Project description:Wildebeests carry asymptomatically Alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing a lethal lymphoproliferative disease named malignant catarrhal fever (MCF) in a number of susceptible species of the Artiodactyla order, including cattle. The local population welfare in eastern Africa is directly endangered by the important but underestimated impact of this disease on their livelihood. Although AlHV-1 genomic DNA is detected in abundance in tissues during MCF, no infectious viral particles and very low viral protein expression levels are observed. This suggests that AlHV-1 might be latent during MCF. Here, we studied the implication of AlHV-1 latency during MCF. We first examined the expression of poly-adenylated RNA from infected (multiplicity of infection, moi = 0.01) MDBK cells at 72h pi. This late time point was chosen as we expect the majority of viral genes to be expressed. The expression was obtained from two-color dye-swap analyses of 4 independent biological repeats. To determine cellular and viral gene expression during MCF, we extracted RNA from the inguinal LN (iLN) of each calf for analysis on a custom designed array. The arbitrary choice of the iLN as the selected tissue was based on the fact that AlHV-1 viral genomic load are the highest in the LN. Cellular and viral RNA transcription profiles were analyzed with two-color dye-swap analyses of 4 independent biological repeats.
Project description:Wildebeests carry asymptomatically Alcelaphine herpesvirus 1 (AlHV-1), a M-NM-3-herpesvirus inducing a lethal lymphoproliferative disease named malignant catarrhal fever (MCF) in a number of susceptible species of the Artiodactyla order, including cattle. The local population welfare in eastern Africa is directly endangered by the important but underestimated impact of this disease on their livelihood. Although AlHV-1 genomic DNA is detected in abundance in tissues during MCF, no infectious viral particles and very low viral protein expression levels are observed. This suggests that AlHV-1 might be latent during MCF. Here, we studied the implication of AlHV-1 latency during MCF. We first examined the expression of poly-adenylated RNA from infected (multiplicity of infection, moi = 0.01) MDBK cells at 72h pi. This late time point was chosen as we expect the majority of viral genes to be expressed. The expression was obtained from two-color dye-swap analyses of 4 independent biological repeats. To determine cellular and viral gene expression during MCF, we extracted RNA from the inguinal LN (iLN) of each calf for analysis on a custom designed array. The arbitrary choice of the iLN as the selected tissue was based on the fact that AlHV-1 viral genomic load are the highest in the LN. Cellular and viral RNA transcription profiles were analyzed with two-color dye-swap analyses of 4 independent biological repeats. Cellular and viral gene expression were analysed in Mock- and AlHV-1-infected MDBK cells (in vitro) as well as in the inguinal lymphnodes of Mock- and AlHV-1-infected calves (in vivo). Each experiment (in vitro and in vivo) was carried out with 4 biological replicates for each conditon (mock- and AlHV-1-infected). The 4 samples for each experiment were hybridized in a one-to-one dye-swap design without pooling the Mock-infected samples, and yielding 8 arrays per experiment.
Project description:We hypothesized that the gene expression pattern of OvHV-2 may be important to understand the pathogenesis of malignant catarrhal fever (MCF). Therefore, RNA was extracted from lymph nodes of animals with MCF and healthy controls to be analyzed by a custom-made microarray. Two regions on the viral genome were transcriptionally active, one encoding a homologue to the latency-associated nuclear antigen (ORF73) of other gamma herpesviruses, the other with no predicted open reading frame. Keywords: Disease state analysis
Project description:We hypothesized that the gene expression pattern of OvHV-2 may be important to understand the pathogenesis of malignant catarrhal fever (MCF). Therefore, RNA was extracted from lymph nodes of animals with MCF and healthy controls to be analyzed by a custom-made microarray. Two regions on the viral genome were transcriptionally active, one encoding a homologue to the latency-associated nuclear antigen (ORF73) of other gamma herpesviruses, the other with no predicted open reading frame. Keywords: Disease state analysis Two biological replicates with two technical replicates each, with one of the technical replicates being a dye-swap.
Project description:Alcelaphine herpesvirus 1 (AlHV-1) is a ?-herpesvirus (?-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces an acute and fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+ T cells are unknown. Many ?-HVs express microRNAs (miRNAs). These small noncoding RNAs can suppress host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, we have cloned small RNAs and sequenced 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and qRT-PCR in lymphoid organs of MCF-developing calves or rabbits. To determine the concerted contribution in MCF of 28 viral miRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro nor MCF induction in rabbits, demonstrating that AlHV-1 miRNAs clustered in the non-protein-coding genomic region are not essential for MCF induction. Small RNA sequencing from total RNA from AlHV-1-infected bovine lymphoblastoid cell line propagated with interleukin 2
Project description:Changes in gene expression in lymph node and kidney of cattle infected with alcelaphine herpesvirus-1. All animals in the group infected with AlHV-1 showed clinical signs of Malignant catarrhal fever (MCF) within one month. Diagnosis of MCF in these animals was confirmed by diagnostic PCR. No clinical signs were seen in the control animals, which were subject to post-mortem examination separately from the MCF-affected animals. RNA samples from kidney and lymph node of 4 MCF-affected and 4 control animals that passed the RNA quality criteria were used for array analysis. Quality assessments of probe-level Affymetrix chip data and normalised data were carried out using statistical and graphical methods including MA plots and smoothed histogram plots. The MA-plots showed normalisation was effective; while smoothed histograms indicated there was no systematic bias in the data.
Project description:We hypothesized that the relative abundances of host cell transcripts in lymph nodes of animals with malignant catarrhal fever (MCF), compared to healthy controls, may be used to identify pathways that may help to explain the pathogenesis of MCF. Therefore, an abundance of host cell gene expression patterns in lymph nodes of animals with MCF and healthy controls were analyzed by microarray. Indeed, a vast number of genes related to inflammatory processes, lymphocyte activation, cell proliferation and apoptosis were detected at different abundances. However, the IL-2 transcript was eminent among the transcripts, which were, compared to healthy controls, less abundant in animals with MCF. Compared to healthy cattle, bovines with MCF appear to mimic an IL-2 knockout phenotype that has been described in mice. This supports the hypothesis that immunopathogenic events are linked to the pathogenesis of MCF. IL-2-deficiency may play an important role in the process. Keywords: disease state analysis
Project description:Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces an acute and fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+ T cells are unknown. Many γ-HVs express microRNAs (miRNAs). These small noncoding RNAs can suppress host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, we have cloned small RNAs and sequenced 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and qRT-PCR in lymphoid organs of MCF-developing calves or rabbits. To determine the concerted contribution in MCF of 28 viral miRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro nor MCF induction in rabbits, demonstrating that AlHV-1 miRNAs clustered in the non-protein-coding genomic region are not essential for MCF induction.
Project description:We hypothesized that the relative abundances of host cell transcripts in lymph nodes of animals with malignant catarrhal fever (MCF), compared to healthy controls, may be used to identify pathways that may help to explain the pathogenesis of MCF. Therefore, an abundance of host cell gene expression patterns in lymph nodes of animals with MCF and healthy controls were analyzed by microarray. Indeed, a vast number of genes related to inflammatory processes, lymphocyte activation, cell proliferation and apoptosis were detected at different abundances. However, the IL-2 transcript was eminent among the transcripts, which were, compared to healthy controls, less abundant in animals with MCF. Compared to healthy cattle, bovines with MCF appear to mimic an IL-2 knockout phenotype that has been described in mice. This supports the hypothesis that immunopathogenic events are linked to the pathogenesis of MCF. IL-2-deficiency may play an important role in the process. Experiment Overall Design: Comparison of lymph node samples from 3 infected animals with lymph node samples from 6 uninfected animals.
Project description:The Toll-like receptor (TLR) and peptidoglycan recognition protein 1 (PGLYRP1) genes play key roles in the innate immune systems of mammals. While the TLRs recognize a variety of invading pathogens and induce innate immune responses, PGLYRP1 is directly microbicidal. We used custom allele-specific assays to genotype and validate 220 diallelic variants, including 54 nonsynonymous SNPs in 11 bovine innate immune genes (TLR1-TLR10, PGLYRP1) for 37 cattle breeds. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and we were unable to differentiate between the specialized B. t. taurus beef and dairy breeds, despite an average polymorphism density of one locus per 219 bp. Ninety-nine tagSNPs and one tag insertion-deletion polymorphism were sufficient to predict 100% of the variation at all 11 innate immune loci in both subspecies and their hybrids, whereas 58 tagSNPs captured 100% of the variation at 172 loci in B. t. taurus. PolyPhen and SIFT analyses of nonsynonymous SNPs encoding amino acid replacements indicated that the majority of these substitutions were benign, but up to 31% were expected to potentially impact protein function. Several diversity-based tests provided support for strong purifying selection acting on TLR10 in B. t. taurus cattle. These results will broadly impact efforts related to bovine translational genomics.