ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology

• Published in: Journal of virological methods 2023-02, Vol.312, p.114652-114652, Article 114652
• Main Authors: Yu, YongZhong, Zhang, Fan, Duan, Xuyang, Yang, ChaoQun, Cui, YuDong, Yu, Li
• Format: Article
• Language: English
• Subjects: Animals; CRISPR-Cas Systems; CRISPR/Cas9 technique; Ecthyma, Contagious; Gene Editing; Gene Expression; Humans; Orf virus; Orf virus - genetics; ORFV-TRAP recombinant virus; Sheep; Staphylococcus aureus; Staphylococcus aureus - genetics; Staphylococcus aureus - metabolism; TRAP; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2022.114652

Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease. •This study provides a feasible approach for manipulating non-essential genes of Parapoxvirus in cells using CRISPR/Cas9 gene editing techniques.•The viral VEGF gene of ORFV was modified successfully by the targeted-inserted TRAP gene of Staphylococcus aureus.•The efficiency of VEGF gene editing was generally consistent with the results of endonuclease digestion in vitro.•It was identified that recombinant ORFV could effectively deliver TRAP gene and express it in S. aureus non-infected cells.