Molecular based detection, Validation of LAMP assay and Phylogenetic analysis of Capripoxvirus in Kenya

Abstract

The genus Capripoxvirus (CaPVs) comprises of sheeppox virus (SPV), goatpox virus (GPV) and Lumpy skin disease virus (LSDV) that cause sheep pox (SP), goat pox (GP) and Lumpy skin disease (LSD) in sheep, goats and cattle respectively. They are serious poxviruses of ruminants and are of significant economic impact for the livestock industry and food security. These diseases are contagious and are endemic in parts of Africa and the Middle East. Being viral diseases, the mainly employed methods of management and control of spread is by vaccination and quarantine which are only effective with rapid detection of the disease. These diseases are transboundary and notifiable. There has always been challenges in diagnosis when using traditional methods like ELISA and Cell cultures due to time factor and cross reactivity of the Pox virus antigen with other closely related viruses like Orf virus. Therefore, a need to employ more rapid, simple, specific and sensitive detection methods for diagnosis which will ensure early detection of the disease and therefore effective implementation of control measures. The objective of this study was to compare the available molecular techniques, validate LAMP assay and evaluate CaPVs genome variability, diversity and evolutionary relationships. A cross-sectional study of 130 samples that included skin scrapings, whole blood and cell cultures from sheep, goats and cattle were analyzed by LAMP assay, real time PCR as the gold standard method and Conventional PCR and the results compared. Genomic DNA libraries were prepared from extracted DNA, deep sequenced and the obtained data analyzed. The sensitivity of LAMP assay was 97% and a proportion of detection of 61% was realized in the studied population. The specificity of the LAMP assay was at 100% and successfully detected CaPVs DNA extracted from all samples without cross reacting with closely related virus like Orf virus and Pestes des Petit Ruminants. The deep sequenced LAMP positive samples confirmed the presence of the detected CaPVs after blast analysis against the NCBI database. The phylogenetic analysis confirmed the 3 distinct capripoxviruses lineages: LSDV, GPV and SPV lineages. The Multiple sequence analysis of the P32, GPCR and RP030 genes revealed the virus specific signature found in specific viruses and therefore used to differentiate them. The study therefore supports the adoption of LAMP assays for rapid CaPVs diagnosis and also provides a simpler, effective and rapid method of detection, monitoring and controlling outbreaks xvi and the spread of disease. This study also forms a basis for molecular epidemiological studies and a reference in CaPVs genomic research in Kenya as a result of determined diversity and evolutionary relationships.