The Characterization and Differentiation of Recombinant Lumpy Skin Disease Isolates Using a Region within ORF134

The recent description and characterization of several novel and unique lumpy skin disease virus (LSDV) strains have revealed the inadequacy of current techniques for differentiating between vaccine- and wild-type viruses. The lack of reliable sequencing targets for promptly distinguishing circulating recombinant vaccine-like strains (RVLSs) highlights the need to develop a single and simple differentiation tool. In this study, we analyzed the available LSDV whole-genome sequences and identified a 705-bp region in open reading frame (ORF) LW134. Based on a single run of nucleotide sequencing and phylogenetic analysis, the region with 13 informative single nucleotide polymorphisms (SNPs) was capable of accurately segregating the novel RVLSs into the same five clusters previously confirmed by whole-genome sequencing. In addition, archived RVLSs from Russia were analyzed for further characterization using the newly described single PCR and sequencing assay. The ORF LW134 assay identified one archived RVLS as a novel cluster distinct from the previously described five clusters, while clustering the remaining samples into previously designated lineages, demonstrating the reliability of the assay. The novel PCR and sequencing assays described in this study have great potential for accurately delineating the molecular and evolutionary affiliation of circulating RVLSs.