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A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

No abstract

Capripoxvirus belongs to poxviridae family and causes three economically important diseases in ruminants namely, lumpy skin disease (LSD) in cattle, sheeppox in sheep, and goatpox in goats. Albeit non-zoonotic in nature, they have potential to cause high economic loss among the farmers. Capripoxvirus members share common structural proteins and can rise cross-immunity among them. The present study aimed to design a recombinant chimeric-vaccine from immunogenic proteins of these three members to protect all the host species by using immunoinformatics analysis. The palmitoylated EEV (Extracellular Enveloped Virion) membrane glycoprotein of LSD virus, SPPV-ORF 117 of sheeppox virus, B5R (EEV host range protein) of goatpox virus, and a common protein to all the members, P32, were the major immunodominant proteins used in the present chimeric vaccine construction. Several computational tools were applied to define the most immunogenic regions and different possible adjuvants and universal T-helper agonists were linked to the new construct. The designed vaccine construct was examined for physicochemical properties, immunogenicity, 3D model, Docking analysis and Molecular Dynamics simulations, by using reliable software. After evaluation of the results, the final designed vaccine is expected to have potential in stimulating the humoral response in addition to the cellular responses with acceptable stability.

The present study was aimed to elucidate the host–virus interactions using RNA‐Seq analysis at 1 h and 8 h of post‐infection of sheeppox virus (SPPV) in lamb testis cell. The differentially expressed genes (DEGs) and the underlying mechanisms linked to the host immune responses were obtained. The protein–protein interaction (PPI) network analysis and ingenuity pathway analysis (IPA) illustrated the interaction between the DEGs and their involvement in cell signalling responses. Highly connected hubs viz. AURKA, CHEK1, CCNB2, CDC6 and MAPK14 were identified through PPI network analysis. IPA analysis showed that IL‐6‐ and ERK5‐mediated signalling pathways were highly enriched at both time points. The TP53 gene was identified to be the leading upstream regulator that directly responded to SPPV infection, resulting in downregulation at both time points. The study provides an overview of how the lamb testis genes and their underlying mechanisms link to growth and immune response during SPPV infection.