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Transcriptional analysis of Bos taurus and AlHV-1 in MDBK infected cells and in lymph nodes of infected calves

ABSTRACT: 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.

ORGANISM(S): Bos taurus Alcelaphine gammaherpesvirus 1

PROVIDER: GSE40644 | GEO | 2013/03/21

SECONDARY ACCESSION(S): PRJNA174536

REPOSITORIES: GEO

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Transcriptional analysis of Bos taurus and AlHV-1 in MDBK infected cells and in lymph nodes of infected calves

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.

2013-03-21 | E-GEOD-40644 | biostudies-arrayexpress

Small RNA deep sequencing identifies viral microRNAs during malignant catarrhal fever induced by alcelaphine herpesvirus 1

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

2015-08-27 | E-GEOD-71056 | biostudies-arrayexpress

Small RNA deep sequencing identifies viral microRNAs during malignant catarrhal fever induced by alcelaphine herpesvirus 1

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.

2015-08-27 | GSE71056 | GEO

Deep sequencing after alcelaphine gammaherpesvirus 1 infection reveals the nature of CD8+ T cell expansion and identify an essential viral protein for fatal bovine malignant catarrhal fever [RNA-seq]

Project description:Alcelaphine gammaherpesvirus 1 (AlHV-1) is a member of the Gammaherpesvirinae subfamily and establishes asymptomatic latent infection in its natural host species, the wildebeest. Cross-species transmission to various ruminant species including cattle can occur, resulting in the induction of malignant catarrhal fever (MCF), a deadly peripheral T cell lymphoproliferative disease. Here, we experimentally infected calves to confirm that AlHV-1 latency-associated gene expression is essential for persistent infection of CD8+ T cells and MCF development. Then, deep sequencing of the T cell receptor repertoire revealed an oligoclonal expansion of peripheral CD8+ T cells during bovine MCF, associated with transcriptomic and epigenetic changes identified by (sc)RNA-seq and ATAC-seq analyses which indicated a mixed effector/memory and exhaustion phenotype of infected cells in vivo. Analysis of the viral genome transcription identified viral genomic regions being expressed in infected CD8+ T cells, such as the region predicted to encode the gene A10. A10 encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. We could demonstrate that impaired expression of A10 did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF in the rabbit experimental model, and we showed that A10 is phosphorylated in T lymphocytes in vitro and affects T cell signaling. Finally, while AlHV-1 viruses expressing mutated forms of A10 devoid of ITAM and/or SH3 motifs could induce MCF, an A10 knock-in viral mutant unable to phosphorylate tyrosine residues resulted in the absence of MCF development. Overall, we identified AlHV-1-induced phenotypic changes in CD8+ T cells during MCF and demonstrated that A10 expression in infected CD8+ T lymphocytes results in the dysregulation of T cell signaling and MCF.

2024-05-01 | GSE253728 | GEO

Deep sequencing after alcelaphine gammaherpesvirus 1 infection reveals the nature of CD8+ T cell expansion and identify an essential viral protein for fatal bovine malignant catarrhal fever [scRNA-seq]

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