Contagious ecthyma virus(CEV),commonly known as Orf virus(ORFV),mainly causes characteristic "mouth sore" lesions in sheep and goats,and can also be infected by human contact.It is an important infectious pathogen of zoonotic diseases.It seriously affects the development of sheep farming and the safety of public health.The ORFV genome is large and it encodes many proteins,and the functions of many proteins are still unclear,which seriously restricts the development of vaccines.At present,there is no effective vaccine for the prevention and control of this disease.Live attenuated vaccine has become an important development direction of new vaccines because of its advantages of low dose and safety.Recent studies on the function of the proteins encoded by ORFV ORF120 and ORF121 genes indicate that ORF120 and ORF121 genes are important virulence factors and therefore can be used as targets of attenuated vaccines.In this study,the recombinant shuttle plasmid p UC57-LFΔORFs120-121-e GFP-RFΔORFs 120-121 carrying green fluorescent protein was constructed by homologous recombination technology,and transfected into ORFV wild virus strain infected cells.Fluorescence screening,plaque purification,PCR identification and other methods were used.The ORFV ORF120 and ORF121 double gene deletion attenuated strains were successfully screened and identified and named ORFV-SYΔORFs 120-121 double gene deletion attenuated strains.The in vitro reproductive capacity of the recombinant ORFV-SYΔORFs 120-121 strain was evaluated by one-step and multi-step growth curves.The results showed that the replication and proliferation capacity of the recombinant strain was basically the same as that of the wild strain,and the virus titer did not change significantly,indicating that the deletion of ORF120 and ORF121 genes did not affect the replication and proliferation of the recombinant strain in host cells.In order to clarify the safety and immune protection effect of ORFV-SYΔORFs120-121 recombinant strain,and evaluate its application prospect as a vaccine candidate strain,this study immunized lambs with ORFV-SYΔORFs 120-121 recombinant strain by subcutaneous/scratch route respectively,and the results showed that,Both subcutaneous and scratch inoculation can induce strong immune protection,mainly Th1 immune response.The antibody level was found to be the highest at 2 w after inoculation,but the duration was short;This result is consistent with the characteristic that ORFV mainly induces cellular immunity.The results of animal challenge protection test showed that subcutaneous and scratch inoculation of ORFV-SYΔORFs120-121 recombinant strain could provide good immune protection for homologous parent strain.In conclusion,the ORFV-SYΔORFs 120-121 recombinant strain is expected to be a candidate vaccine strain of live attenuated ORFV vaccine,which provides an important technology basis for the prevention and control of infectious sheep impetigo disease in clinical practice. |