A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses

• The current study describes an improved HRM assay able to distinguish between virulent and field types of Lumpy skin disease virus.
• The HRM test is based on a 27 bp deletion in the LSDV126 gene of the vaccine strain. The vaccine virus displays ∼0.50 °C lower Tm compare to field viruses.
• Viruses isolated from immunize cattle that developed disease symptoms behave in the assay identically to the vaccine strain.
• Sequencing of the LSDV126 gene verified that the 27 bp deletion is present only in vaccinated cattle isolates, validating the HRM results.

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.