Construction Of 002 And 121 Double Gene-deleted Orf Virus And Evaluation Of Their Protective Efficacy

ORFV is a linear double-stranded DNA virus,which belongs to the Poxviridae and Parapoxvirus genus.It causes a highly contagious infectious disease of goats,sheep,and human beings,also known as CE or"Orf".ORFV is epithelial and can cause clinical symptoms of erythema,papule,blisters,and pustules.Lambs are mainly infected by ORFV and the infected lambs often suffer from eating disorders due to pain in the corners of the mouth,leading to weight loss.This pathogen is able to infect sheep flock continuously,and it is difficult to be cleared.If an animal is infected ORFV alone,the mortality rate is low,the clinical symptoms are not obvious,and it is easy to be ignored.However,the mortality will increase after secondary infection,which is harmful to sheep industry.At present,this disease has been listed as a legally reported infectious disease by the World Organization for Animal Health?OIE?.So far,there is no effective treatment for sheep infected by ORFV.Vaccination is the only way to prevent and control this disease.However,the traditional inactivated vaccines and attenuated vaccines on the market have disadvantages such as short duration of protection,virulence reversion,and spread of virus,which have limited their clinical application and cannot meet the prevention and control of the disease under the current situation.Therefore,there is an urgent need to develop new vaccines to prevent the disease.ORFV's 002 and 121 genes are located in the variable regions at both ends of the genome,and the proteins encoded by 002 and 121 genes inhibit NF-?B activity;It is speculated that the 002 and 121 genes of ORFV may be related to virus virulence.In this study,the ORFV virulent strain"ORFV-SY17"stored in the laboratory was used as the original strain.The homelogens recombination technology was used to construct the 002/121 double gene deletion strain of ORFV-SY17 strain based on the ORFV 002 single gene deletion strain?ORFV-SY17?002?.The transfer plasmid pSPV-121LEGFP-121R was constructed,which was followed by cell transfection into OFTU cells infected with ORFV-SY17?002.After screening,recombinant virus strains ORFV-SY17?002/?121 were obtained.ORFV-SY17?002/?121 has stable heredity and TCID₅₀0 of 10^-5.625/0.1ml.The deletion of the 002 and 121 genes does not affect the ability of the strain to replicate in the cell.15 3 months old lambs were selected for vaccination and divided into three groups randomly:ORFV-SY17?002/?121 immunization group?M-002/121?,ORFV-SY17 immunization group?M-SY17?and PBS control group.All groups of lambs were vaccinated through lip scratches.The M-002/121 and M-SY17 groups were vaccinated with the corresponding virus,and the control group was vaccinated with sterile PBS.After 3 weeks of immunization,boosters were administered in the same manner and dose.Blood samples of the lambs in each immunization group were collected on day 1,day 3,day 5,day 7,day 14,day 21,day 28,day 35,and day 42after immunization,respectively,and the samples were subjected to anticoagulant and serum isolation treatment.T lymphocytes were also isolated after anticoagulation treatment.FACS assay was used to quantity the population of CD4⁺and CD8⁺T lymphocytes.Serum was separated for,ELISA detection assay to detect the level of IL-4 and IFN-?cytokines.The results showed that both ORFV-SY17 and ORFV-SY17?002/?121 were able to induce cellular and humoral immunity,but the level of humoral immunity is not well induced,while the cellular immune response is the main response.The fifth week after the first immunization,9 lambs were randomly selected from each experimental group for challenge experiments.The strain used for challenge was the ORFV-SY17 wild strain.After challenging,all the lambs were observed daily and corresponding records were made together.Positive infections were observed in the PBS control group,but no observed infection were occurred in the experimental group.The results showed that the immunological protection rate of the wild-type strain ORFV-SY17 and the attenuated strain of ORFV-SY17?002/?121 reached 100%in the present study.Moreover,the ORFV-SY17?002/?121 attenuated strain has a limited feeding disturbance when inoculated when compared with ORFV-SY17 strain.This is of great significance for Orf prevention and control.