An HRM Assay to Differentiate Sheeppox Virus Vaccine Strains from Sheeppox Virus Field Isolates and other Capripoxvirus Species

Abstract

Sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates.

Evaluation of Serological Tests for Detection of Antibodies against Lumpy Skin Disease Virus

“…Lumpy skin disease (LSD) is a highly contagious transboundary disease in cattle of all ages and breeds ( 1 ) characterized by fever, lymphadenopathy, and nodular skin lesions which can be observed also in the mucous membranes of the eyes and in the respiratory and gastrointestinal tracts ( 2 ). Until recently, the endemic geographic range of LSD was limited to the African continent, including Madagascar ( 3 ). The disease has spread widely throughout the Middle East ( 4 – 7 ) and Balkan regions ( 8 ), southern Caucasus, and parts of the Russian Federation ( 9 ), attracting huge attention.…”

Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species

“…Other gel-based assays focus on a species-specific deletion in the genome of SPPV, leading to a shorter PCR fragment without the additional digestion step [ 42 ], or target one species specifically [ 43 , 44 , 45 ]. Another diagnostic PCR method is conventional PCR followed by either a high-resolution melting curve analysis [ 24 ] or performance of a fluorescence melting curve analysis [ 29 , 30 ]. Similar tools have been published for the differentiation of field and vaccine strains of capripox viruses (so-called genetic DIVA).…”

“…Similar tools have been published for the differentiation of field and vaccine strains of capripox viruses (so-called genetic DIVA). In detail, gel-based systems with a different amplicon length analyzed by agarose gel electrophoresis [ 20 ], RFLP [ 46 ], or high-resolution melting (HRM) curve [ 24 , 47 , 48 ] as well as a nested PCR assay [ 49 ] have been described. What all these assays have in common is the need for an additional processing step following the PCR.…”

“…Next to virus isolation in primary cell cultures [ 12 , 13 ] or continuous cell lines [ 14 ], and virus detection via antigen capture ELISAs [ 15 , 16 ] or electron microscopy [ 17 , 18 ], detection of CaPV viral genomes [ 19 ] is an important tool for the confirmation of CaPV cases and outbreaks. Here, different PCR detection systems have been developed over the years, ranging from conventional gel-based assays combined with agarose gel electrophoresis [ 20 ] or with restriction fragment length polymorphism (RFLP) analyses [ 21 ] through loop-mediated isothermal amplification (LAMP) assays [ 22 , 23 ] and high-resolution melting curve analyses [ 24 ] to real-time quantitative PCR (qPCR) assays using intercalating dyes [ 20 ] or fluorescence-labelled probes [ 6 , 25 , 26 ].…”

“…Whereas PCR systems using intercalating dyes are combined with melting curve analysis [ 29 , 30 ], probe-based assays do not necessarily need additional working steps [ 6 , 25 , 26 ]. Nevertheless, all mentioned methods allow various issues to be analyzed reliably: the presence of CaPV genome in general (pan capripox assays) [ 6 , 21 ], the differentiation between the three CaPV species [ 23 , 24 ], and the genetic differentiation of infected from vaccinated animals (DIVA) analyses [ 20 , 25 , 26 ].…”