Evaluation Of Immune Effects Of ORFV-SY17Δ120 Gene Deletion Mutant

Contagious ecthyma（CE）also known as Orf virus（ORFV）,is an acute,highly contagious infectious disease caused by ORFV,mainly in some small ruminants,including sheep and goats.Although ORFV could induce a strong immune response,it developed immune escaping ability in its interaction with the host.Due to its immune evasion,animals might be infected repeatedly.The disease has posed a threat to sheep industry,which has become an important epidemic disease in flocks.Vaccination is still an important means to prevent and control the disease,but there is still a lack of effective vaccines for the prevention and control of the disease,which makes the poor immune effect of vaccines and even immune failure occur from time to time.The live attenuated ORFV vaccines have the advantages of low vaccination dose,stronger immunity,and longer maintenance of vaccine effects,making them a promising candidate for the future development of new and effective ORFV vaccines.In this study,the biological characteristics including the virulence,the ability of replication and the genetic stability,and the safety of the ORFV-SY17（35）120 gene deletion mutant constructed in our previous study were assayed by CPE observation,electron microscopy observation,PCR detection,viral plaque assay,TCID₅₀ assay,animal growth performance assay,pathogen detection,and pathological analysis,in order to evaluate its potential as a live attenuated vaccine candidate.ORFV-SY17（35）120gene deletion mutants had a similar virion morphology to the ORFV-SY17 parental strain,and infected cells with both virus strains also exhibited similar CPEs,such as semicircles and shrinkage.Viral plaque assays and TCID₅₀ showed that the ORFV-SY17（35）120 gene deletion strain had slightly less plaque formation and proliferative capacity than the ORFV-SY17 parental strain.The one-step growth curve（MOI=10）and multi-step growth curve（MOI=0.1）of the ORFV-SY17（35）120 gene deletion mutants and the ORFV-SY17 parental strain were similar,indicating that the ORF120 deletion had no significant effect on the replication ability of the ORFV-SY17（35）120 gene deletion mutants.The ORFV-SY17（35）120 gene deletion mutants were serially passaged for 12 passages,and the ORF120,as well as ORF011 genes,were tested by PCR to identify its genetic stability.The results showed that the ORF011 gene was stably inherited in the ORFV-SY17（35）120 gene deletion mutants.In addition,the virus titers of the ORFV-SY17（35）120 gene deletion mutant at each passage were determined by TCID₅₀assay.The results indicated that the virus titers of the ORFV-SY17（35）120 gene deletion mutant were relatively stable Finally,the safety of the ORFV-SY17（35）120 gene deletion mutant was evaluated by experimental infection of healthy lambs.The results of the growth performance,the observation of clinical signs,and the pathological changes of lambs indicated that the ORFV-SY17（35）120 gene deletion mutant had a good safety.In summary,the above research results indicate that the ORFV-SY17（35）120 gene deletion mutants are expected to be a new candidate for ORFV attenuated vaccines.Lived vaccines are usually administered in a similar route to natural infections.The traditional ORFV lived vaccines were usually inoculated by epidermal scratching,which has been confirmed to stimulate a strong immune response and effective immunological protection.However,there are some disadvantages for scratching inoculation,such as a relatively difficult manipulation,the strong stress response in animals,and the biological hazard Thus,this study focuses on the optimization of the inoculation route of the ORFV-SY17（35）120 gene deletion mutant.Three-four month-old lambs were immunized by scratching and subcutaneous injections with the ORFV-SY17（35）120 gene deletion mutants.The immunization procedure was applied as follows:the primary immunization on the 0th day,the booster immunization on the 14 day,and the challenge protection test on the 28 day.The Ig M,Ig G specific antibodies,neutralization antibodies,ORFV 059 virus protein antibodies,as well as cytokines and T lymphocyte subsets in serum samples of immunized animals at different time points were detected by ELISA,Western blot,and FACS.The results indicated that both scratch immunization and subcutaneous immunization with ORFV-SY17（35）120 gene deletion mutants could induce the production of ORFV-specific antibodies,ORFV 059protein antibodies,Th1/Th2 cytokines,and CD4⁺and CD8⁺T lymphocytes in lambs.The animal regression experiment using the virulent strain ORFV-SY17 strain proved that both scratch and subcutaneous immunization with ORFV-SY17（35）120 gene deletion mutants can provide good immune protection.The above research results imply that the deletion of ORF120 gene did not affect the replication and proliferation of ORFV-SY17（35）120 gene deletion strain on OFTu cells and was genetically stable.ORFV-SY17（35）120 gene deletion strain was relatively safe for vaccinated lambs,and the safe immunization dose was determined to be 1.5×10^6.5 TCID50 per lamb.In addition,the ORFV-SY17（35）120 gene deletion mutants may be a new candidate for ORFV attenuated vaccines,and that subcutaneous immunization is an alternative to the traditional scratch immunization route,providing a valuable technical mean for the clinical management of contagious ecthyma.