Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus

• Published in: Veterinary microbiology Vol. 294; p. 110122
• Main Authors: Ren, Shanhui, Zhang, Yuzhe, Gao, Xiaolong, Wang, Xiangwei, Tong, Lina, Wang, Shasha, Sun, Yuefeng, Yin, Xiangping, Chen, Haotai
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2024
• Subjects: Cre-loxP system; Lumpy skin disease virus; MDBK-Cre cell line; Recombinant virus; Cre-loxP system; Lumpy skin disease virus; Recombinant virus; MDBK-Cre cell line
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2024.110122

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine. •Establish a Cre recombinase enzyme-stable MDBK cell line.•Establish a platform for the Cre-loxP recombination system under a pmH5 promoter.•Construct recombinant LSDV-EGFP-ΔTK and LSDV-ΔTK viruses.