The Safety Evaluation And Immunological Effects Of Orf Virus Gene Deletion Attenuated Strain

Contagious Ecthyma known as"Orf",is an acute and highly contagious infectious disease of sheep and goats caused by Orf virus?ORFV?infection.The disease is also an important zoonotic disease.At present,the disease has been listed as a legally reported infectious disease by the world animal health organization.Vaccination is still an important way to the prevention and control of the disease,but due to the existence of ORFV immune evasion phenomenon,causing the existing vaccines exist immune protection period is short,immune the protective effect of uncertain problems.In view of the frequent occurrence of this disease in sheep and the lack of ideal vaccine for prevention and control of the disease,it is urgent to develop an effective and safe new vaccine.Based on our previous study of 2 strains of ORFV attenuated virulence gene deletion?ORFV-NA1/11?002 and ORFV-NA1/11?121?,firstly,by inoculating OFTu propagation culture,and identify the characteristics of proliferation in OFTu cells using the method of determination of TCID50,analysis showed that the strain compared with the wild type?ORFV-NA1/11?,ORFV-NA1/11?002 and?121reduction in gene deletion had no significant difference in virus replication in OFTu cells and the kinetics of virus production?P>0.05?,showed that the deletion of 002and 121 genes does not affect its replication in OFTu cells.After that,the ORFV-NA1/11?002 and ORFV-NA1/11?121 gene deleted attenuated strains were inoculated with 3 month old lambs at different dose.The safety of ORFV-NA1/11delta 002 and delta 121 gene deletion attenuated strain on lambs was evaluated through detection and analysis of the clinical symptoms and pathological changes of the lambs and the detection of the distribution of the virus.The results confirm that ORFV-NA1/11?002 and ORFV-NA1/11?121 gene deleted attenuated strains can be successfully inoculated,and the safety dose of lamb vaccination is 10⁵ TCID₅₀.30 sheep of 3 month old lambs without specific antibodies were randomly divided into three groups,named as,ORFV-NA1/11?002 immunization group?M002?,ORFV-NA1/11?121 immunization group?M121?and control group.In group M002 and group M121,the lambs were inoculated through the scratch of the medial part of the lip and the right hind limb respectively.The inoculation volume was 10⁵ TCID₅₀,and the control group was inoculated with sterile PBS in the same way.3 weeks after the first immunization was strengthened in the same way and dose.After the inoculation,the blood was collected weekly and the lambs serum was separated.The antibody level was detected by ELISA.At the same time,we collected weekly lambs for anticoagulation and separated peripheral blood lymphocytes,and detected the expression of CD4+and CD8+T lymphocytes by FACS.The levels of IFN-?,IL-2,IL-6,IL-10 and other Th1 and Th2 cytokines in serum were detected by cytokine detection kit.The results show that ORFV-NA1/11?002 and ORFV-NA1/11?121 gene deletion strains can induce the proliferation of lymphocyte subclasses in animal body,and mainly cause the level of Th1 cell factor,that is,the immune response that stimulates the body is mainly cellular immunity.In 2 weeks after Strengthen immunity,from M002 group,from the M002 group,the M121 group and the PBS group,8 samples were randomly selected,and the ORFV-sy wild strains isolated from the laboratory were used for the attack test.Observe and record the daily incidence of lamb,M002 group,M121 group inoculated lamb had no infection,and all of the PBS groups were infected.The results showed that the immune protection rate of ORFV-NA1/11?002?ORFV-NA1/11?121 gene deletion attenuated strains were 100%.The above findings provide important evidence for the formulation of effective prevention and control techniques for the disease,and will have broad application prospects.