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Elucidation of the open chromatin regions in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus, using ATAC-Seq

ABSTRACT: Bovine Respiratory Syncytial Virus (BRSV) is a cause of Bovine Respiratory Disease (BRD). The objective of this study was to elucidate the chromatin regions which were differentially open in the bronchial lymph nodes (BLN) of dairy calves experimentally challenged with BRSV, relative to unchallenged control calves. Holstein-Friesian calves were either challenged with BRSV inoculum (n=12) or mock challenged with PBS (n=6). Calves were euthanised on day 7 post-challenge. ATAC-Seq libraries were prepared from fresh BLN tissues and sequenced (75 bp paired-end). Sequence reads were aligned to the UMD3.1 reference genome and peak calling (q < 0.01) was performed with MACS2, employing the BAMPE model. Using Diffbind, 9,144 and 5,096 differentially accessible peaks were identified between BRSV challenged and control calves (P < 0.05, FDR < 0.05) using DeSeq2 and EdgeR, respectively. There were 169 and 110 genes previously found to be differentially expressed using RNA-Seq located within or up to 2kb upstream of the differentially accessible peaks identified by the DeSeq 2 and EdgeR analyses, respectively. There were 237 differentially accessible peaks positioned within 40 previously identified susceptibility loci for BRD. These open chromatin regions are likely involved in the gene transcriptional and regulatory response to infection by BRSV.

ORGANISM(S): Bos taurus

PROVIDER: GSE148056 | GEO | 2021/01/13

REPOSITORIES: GEO

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Project description:Bovine Respiratory Disease (BRD) is responsible for the majority of calf morbidity and mortality. One of the main causes of BRD is initial infection with Bovine Respiratory Syncytial Virus (BRSV). The objective of this study was to elucidate the changes in the host's bronchial lymph node miRNA transcriptome in response to BRSV in a controlled viral challenge. Holstein-Friesian calves were either inoculated with BRSV (103.5 TCID50/ml x 15 ml) (n=12) or mock challenged with phosphate buffered saline (n=6). Clinical signs were scored daily until euthanasia at day 7 post-challenge. Total RNA was extracted and sequenced (75 bp) from bronchial lymph nodes. Sequences were filtered for contaminant short RNA species, and subsequently read counts for known miRNAS were generated using the miRDeep2 package with the UMD 3.1 version of the genome and the bovine mature miRNA sequences from the miRBase database (release 22). EdgeR was used for differential expression analysis and Targetscan was used to identify target genes for the differentially expressed (DE) miRNAs. Pathway and gene ontology analyses on target genes were carried out using Ingenuity Pathway analysis (Qiagen). There was a clear separation between BRSV challenged and control calves based on miRNA gene expression changes, despite an observed mild clinical manifestation of the disease. There were 119 DE miRNAs (p<0.05, FDR<0.1, fold change>1.5) between the BRSV challenged and control calves. The DE miRNAs were predicted to target 465 genes which were previously found to be DE in bronchial lymph node tissue, between these BRSV challenged and control calves. Out of the DE predicted target genes, 455 genes had inverse fold changes to that of the DE miRNAs. There were 8 enriched pathways among the DE predicted target genes with inverse fold changes to that of the DE miRNA including: granulocyte and agranulocyte adhesion and diapedesis, interferon signaling and role of pathogen recognition receptors in recognition of bacteria and viruses. Functions predicted to be increased included: T cell response, apoptosis of leukocytes, immune response of cells and stimulation of cells. Pathogen recognition and proliferation of cytotoxic T cells are vital for the recognition and subsequent elimination of the virus.

Project description:Background: Weaning of beef calves is a necessary husbandry practice and involves separating the calf from its mother, resulting in numerous stressful events including dietary change, social reorganisation and the cessation of the maternal-offspring bond and is often accompanied by housing. While much recent research has focused on the physiological response of the bovine immune system to stress in recent years, little is known about the molecular mechanisms modulating the immune response. Therefore, the objective of this study was to provide new insights into the molecular mechanisms underlying the physiological response to weaning at housing in beef calves using Illumina RNA-seq. Results: The leukocyte transcriptome was significantly altered for at least 7 days following either housing or weaning at housing. Analysis of differentially expressed genes revealed that four main pathways, cytokine signalling, transmembrane transport, haemostasis and G-protein-coupled receptor (GPRC) signalling, were differentially regulated between control and weaned calves and underwent significant transcriptomic alterations in response to weaning stress on day 1, 2 and 7. Of particular note, chemokines, cytokines and integrins were consistently found to be up-regulated on each day following weaning. Evidence for alternative splicing of genes was also detected, indicating that a number of genes involved in the innate and adaptive immune response may be alternatively transcribed, including those responsible for toll receptor cascades and T cell receptor signalling. Conclusions: This study represents the first application of RNA-Seq technology for genomic studies in bovine leukocytes in response to weaning stress. Weaning stress induces the activation of a number of cytokine, chemokine and integrin transcripts and may alter the immune system whereby the ability of a number of cells of the innate and adaptive immune system to locate and destroy pathogens is transcriptionally enhanced. Stress alters the homeostasis of the transcriptomic environment of leukocytes for at least 7 days following weaning, indicating long-term effects of stress exposure in the bovine. The identification of gene signature networks that are stress activated provides a mechanistic framework to characterise the multifaceted nature of weaning stress adaptation in beef calves. Thus, capturing subtle transcriptomic changes provides insight into the molecular mechanisms that underlie the physiological response to weaning stress. Examination of a time course (day 0, 1, 2 and 7) for 2 treatments, calves either housed with their dam (control) or housed and simultaneously weaned, using RNA-seq. The supplementary processed data file 'read_counts.txt' contains unnormalized read counts for each Ensembl bovine gene in each of the 48 samples. Unnormalized counts are required for input to EdgeR. Genome build: Btau4.0

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Elucidation of the open chromatin regions in the bronchial lymph nodes of dairy calves experimentally challenged with bovine respiratory syncytial virus, using ATAC-Seq

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