Abstract
Background: Orf is a zoonotic infectious disease caused by the Orf virus (ORFV), posing a serious threat to the health of ruminants and humans. Vaccination remains the most effective strategy for disease control. However, the limitations of conventional vaccines necessitate the development of safer and more efficient alternatives. Methods: Based on the previously attenuated Orf vaccine strain (HB-TS09F65) as the parental virus, two gene-deletion strains, rHB09-ΔORF127 and rHB09-ΔORF130, were successfully constructed by deleting the ORF130 and ORF127 genes via homologous recombination. Results: In vitro experiments demonstrated that both gene-deletion strains exhibited replication capacity and genetic stability comparable to their parental strains (vaccine and virulent strains). Compared with the parental strains, the two gene-deletion viruses showed reduced virulence. Immunogenicity studies of two gene-deleted virus strains demonstrated that they elicited humoral immune responses comparable to the parental virus, and significantly enhanced Th1-type cellular immune responses. Challenge experiments in a rabbit model demonstrated that rHB09-ΔORF127 and rHB09-ΔORF130 provided protection rates of 100% and 85.7%, respectively. Conclusion: The two gene-deletion strains exhibit efficient antigen production, favorable immunogenicity, improved safety, and effective protection, making them promising candidate strains for the development of a gene-deleted vaccine against Orf.