title: Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants.
abstract: 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 beta-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.
authors.affiliation: Onderstepoort Veterinary Institute, P/Bag X5, Onderstepoort 0110, South Africa. WallaceD@arc.agric.za
authors.forename: David Brian
authors.initials: DB
authors.lastname: Wallace
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Weyer
authors.forename: Louis Hendrik
authors.initials: LH
authors.lastname: Nel
authors.forename: Gerrit Johannes
authors.initials: GJ
authors.lastname: Viljoen
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D018115:Ephemeral Fever Virus, Bovine / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008167:Lumpy skin disease virus / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D011820:Rabies virus / Q000235:genetics; D011995:Recombination, Genetic; D012296:Rift Valley fever virus / Q000235:genetics; D014614:Vaccines, Synthetic; D014765:Viral Vaccines
chemical_list: D014614:Vaccines, Synthetic; D014765:Viral Vaccines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins