No abstract

In this study, transcriptome analysis of PPRV infected PBMC subsets—T helper cells, T cytotoxic cells, monocytes, and B lymphocytes was done to delineate their role in host response. PPRV was found to infect lymphocytes and not monocytes. The established receptor for PPRV—SLAM was found downregulated in lymphocytes and non-differentially expressed in monocytes. A profound deviation in the global gene expression profile with a large number of unique upregulated genes (851) and downregulated genes (605) was observed in monocytes in comparison to lymphocytes. ISGs—ISG15, Mx1, Mx2, RSAD2, IFIT3, and IFIT5 that play a role in antiviral response and the genes for viral sensors—MDA5, LGP2, and RIG1, were found to be upregulated in lymphocytes and downregulated in monocytes. The transcription factors—IRF-7 and STAT-1 that regulate expression of most of the ISGs were found activated in lymphocytes and not in monocytes. Interferon signaling pathway and RIG1 like receptor signaling pathway were found activated in lymphocytes and not in monocytes. This contrast in gene expression profiles and signaling pathways indicated the predominant role of lymphocytes in generating the antiviral response against PPRV in goats, thus, giving us new insights into host response to PPRV.

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.

Cancer remains one of the leading causes of death worldwide in humans and animals. Conventional treatment regimens often fail to produce the desired outcome due to disturbances in cell physiology that arise during the process of transformation. Additionally, development of treatment regimens with no or minimum side-effects is one of the thrust areas of modern cancer research. Oncolytic viral gene therapy employs certain viral genes which on ectopic expression find and selectively destroy malignant cells, thereby achieving tumor cell death without harming the normal cells in the neighborhood. Apoptin, encoded by Chicken Infectious Anemia Virus’ VP3 gene, is a proline-rich protein capable of inducing apoptosis in cancer cells in a selective manner. In normal cells, the filamentous Apoptin becomes aggregated toward the cell margins, but is eventually degraded by proteasomes without harming the cells. In malignant cells, after activation by phosphorylation by a cancer cell-specific kinase whose identity is disputed, Apoptin accumulates in the nucleus, undergoes aggregation to form multimers, and prevents the dividing cancer cells from repairing their DNA lesions, thereby forcing them to undergo apoptosis. In this review, we discuss the present knowledge about the structure of Apoptin protein, elaborate on its mechanism of action, and summarize various strategies that have been used to deliver it as an anticancer drug in various cancer models.