Abstract
Bovine respiratory disease (BRD) causes substantial morbidity and mortality, affecting cattle of all ages. One of the main causes of BRD is an initial inflammatory response to bovine respiratory syncytial virus (BRSV). MicroRNAs are novel and emerging non-coding small RNAs that regulate many biological processes and are implicated in various inflammatory diseases. The objective of the present study was to elucidate the changes in the bovine bronchial lymph node miRNA transcriptome in response to BRSV following an experimental viral challenge. Holstein-Friesian calves were either administered a challenge dose of BRSV (103.5 TCID50/ml × 15 ml) (n = 12) or were mock inoculated with sterile phosphate buffered saline (n = 6). Daily scoring of clinical signs was performed and calves were euthanized at day 7 post-challenge. Bronchial lymph nodes were collected for subsequent RNA extraction and sequencing (75 bp). Read counts for known miRNAs were generated using the miRDeep2 package using the UMD3.1 reference 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. Target genes were examined for enriched pathways and gene ontologies using Ingenuity Pathway Analysis (Qiagen). Multi-dimensional scaling (MDS) based on miRNA gene expression changes, revealed a clearly defined separation between the BRSV challenged and control calves, although the clinical manifestation of disease was only mild. One hundred and nineteen DE miRNAs (P < 0.05, FDR < 0.1, fold change > 1.5) were detected 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. Of the DE predicted target genes, 455 had fold changes that were inverse to the corresponding DE miRNAs. There were eight enriched pathways among the DE predicted target genes with inverse fold changes to their corresponding DE miRNA including: granulocyte and agranulocyte adhesion and diapedesis, interferon signalling 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 of the virus and its subsequent elimination.
Highlights
The majority of morbidity and mortality reported in calves between 1 and 6 months of age is associated with bovine respiratory disease (BRD) (Taylor et al, 2010; Curtis et al, 2016; Johnston et al, 2016; Murray et al, 2017)
Holstein-Friesian bull calves with low Bovine respiratory syncytial virus (BRSV) maternally derived antibodies and a negative BRSV PCR result were either challenged with 103.5 TCID50/ml × ml inoculum of BRSV strain SVA 274/9220 (Graham et al, 1999) (n = 12; BRSV challenged) or were mock challenged with sterile phosphate buffered saline (PBS) (n = 6; control), by aerosol inhalation, at the Agri-Food Biosciences Institute (AFBI), Stormont, Northern Ireland
There were no significant differences in clinical scores between the BRSV challenged and the control calves at any of the time-points (P > 0.05), analysed with a repeated measures mixed model procedure in SAS v 9.4
Summary
The majority of morbidity and mortality reported in calves between 1 and 6 months of age is associated with bovine respiratory disease (BRD) (Taylor et al, 2010; Curtis et al, 2016; Johnston et al, 2016; Murray et al, 2017). Bovine respiratory syncytial virus (BRSV) is an enveloped, non-segmented, negative-stranded RNA virus of the Orthopneumovirus genus from the family Pneumoviridae, and is one of the leading infectious viral causes of BRD (Valarcher and Taylor, 2007; Rima et al, 2017; Sudaryatma et al, 2018). Morbidity resulting from BRSV infections ranges from 60 to 80%, and mortality has been reported to reach 20% during disease outbreaks (Valarcher and Taylor, 2007). BRSV is capable of interfering with the host’s anti-viral interferon based response and inducing immunomodulation by shifting the adaptive immune response towards a Th2 dominated response, rather than an effective cytotoxic cell mediated response, which enables establishment and maintenance of the virus (Gershwin, 2007; Valarcher and Taylor, 2007)
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