Performance of the currently available DIVA real-time PCR assays in classical and recombinant lumpy skin disease viruses.

The use of live homologous vaccines to protect against lumpy skin disease virus (LSDV) infection requires the use of molecular tools to differentiate between infected and vaccinated animals (DIVA). In this study, the commercial real-time PCR assays; ID Gene™ LSD DIVA Triplex kit and Bio-T kit® LSD - DIVA, as well as published assays targeting the GPCR gene (Journal of Virological Methods, 249, 48-57) and ORF008 and ORF126 (Sel'skokhozyaistvennaya Biologiya, 54, 347-358) were evaluated. These assays correctly identified classical field isolates (European lineage) and vaccine (Neethling vaccine). In contrast, when tested using vaccine-like recombinant viruses, the commercial and published assays were not able to correctly identify recombinant isolates. At the same time, the recombinant viruses were detected as either field and/or vaccine, or not detected at all depending on the assay. The different gene sequences present in recombinant viruses cause these DIVA assays to incorrectly assign recombinant viruses as either a field or vaccine virus. This observation has implications for using these assays and for identification of LSDV vaccine.

Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease.

Vaccination against lumpy skin disease (LSD) is crucial for maintaining the health of animals and the economic sustainability of farming. Either homologous vaccines consisting of live attenuated LSD virus (LSDV) or heterologous vaccines consisting of live attenuated sheeppox or goatpox virus (SPPV/GPPV) can be used for control of LSDV. Although SPPV/GTPV-based vaccines exhibit slightly lower efficacy than live attenuated LSDV vaccines, they do not cause vaccine-induced viremia, fever, and clinical symptoms of the disease following vaccination, caused by the replication capacity of live attenuated LSDVs. Recombination of capripoxviruses in the field was a long-standing hypothesis until a naturally occurring recombinant LSDV vaccine isolate was detected in Russia, where the sheeppox vaccine alone is used. This occurred after the initiation of vaccination campaigns using LSDV vaccines in the neighboring countries in 2017, when the first cases of presumed vaccine-like isolate circulation were documented with concurrent detection of a recombinant vaccine isolate in the field. The follow-up findings presented herein show that during the period from 2015 to 2018, the molecular epidemiology of LSDV in Russia split into two independent waves. The 2015-2016 epidemic was attributable to the field isolate. Whereas the 2017 epidemic and, in particular, the 2018 epidemic represented novel disease importations that were not genetically linked to the 2015-2016 field-type incursions. This demonstrated a new emergence rather than the continuation of the field-type epidemic. Since recombinant vaccine-like LSDV isolates appear to have entrenched across the country's border, the policy of using certain live vaccines requires revision in the context of the biosafety threat it presents.

Complete Genome Sequence of the Lumpy Skin Disease Virus Recovered from the First Outbreak in the Northern Caucasus Region of Russia in 2015.

We report here the complete genome sequence of a lumpy skin disease virus (LSDV) isolate obtained in the Northern Caucasus region of Russia in 2015. The LSDV/Russia/Dagestan/2015 genome sequence grouped with field LSDV isolates found in Serbia and Greece, suggesting the monophyletic origin of LSDV isolates that recently affected countries in the Northern Hemisphere.

Analysis and insights into recombination signals in lumpy skin disease virus recovered in the field.

Wide spread incidences of vaccine-like strains of lumpy skin disease virus (LSDV) have recently been reported in a Russian region with a neighboring country that actively vaccinate with a live attenuated LSD vaccine. The use of live-attenuated viruses (LAVs) as vaccines during an active outbreak, creates potential ground for coinfection of hosts and emergence of a strain combining genetic fragments of both parental vaccine and field strains. In this study, we analyse the vaccine-like strain LSDV RUSSIA/Saratov/2017 detected in Saratovskaya oblast, a region sharing border with Kazakhstan. To gain insight into possible recombination signals, a full-genome next-generation sequencing of the viral genome was performed using the Illumina platform. The genome contains the backbone of a live-attenuated vaccine with a patchwork of wild-type field virus DNA fragments located throughout. A total of 27 recombination events were identified. The average distance between the recombination sites was 3400 base pairs (bp). The impact of the recombination events on the virulence and transmission capacity of the identified virus remains to be clarified. These findings provide evidence for the first time of genetic exchanges between closely related strains of capripoxviruses in the field and a vaccine strain, and prompt a revisiting of the vaccination issue for a safe and efficacious prevention and control strategy of LSD.

A real time high-resolution melting PCR assay for detection and differentiation among sheep pox virus, goat pox virus, field and vaccine strains of lumpy skin disease virus.

In this paper, we report on the development of a real time high-resolution melting (HRM) PCR assay for detection and differentiation among sheep pox virus (SPPV), goat pox virus (GTPV), field isolates and vaccine strains of lumpy skin disease virus (LSDV) based on high-resolution melting curve analysis of their target PCR amplicons. A 111 bp region of LSDV010 ORF, which harbors unique genetic differences for each of these viral species, was selected as the PCR target in this study. During the validation of this assay using DNA from clinical isolates originated from naturally infected animals from the different geographic locations and reference strains, the obtained PCR amplicons demonstrated that the melting temperature picks were specific for each tested viral species, i.e., 74.56 ±â€¯0.04 °C for field LSDV, 74.95 ±â€¯0.08 °C for vaccine LSDV, 74.24 ±â€¯0.06 °C for SPPV and 73.61 ±â€¯0.04 °C for GTPV. The assessment of the assay sensitivity utilizing a LSDV field strain as a PCR template revealed the assay detection limit as low as 0.1 TCD50 lg/ml. Overall, this assay based on Rotor-Gene Q (QIAGEN) platform was shown to be reproducible across replicates and operators and can be recommended as an additional diagnostic tool to the currently available molecular assays for detection and differentiation of the genus Capripoxvirus species, including the differentiation of vaccine strains of LSDV from field isolates. The assay can be used for detection of these viruses in animal- and insect-derived field specimens.