VARV B22R homologue as phylogenetic marker gene for Capripoxvirus classification and divergence time dating.

Sheeppox disease is associated with significant losses in sheep production world over. The sheep pox virus, the goatpox virus, and the lumpy skin disease virus cannot be distinguished by conventional serological tests. Identification of these pathogens needs molecular methods. In this study, seven genes viz. EEV maturation protein-F12L, Virion protein-D3R, RNA polymerase subunit-A5R, Virion core protein-A10L, EEV glycoprotein-A33R, VARV B22R homologue, and Kelch like protein-A55R that cover the start, middle, and end of the genome were selected. These genes were amplified from Roumanian-Fanar vaccine strain and Jaipur virulent strain, cloned, and sequenced. On analysis with the available database sequences, VARV B22R homologue was identified as a marker for phylogenetic reconstruction for classifying the sheeppox viruses of the ungulates. Further, divergence time dating with VARV B22R gene accurately predicted the sheeppox disease outbreak involving Jaipur virulent strain.

Expression kinetics of ISG15, IRF3, IFNγ, IL10, IL2 and IL4 genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in PPRV vaccinated, challenged, infected sheep and goats.

Here, we studied the in vivo expression of Th1 (IL2 and IFN gamma) and Th2 (IL4 and IL10) - cytokines and antiviral molecules - IRF3 and ISG15 in peripheral blood mononuclear cells in relation to antigen and antibody dynamics under Peste des petits ruminants virus (PPRV) vaccination, infection and challenge in both sheep and goats. Vaccinated goats were seropositive by 9 days post vaccination (dpv) while in sheep idiosyncratic response was observed between 9 and 14 dpv for different animals. Expression of PPRV N gene was not detected in PBMCs of vaccinated and vaccinated challenged groups of both species, but was detected in unvaccinated infected PBMCs at 9 and 14 days post infection. The higher viral load at 9 dpi coincided with the peak clinical signs of the disease. The peak in viral replication at 9 dpi correlated with significant expression of antiviral molecules IRF3, ISG15 and IFN gamma in both the species. With the progression of disease, the decrease in N gene expression also correlated with the decrease in expression of IRF3, ISG15 and IFN gamma. In the unvaccinated infected animals ISG15, IRF3, IFN gamma and IL10 expression was higher than vaccinated animals. The IFN gamma expression predominated over IL4 in both vaccinated and infected animals with the infected exhibiting a stronger Th1 response. The persistent upregulation of this antiviral molecular signature - ISG15 and IRF3 even after 2 weeks post vaccination, presumably reflects the ongoing stimulation of innate immune cells.

Genome sequencing of an Indian peste des petits ruminants virus isolate, Izatnagar/94, and its implications for virus diversity, divergence and phylogeography.

Peste des petits ruminants is an important transboundary disease infecting small ruminants. Genome or gene sequence analysis enriches our knowledge about the evolution and transboundary nature of the causative agent of this disease, peste des petits ruminants virus (PPRV). Although analysis using whole genome sequences of pathogens leads to more precise phylogenetic relationships, when compared to individual genes or partial sequences, there is still a need to identify specific genes/genomic regions that can provide evolutionary assessments consistent with those predicted with full-length genome sequences. Here the virulent Izatnagar/94 PPRV isolate was assembled and compared to all available complete genome sequences (currently in the NCBI database) to estimate nucleotide diversity and to deduce evolutionary relationships between genes/genomic regions and the full length genomes. Our aim was to identify the preferred candidate gene for use as a phylogenetic marker, as well as to predict divergence time and explore PPRV phylogeography. Among all the PPRV genes, the H gene was identified to be the most diverse with the highest evolutionary relationship with the full genome sequences. Hence it is considered as the most preferred candidate gene for phylogenetic study with 93% identity set as a nucleotide cutoff. A whole genome nucleotide sequence cutoff value of 94% permitted specific differentiation of PPRV lineages. All the isolates examined in the study were found to have a most recent common ancestor in the late 19th or in the early 20th century with high posterior probability values. The Bayesian skyline plot revealed a decrease in genetic diversity among lineage IV isolates since the start of the vaccination program and the network analysis localized the ancestry of PPRV to Africa.

Differential expression of long non-coding RNAs under Peste des petits ruminants virus (PPRV) infection in goats.

Peste des petits ruminants (PPR) characterized by fever, sore mouth, conjunctivitis, gastroenteritis, and pneumonia, is an acute, highly contagious viral disease of sheep and goats. The role of long non-coding RNAs (lncRNAs) in PPRV infection has not been explored to date. In this study, the transcriptome profiles of virulent Peste des petits ruminants virus (PPRV) infected goat tissues - lung and spleen were analyzed to identify the role of lncRNAs in PPRV infection. A total of 13,928 lncRNA transcripts were identified, out of which 170 were known lncRNAs. Intergenic lncRNAs (7625) formed the major chunk of the novel lncRNA transcripts. Differential expression analysis revealed that 15 lncRNAs (11 downregulated and 4 upregulated) in the PPRV infected spleen samples and 16 lncRNAs (13 downregulated and 3 upregulated) in PPRV infected lung samples were differentially expressed as compared to control. The differentially expressed lncRNAs (DElncRNAs) possibly regulate various immunological processes related to natural killer cell activation, antigen processing and presentation, and B cell activity, by regulating the expression of mRNAs through the cis- or trans-regulatory mechanism. Functional enrichment analysis of differentially expressed mRNAs (DEmRNAs) revealed enrichment of immune pathways and biological processes in concordance with the pathways in which correlated lncRNA-neighboring genes were enriched. The results suggest that a coordinated immune response is raised in both lung and spleen tissues of the goat through mRNA-lncRNA crosstalk.

Proteome Modulation in Peripheral Blood Mononuclear Cells of Peste des Petits Ruminants Vaccinated Goats and Sheep.

In the present study, healthy goats and sheep (n = 5) that were confirmed negative for peste des petits ruminants virus (PPRV) antibodies by monoclonal antibody-based competitive ELISA and by serum neutralization test and for PPRV antigen by s-ELISA were vaccinated with Sungri/96. A quantitative study was carried out to compare the proteome of peripheral blood mononuclear cells (PBMCs) of vaccinated goat and sheep [5 days post-vaccination (dpv) and 14 dpv] vs. unvaccinated (0 day) to divulge the alteration in protein expression following vaccination. A total of 232 and 915 proteins were differentially expressed at 5 and 14 dpv, respectively, in goats. Similarly, 167 and 207 proteins were differentially expressed at 5 and 14 dpv, respectively, in sheep. Network generated by Ingenuity Pathway Analysis was "infectious diseases, antimicrobial response, and inflammatory response," which includes the highest number of focus molecules. The bio functions, cell-mediated immune response, and humoral immune response were highly enriched in goats at 5 dpv and at 14 dpv. At the molecular level, the immune response produced by the PPRV vaccine virus in goats is effectively coordinated and stronger than that in sheep, though the vaccine provides protection from virulent virus challenge in both. The altered expression of certain PBMC proteins especially ISG15 and IRF7 induces marked changes in cellular signaling pathways to coordinate host immune responses.

Systems biology under heat stress in Indian cattle.

Transcriptome profiling of Vrindavani and Tharparkar cattle (n = 5 each) revealed that more numbers of genes were dysregulated in Vrindavani than in Tharparkar. A contrast in gene expression was observed with 18.9 % of upregulated genes in Vrindavani downregulated in Tharparkar and 17.8% upregulated genes in Tharparkar downregulated in Vrindavani. Functional annotation of genes differentially expressed in Tharparkar and Vrindavani revealed that the systems biology in Tharparkar is moving towards counteracting the effects due to heat stress. Unlike Vrindavani, Tharparkar is not only endowed with higher expression of the scavengers (UBE2G1, UBE2S, and UBE2H) of misfolded proteins but also with protectors (VCP, Serp1, and CALR) of naïve unfolded proteins. Further, higher expression of the antioxidants in Tharparkar enables it to cope up with higher levels of free radicals generated as a result of heat stress. In this study, we found relevant genes dysregulated in Tharparkar in the direction that can counter heat stress.

Early transcriptome profile of goat peripheral blood mononuclear cells (PBMCs) infected with peste des petits ruminant's vaccine virus (Sungri/96) revealed induction of antiviral response in an interferon independent manner.

Sungri/96 vaccine strain is considered the most potent vaccine providing long-term immunity against peste des petits ruminants (PPR) in India. Previous studies in our laboratory highlighted induction of robust antiviral response in an interferon independent manner at 48 h and 120 h post infection (p.i.). However, immune response at the earliest time point 6 h p.i. (time taken to complete one PPRV life cycle), in PBMCs infected with Sungri/96 vaccine virus has not been investigated. This study was taken up to understand the global gene expression profiling of goat PBMCs after Sungri/96 PPRV vaccine strain infection at 6 h post infection (p.i.). A total of 1926 differentially expressed genes (DEGs) were identified with 616 - upregulated and 1310 - downregulated. TLR7/TLR3, IRF7/IRF1, ISG20, IFIT1/IFIT2, IFITM3, IL27 and TREX1 were identified as key immune sensors and antiviral candidate genes. Interestingly, type I interferons (IFNα/ß) were not differentially expressed at this time point as well. TREX1, an exonuclease which inhibits type I interferons at the early stage of virus infection was found to be highly upregulated. IL27, an important antiviral host immune factor was significantly upregulated. ISG20, an antiviral interferon induced gene with exonuclease activity specific to ssRNA viruses was highly expressed. Functional profiling of DEGs showed significant enrichment of immune system processes with 233 genes indicating initiation of immune defense response in host cells. Protein interaction network showed important innate immune molecules in the immune network with high connectivity. The study highlights important immune and antiviral genes at the earliest time point.

Contrasting Gene Expression Profiles of Monocytes and Lymphocytes From Peste-Des-Petits-Ruminants Virus Infected Goats.

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.

Dysregulated miRNAome and Proteome of PPRV Infected Goat PBMCs Reveal a Coordinated Immune Response.

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.

Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection.

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.

Transcriptome analysis reveals common differential and global gene expression profiles in bluetongue virus serotype 16 (BTV-16) infected peripheral blood mononuclear cells (PBMCs) in sheep and goats.

Bluetongue is an economically important infectious, arthropod borne viral disease of domestic and wild ruminants, caused by Bluetongue virus (BTV). Sheep are considered the most susceptible hosts, while cattle, buffalo and goats serve as reservoirs. The viral pathogenesis of BTV resulting in presence or absence of clinical disease among different hosts is not clearly understood. In the present study, transcriptome of sheep and goats peripheral blood mononuclear cells infected with BTV-16 was explored. The differentially expressed genes (DEGs) identified were found to be significantly enriched for immune system processes - NFκB signaling, MAPK signaling, Ras signaling, NOD signaling, RIG signaling, TNF signaling, TLR signaling, JAK-STAT signaling and VEGF signaling pathways. Greater numbers of DEGs were found to be involved in immune system processes in goats than in sheep. Interestingly, the DEHC (differentially expressed highly connected) gene network was found to be dense in goats than in sheep. Majority of the DEHC genes in the network were upregulated in goats but down-regulated in sheep. The network of differentially expressed immune genes with the other genes further confirmed these findings. Interferon stimulated genes - IFIT1 (ISG56), IFIT2 (ISG54) and IFIT3 (ISG60) responsible for antiviral state in the host were found to be upregulated in both the species. STAT2 was the TF commonly identified to co-regulate the DEGs, with its network showing genes that are downregulated in sheep but upregulated in goats. The genes dysregulated and the networks perturbed in the present study indicate host variability with a positive shift in immune response to BTV in goats than in sheep.

Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells.

Peste des petits ruminanats virus (PPRV), a morbillivirus causes an acute, highly contagious disease - peste des petits ruminants (PPR), affecting goats and sheep. Sungri/96 vaccine strain is widely used for mass vaccination programs in India against PPR and is considered the most potent vaccine providing long-term immunity. However, occurrence of outbreaks due to emerging PPR viruses may be a challenge. In this study, the temporal dynamics of immune response in goat peripheral blood mononuclear cells (PBMCs) infected with Sungri/96 vaccine virus was investigated by transcriptome analysis. Infected goat PBMCs at 48h and 120h post infection revealed 2540 and 2000 differentially expressed genes (DEGs), respectively, on comparison with respective controls. Comparison of the infected samples revealed 1416 DEGs to be altered across time points. Functional analysis of DEGs reflected enrichment of TLR signaling pathways, innate immune response, inflammatory response, positive regulation of signal transduction and cytokine production. The upregulation of innate immune genes during early phase (between 2-5 days) viz. interferon regulatory factors (IRFs), tripartite motifs (TRIM) and several interferon stimulated genes (ISGs) in infected PBMCs and interactome analysis indicated induction of broad-spectrum anti-viral state. Several Transcription factors - IRF3, FOXO3 and SP1 that govern immune regulatory pathways were identified to co-regulate the DEGs. The results from this study, highlighted the involvement of both innate and adaptive immune systems with the enrichment of complement cascade observed at 120h p.i., suggestive of a link between innate and adaptive immune response. Based on the transcriptome analysis and qRT-PCR validation, an in vitro mechanism for the induction of ISGs by IRFs in an interferon independent manner to trigger a robust immune response was predicted in PPRV infection.

Global gene expression profile of peripheral blood mononuclear cells challenged with Theileria annulata in crossbred and indigenous cattle.

Bovine tropical theileriosis is an important haemoprotozoan disease associated with high rates of morbidity and mortality particularly in exotic and crossbred cattle. It is one of the major constraints of the livestock development programmes in India and Southeast Asia. Indigenous cattle (Bos indicus) are reported to be comparatively less affected than exotic and crossbred cattle. However, genetic basis of resistance to tropical theileriosis in indigenous cattle is not well documented. Recent studies incited an idea that differentially expressed genes in exotic and indigenous cattle play significant role in breed specific resistance to tropical theileriosis. The present study was designed to determine the global gene expression profile in peripheral blood mononuclear cells derived from indigenous (Tharparkar) and cross-bred cattle following in vitro infection of T. annulata (Parbhani strain). Two separate microarray experiments were carried out each for cross-bred and Tharparkar cattle. The cross-bred cattle showed 1082 differentially expressed genes (DEGs). Out of total DEGs, 597 genes were down-regulated and 485 were up-regulated. Their fold change varied from 2283.93 to -4816.02. Tharparkar cattle showed 875 differentially expressed genes including 451 down-regulated and 424 up-regulated. The fold change varied from 94.93 to -19.20. A subset of genes was validated by qRT-PCR and results were correlated well with microarray data indicating that microarray results provided an accurate report of transcript level. Functional annotation study of DEGs confirmed their involvement in various pathways including response to oxidative stress, immune system regulation, cell proliferation, cytoskeletal changes, kinases activity and apoptosis. Gene network analysis of these DEGs plays an important role to understand the interaction among genes. It is therefore, hypothesized that the different susceptibility to tropical theileriosis exhibited by indigenous and crossbred cattle is due to breed-specific differences in the dealing of infected cells with other immune cells, which ultimately influence the immune response responded against T. annulata infection.

Systems biology approach: Panacea for unravelling host-virus interactions and dynamics of vaccine induced immune response.

Systems biology is an interdisciplinary research field in life sciences, which involves a comprehensive and quantitative analysis of the interactions between all of the components of biological systems over time. For the past 50 years the discipline of virology has overly focused on the pathogen itself. However, we now know that the host response is equally or more important in defining the eventual pathological outcome of infection. Systems biology has in recent years been increasingly recognised for its importance to infectious disease research. Host-virus interactions can be better understood by taking into account the dynamical molecular networks that constitute a biological system. To decipher the pathobiological mechanisms of any disease requires a deep knowledge of how multiple and concurrent signal-transduction pathways operate and are deregulated. Hence the intricacies of signalling pathways can be dissected only by system level approaches.