# Development of a Multi-Locus Real-Time PCR with a High-Resolution Melting Assay to Differentiate Wild-Type, Asian Recombinant, and Vaccine Strains of Lumpy Skin Disease Virus

## TL;DR

Scientists developed a fast and affordable test to distinguish between wild, vaccine, and recombinant strains of a cattle virus causing economic losses.

## Contribution

A novel multi-locus HRM assay was developed to differentiate LSDV strains in under 2 hours.

## Key findings

- The HRM assay can distinguish wild-type, vaccine, and recombinant LSDV strains using three target genes.
- The method successfully identified recombinant strains with wild-type and vaccine-type gene combinations.
- The assay was validated using synthetic DNA and clinical samples.

## Abstract

Lumpy skin disease (LSD) is a transboundary viral disease of cattle, the clinical signs of which are fever, anorexia, skin nodules, decreased milk production, infertility, and abortion, leading to severe economic losses. A live-attenuated vaccine is available to control this disease, but a strategy for the differentiation of infected from vaccinated animals (DIVA) is needed because of the side effects of the vaccine. Recently, a recombinant strain, which is a hybrid of the wild-type field and vaccine strains, has spread throughout Asian countries. Therefore, a rapid and low-cost method to differentiate field, vaccine, and recombinant strains is needed. We chose the high-resolution melting (HRM) assay, which is a post-PCR assay capable of discriminating gene variations without sequencing, and selected three genes (ORF095, ORF126, and ORF145) to establish the differentiation assay. In this study, we found that each gene can be distinguished as the field or vaccine type by each HRM assay, and the multi-locus HRM assay can identify the recombinant viruses that have the wild-type ORF095 and ORF145 genes and the vaccine-type ORF126 gene within 2 h of the PCR run. We believe that this method will be useful to control LSD in many countries.

Lumpy skin disease virus (LSDV) affects cattle and causes significant economic damage. The live vaccine derived from an attenuated strain is effective but is associated with mild disease and skin lesions in some vaccinated cattle. Moreover, recombinant LSDV strains, particularly one with wild-type field and vaccine strains, have recently emerged and spread throughout Asian countries. A cost-effective LSDV typing method is required. We developed a multi-locus real-time PCR with a high-resolution melting (HRM) assay to differentiate between the wild-type, vaccine, and dominant Asian recombinant strains. Based on a multiple alignment analysis, we selected three target genes for the HRM assay, ORF095, ORF126, and ORF145, in which there are insertions/deletions and nucleotide substitutions between wild-type and vaccine strains, and designed primer sets for the assay. Using the synthetic DNA encoding these genes for the two strains, it was shown that the PCR amplicons intercalated with a saturating fluorescent dye could clearly differentiate between wild-type and vaccine strains in the HRM analysis for all three target genes. Further, using clinical samples, our method was able to identify recombinant strains harboring the wild-type ORF095 and ORF145 and the vaccine strain ORF126 genes. Thus, our HRM assay may provide rapid LSDV typing.