MiRNAs Predicted to Regulate Host Anti-viral Gene Pathways in IPNV-Challenged Atlantic Salmon Fry Are Affected by Viral Load, and Associated With the Major IPN Resistance QTL Genotypes in Late Infection.

Nardos Tesfaye Woldemariam,Bjørn Høyheim,Hilde Sindre,Rune Andreassen,Oleg Agafonov,James E Bron,Diego Robledo,Ross D Houston

doi:10.3389/fimmu.2020.02113

Abstract

Infectious pancreatic necrosis virus (IPNV) infection has been a major problem in salmonid aquaculture. Marker-assisted selection of individuals with resistant genotype at the major IPN quantitative trait locus (IPN-QTL) has significantly reduced mortality in recent years. We have identified host miRNAs that respond to IPNV challenge in salmon fry that were either homozygous resistant (RR) or homozygous susceptible (SS) for the IPN-QTL. Small RNA-sequenced control samples were compared to samples collected at 1, 7, and 20 days post challenge (dpc). This revealed 72 differentially expressed miRNAs (DE miRNAs). Viral load (VL) was lower in RR vs. SS individuals at 7 and 20 dpc. However, analysis of miRNA expression changes revealed no differences between RR vs. SS individuals in controls, at 1 or 7 dpc, while 38 “high viral load responding” miRNAs (HVL-DE miRNAs) were identified at 20 dpc. Most of the HVL-DE miRNAs showed changes that were more pronounced in the high VL SS group than in the low VL RR group when compared to the controls. The absence of differences between QTL groups in controls, 1 and 7 dpc indicates that the QTL genotype does not affect miRNA expression in healthy fish or their first response to viral infections. The miRNA differences at 20 dpc were associated with the QTL genotype and could, possibly, contribute to differences in resistance/susceptibility at the later stage of infection. In silico target gene predictions revealed that 180 immune genes were putative targets, and enrichment analysis indicated that the miRNAs may regulate several major immune system pathways. Among the targets of HVL-DE miRNAs were IRF3, STAT4, NFKB2, MYD88, and IKKA. Interestingly, TNF-alpha paralogs were targeted by different DE miRNAs. Most DE miRNAs were from conserved miRNA families that respond to viral infections in teleost (e.g., miR-21, miR-146, miR-181, miR-192, miR-221, miR-462, miR-731, and miR-8159), while eight were species specific. The miRNAs showed dynamic temporal changes implying they would affect their target genes differently throughout disease progression. This shows that miRNAs are sensitive to VL and disease progression, and may act as fine-tuners of both immediate immune response activation and the later inflammatory processes.

Highlights

MicroRNAs are small (20–24 nts) non-coding RNAs that regulate gene expression at the post-transcriptional level by binding to mRNA transcripts in a sequence specific manner [1, 2]
There were no differences in miRNA expression between these six controls (Cq > 37) and the other negative controls (Cq > 40) indicating that these controls had not been exposed to Infectious pancreatic necrosis virus (IPNV) at the initial stage of the challenge
All 11 samples were used as controls in the miRNA expression analyses (see Differentially expressed miRNAs (DE-miRNAs) show dynamic expression patterns post challenge and miRNAs associated with infectious pancreatic necrosis (IPN)-QTL genotype and prolonged high viral loads), five of which were homozygous resistant (RR) and six that were homozygous susceptible (SS) for the IPN-QTL

Summary

Introduction

MicroRNAs (miRNAs) are small (20–24 nts) non-coding RNAs that regulate gene expression at the post-transcriptional level by binding to mRNA transcripts (target genes) in a sequence specific manner [1, 2]. Our previous study on miRNAs and host-virus interactions in Atlantic salmon revealed several evolutionary conserved miRNA families that responded to salmonid alphavirus (SAV) infection [12]. Among these conserved miRNAs were miR-21, miR-146, miR-181, and the clustered miRNAs miR-462 and miR-731. Andreassen and Høyheim [13] proposed a model which suggests a role for miRNAs as fine-tuning modulators of immune responses during viral infections in teleost. When a viral infection occurs, miRNAs can take on a dual role that help to fine-tune the immune response in order to benefit the host. Upregulated expression of miRNAs that target activators of inflammation in the later stages of infection could, on the other hand, balance the magnitude of the response, and help prevent escalation of inflammatory processes to a level that could be harmful to the host [13]

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