Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants

Lumpy skin disease virus (LSDV) is being developed as a vector for recombinant vaccines against diseases of veterinary importance. A strategy for generating viral thymidine kinase (TK) gene-disrupted recombinants which are stable and homogeneous using the South African Neethling vaccine strain of LSDV as vector has been developed. To assist with the selection process, the Escherichia coli β-galactosidase (lacZ) visual marker gene was incorporated into the constructs. However, the use of lacZ has certain limitations. An improved strategy was then devised substituting lacZ with the enhanced green fluorescent protein (EGFP) under control of the vaccinia virus (VV) P11K late promoter. The EGFP marker was found to enhance the selection process, and with the inclusion of additional sonication and filtration steps the number of passages required to select recombinants to homogeneity has been reduced. In support of the improved method for generation and selection of recombinants described, three different LSDV recombinants expressing the glycoprotein genes of bovine ephemeral fever virus, Rift Valley fever virus and rabies virus were prepared and characterised.