Construction and characterization of the ORF131 gene deletion strain of lumpy skin disease virus

Abstract Lumpy Skin Disease (LSD), caused by the Lumpy Skin Disease Virus (LSDV), is a legally reportable disease recognized by the World Organization for Animal Health (WOAH) and has resulted in significant economic losses for the global cattle industry. Although several commercial LSDV vaccines are currently available, safer and more effective gene-deleted versions remain lacking. Therefore, screening key functional genes and developing gene-deleted live-attenuated vaccine strains hold substantial research value. In this study, we focused on ORF131, a gene whose function remains unclear. We successfully constructed an rLSDV-ΔORF131-EGFP gene deletion strain utilizing a homologous recombination, followed by purification using limiting dilution and single-cell subcloning techniques. Polymerase Chain Reaction (PCR) and Sanger sequencing validation confirmed that the deletion strain was successfully purified and free from wild-type virus contamination. Biological characterization indicated that the strain was genetically stable, with the optimal viral harvesting time in Madin-Darby Bovine Kidney (MDBK) cells being 72 h. Furthermore, RNA sequencing analysis of virus-infected cells revealed that rLSDV-ΔORF131-EGFP enhanced the immune and inflammatory responses of host cells compared to wild-type LSDV. This study not only provides a potential candidate strain for the development of an LSDV attenuated vaccine but also offers a theoretical foundation for the prevention and control strategies of LSD.