Construction Of A 115 Gene-deleted Mutant Derived From Orf Virus-SY17 Strain

Contagious Ecthyma is an acute contact zoonosis caused by orf virus,which mainly infects small ruminants such as goats and sheep.Contagious Ecthyma is characterized by the formation of blisters,pustules and scars in the nasolabial,breast,and tail roots of infected animals.Contagious Ecthyma seriously endangers human health and causes huge economic losses worldwide.At present,there is no effective preventive or therapeutic drug for Contagious Ecthyma in the clinic,while the vaccine is the main way to control Contagious Ecthyma.However,the clinical application of conventional inactivated vaccines and attenuated vaccines is limited due to their defects and deficiencies.The development of new vaccines is of great significance for the prevention and treatment of Contagious Ecthyma.Combined with the results of bioinformatics prediction,the potential function of the 115 gene located in the terminal region of the ORFV genome may be related to viral virulence,host range and other functions,which will contribute to the development of a live attenuated vaccine.In this study,an ORFV strain was isolated from the lip-scar of infected sheep from a sheep farm which suspected of having Contagious Ecthyma in Songyuan City,Jilin Province.The ORFV strain was named ORFV-SY17.Whole-genome sequencing of ORFV-SY17 was performed by high-throughput sequencing technology.The whole genome sequence of ORFV-SY17 was obtained by splicing,and the sequence was submitted to Genbank(accession number: MG712417).The analysis of the genomic end region of ORFV-SY17 revealed that the ORF 115 has specific amino acid mutation sites,such as isoleucine(I)at position 64,histidine(H)at position 72,etc.These amino acid positions were not found in the corresponding gene sequences of other ORFV strains.In addition,the study found that some amino acid sites in the ORF 115 are related to host sources,such as the 5th and 8th amino acid positions.Phylogenetic analysis showed that different ORFV strains derived from goats and sheep were divided into two branches.These results indicated that the function of the ORF 115 may be related to the host range.In order to further analyze the function of ORF 115,this study used the homologous recombination technique to construct the 115 gene deletion strain of ORFV-SY17.The homologous left arm sequence and the homologous right arm sequence of ORF 115 were obtained by PCR amplification,and then ligated with the p SPV-EGFP vector to construct the transfer vector p SPV-115L-EGFP-115 R.After the OFTu cells were infected with ORFV-SY17,the transfer vector p SPV-115L-EGFP-115 R was transfected into OFTu cells by cell transfection experiments.The recombinant virus strain was initially screened by fluorescence microscopy.The recombinant virus ORFV-SY17?115 was successfully obtained after multiple rounds of screening and PCR identification,and the biological characteristics such as virus titer and genetic stability of the recombinant virus were determined.The TCID50 assay showed that the TCID50 of recombinant virus was 10-5.625/0.1 ml,which was not significantly different from the parental virus.The cytopathic comparison test and the genetic stability test showed that the recombinant virus can normally infect OFTu cells and stably inherited.The above results not only help to reveal the biological function of ORFV 115 gene,but also provide an important material basis for the development of a safer and more effective ORFV attenuated vaccine.These will provide important technical means for clinical prevention and control of Contagious Ecthyma.