In this study, the miRNAome and proteome of virulent Peste des petits ruminants virus (PPRV) infected goat peripheral blood mononuclear cells (PBMCs) were analyzed. The identified differentially expressed miRNAs (DEmiRNAs) were found to govern genes that modulate immune response based on the proteome data. The top 10 significantly enriched immune response processes were found to be governed by 98 genes. The top 10 DEmiRNAs governing these 98 genes were identified based on the number of genes governed by them. Out of these 10 DEmiRNAs, 7 were upregulated, and 3 were downregulated. These include miR-664, miR-2311, miR-2897, miR-484, miR-2440, miR-3533, miR-574, miR-210, miR-21-5p, and miR-30. miR-664 and miR-484 with proviral and antiviral activities, respectively, were upregulated in PPRV infected PBMCs. miR-210 that inhibits apoptosis was downregulated. miR-21-5p that decreases the sensitivity of cells to the antiviral activity of IFNs and miR-30b that inhibits antigen processing and presentation by primary macrophages were downregulated, indicative of a strong host response to PPRV infection. miR-21-5p was found to be inhibited on IPA upstream regulatory analysis of RNA-sequencing data. This miRNA that was also highly downregulated and was found to govern 16 immune response genes in the proteome data was selected for functional validation vis-a-vis TGFBR2 (TGF-beta receptor type-2). TGFBR2 that regulates cell differentiation and is involved in several immune response pathways was found to be governed by most of the identified immune modulating DEmiRNAs. The decreased luciferase activity in Dual Luciferase Reporter Assay indicated specific binding of miR-21-5p and miR-484 to their target thus establishing specific binding of the miRNAs to their targets.This is the first report on the miRNAome and proteome of virulent PPRV infected goat PBMCs.

Identification of suitable candidate reference genes is an important prerequisite for validating the gene expression data obtained from downstream analysis of RNA sequencing using quantitative real time PCR (qRT-PCR). Though existence of a universal reference gene is myth, commonly used reference genes can be assessed for expression stability to confer their suitability to be used as candidate reference genes in gene expression studies. In this study, we evaluated the expression stability of ten most commonly used reference genes (GAPDH, ACTB, HSP90, HMBS, 18S rRNA, B2M, POLR2A, HPRT1, ACAC, YWHAZ) in fourteen different Peste des petits ruminants virus (PPRV) infected tissues of goats and sheep. RefFinder and RankAggreg software were used to deduce comprehensive ranking of reference genes. Our results suggested HMBS and B2M in goats and HMBS and HPRT1 in sheep can be used as suitable endogenous controls in gene expression studies of PPRV infection irrespective of tissues and condition as a whole, thus eliminating the use of tissue specific/ condition specific endogenous controls. We report for the first time suitable reference genes for gene expression studies in PPRV infected tissues. The reference genes determined here can be useful for future studies on gene expression in sheep and goat infected with PPRV, thus saving extra efforts and time of repeating the reference gene determination and validation.

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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.