The Development of a Real-Time PCR Assay for Specific Detection of the NISKHI Sheep Pox Vaccine Virus Strain DNA

Abstract

Sheep pox (SPP) constitutes a global animal health scourge, despite the numerous efforts targeting the eradication of the disease implemented in affected countries. An efficient control and eradication strategy incorporates the use of live attenuated vaccines, which in turn requires a method for differentiation between vaccinated and infected sheep. The NISKHI live attenuated SPP vaccine (LAV) is abundantly used in Russia, Kazakhstan and other Central Asian countries. This study describes the development and evaluation of a real-time PCR with a high-resolution melting assay, capable of differentiating the NISKHI vaccine virus from circulating virulent field strains. The RNA polymerase subunit RPO132 gene contains a unique single nucleotide polymorphism (SNP) capable of altering the melting curves of amplicons from LAV and virulent field isolates circulating in the region. The melting temperature (Tm) of field isolates ranged from 75.47 °C ± 0.04 to 75.86 °C ± 0.08, while the vaccine strain averaged 76.46 °C ± 0.12. Subsequent evaluation of this assay demonstrated that the recent SPP outbreaks in central Russia may be attributed to virulent field isolates. This robust assay was proven to consistently and differentially detect the NISKHI LAV strain when analyzing clinical samples from affected sheep.