Peste des petits ruminants (PPR), is an acute transboundary viral disease of economic importance, affecting goats and sheep. Mass vaccination programs around the world resulted in the decline of PPR outbreaks. Sungri 96 is a live attenuated vaccine, widely used in Northern India against PPR. This vaccine virus, isolated from goat works efficiently both in sheep and goat. Global gene expression changes under PPR vaccine virus infection are not yet well defined. Therefore, in this study we investigated the host-vaccine virus interactions by infecting the peripheral blood mononuclear cells isolated from goat with PPRV (Sungri 96 vaccine virus), to quantify the global changes in the transcriptomic signature by RNA-sequencing. Viral genome of Sungri 96 vaccine virus was assembled from the PPRV infected transcriptome confirming the infection and demonstrating the feasibility of building a complete non-host genome from the blood transcriptome. Comparison of infected transcriptome with control transcriptome revealed 985 differentially expressed genes. Functional analysis showed enrichment of immune regulatory pathways under PPRV infection. Key genes involved in immune system regulation, spliceosomal and apoptotic pathways were identified to be dysregulated. Network analysis revealed that the protein - protein interaction network among differentially expressed genes is significantly disrupted in infected state. Several genes encoding TFs that govern immune regulatory pathways were identified to co-regulate the differentially expressed genes. These data provide insights into the host - PPRV vaccine virus interactome for the first time. Our findings suggested dysregulation of immune regulatory pathways and genes encoding Transcription Factors (TFs) that govern these pathways in response to viral infection.

Peste des petits ruminants (PPR) is one of the highly contagious viral disease, characterized by fever, sore mouth, conjunctivitis, gastroenteritis and pneumonia, primarily affecting sheep and goats. Reports suggested variable host response in goats and sheep and this host response vis – a – vis the expression of miRNAs has not been investigated. Here, miRNAs were sequenced and proteomics data were generated to identify the role of differentially expressed miRNA in PPRV infected lung and spleen tissues of sheep and goats. In lungs, 67 and 37 differentially expressed miRNAs (DEmiRNAs) have been identified in goats and sheep, respectively. Similarly, in spleen, 50 and 56 DEmiRNAs were identified in goats and sheep, respectively. A total of 20 and 11 miRNAs were found to be common differentially expressed in both the species in PPRV infected spleen and lung, respectively. Six DEmiRNAs - miR-21-3p, miR-1246, miR-27a-5p, miR-760-3p, miR-320a and miR-363 were selected based on their role in viral infections, apoptosis and fold change. The target prediction analysis of these six selected DEmiRNAs from the proteome data generated, revealed involvement of more number of genes in lung and spleen of goats than in sheep. On gene ontology analysis of host target genes these DEmiRNAs were found to regulate several immune response signaling pathways. It was observed that the pathways viz. T cell receptor signaling, Rap1 signaling, Toll-like receptor signaling and B cell receptor signaling governed by DEmiRNAs were more perturbed in goats than in sheep. The data suggests that PPRV-induced miR-21-3p, miR-320a and miR-363 might act cooperatively to enhance viral pathogenesis in the lung and spleen of sheep by downregulating several immune response genes. The study gives an important insight into the molecular pathogenesis of PPR by identifying that the PPRV - Izatnagar/94 isolate elicits a strong host response in goats than in sheep.

Pasteurella multocida causes acute septicemic and respiratory diseases, including haemorrhagic septicaemia, in cattle and buffalo with case fatality of 100%. In the present study, mice were infected with P. multocida (1.6 × 10³ cfu, intraperitoneal) to evaluate host gene expression profile at early and late stages of infection using high throughput microarray transcriptome analyses. Several differentially expressed genes (DEGs) at both the time points were identified in P.multocida infected spleen, liver and lungs. Functional annotation of these DEGs showed enrichment of key pathways such as TLR, NF-κB, MAPK, TNF, JAK-STAT and NOD like receptor signaling pathways. Several DEGs overlapped across different KEGG pathways indicating a crosstalk between them. The predicted protein—protein interaction among these DEGs suggested, that the recognition of P. multocida LPS or outer membrane components by TLR4 and CD14, results in intracellular signaling via MyD88, IRAKs and/or TRAF6 leading to activation of NFκB and MAPK pathways and associated cytokines.

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.

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 ten significantly enriched immune response processes were found to be governed by 98 genes. The top ten 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 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.