emergence of variant pseudorabies virus (PRV) strains has further complicated disease control in China. This study aimed to develop a PRV-based viral vector platform for PRRSV antigen delivery and evaluate its safety, immunogenicity, and protective efficacy. Methods: A five-gene-deleted variant PRV strain, PRV-JM-DF, was used as the vector backbone. Candidate genomic insertion sites and endogenous late promoters were systematically screened, followed by construction of recombinant PRVs expressing PRRSV GP2, GP3, GP4, GP5, and M individually or as tandem antigen constructs. Recombinant viruses were characterized for antigen expression, replication, and genetic stability. Safety and immunogenicity were initially evaluated in female BALB/c mice, and the leading candidates were further assessed for safety, immunogenicity, and protective efficacy in 18 Landrace pigs of either sex following challenge with highly pathogenic PRRSV HuN4. Results: The TK locus supported efficient foreign-gene expression, and pUL39 exhibited the highest activity among the tested endogenous late promoters. The recombinant viruses retained replication competence and genetic stability. Among the candidates, rPRV-C-4T induced robust PRRSV- and PRV-specific neutralizing antibody responses and showed no obvious adverse effects during the experimental observation period in pigs. Following HP-PRRSV HuN4 challenge, all rPRV-C-4T-immunized pigs survived and exhibited reduced viremia, viral shedding and tissue viral loads, together with markedly alleviated pathological lesions. Conclusions: rPRV-C-4T provided protection against homologous HP-PRRSV HuN4 challenge while maintaining PRV-directed immune responses, supporting its potential as a PRV-vectored multivalent vaccine candidate.