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.

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.

Dietary Immunostimulant CpG Modulates MicroRNA Biomarkers Associated with Immune Responses in Atlantic Salmon (Salmo salar).

MicroRNAs (miRNAs) are key regulators in fish immune responses. However, no study has previously characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida (ASAL) on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. To this end, first, we performed small RNA deep sequencing and qPCR analyses to identify and confirm pIC- and/or ASAL-responsive miRNAs in the head kidney of salmon fed a control diet. DESeq2 analyses identified 12 and 18 miRNAs differentially expressed in pIC and ASAL groups, respectively, compared to the controls. Fifteen of these miRNAs were studied by qPCR; nine remained significant by qPCR. Five miRNAs (miR-27d-1-2-5p, miR-29b-2-5p, miR-146a-5p, miR-146a-1-2-3p, miR-221-5p) were shown by qPCR to be significantly induced by both pIC and ASAL. Second, the effect of CpG-containing functional feed on miRNA expression was investigated by qPCR. In pre-injection samples, 6 of 15 miRNAs (e.g., miR-181a-5-3p, miR-462a-3p, miR-722-3p) had significantly lower expression in fish fed CpG diet than control diet. In contrast, several miRNAs (e.g., miR-146a-1-2-3p, miR-192a-5p, miR-194a-5p) in the PBS- and ASAL-injected groups had significantly higher expression in CpG-fed fish. Multivariate statistical analyses confirmed that the CpG diet had a greater impact on miRNA expression in ASAL-injected compared with pIC-injected fish. This study identified immune-relevant miRNA biomarkers that will be valuable in the development of diets to combat infectious diseases of salmon.

Expanding the miRNA Repertoire in Atlantic Salmon; Discovery of IsomiRs and miRNAs Highly Expressed in Different Tissues and Developmental Stages.

MicroRNAs (miRNAs) are important post-transcriptional gene expression regulators. Here, 448 different miRNA genes, including 17 novel miRNAs, encoding for 589 mature Atlantic salmon miRNAs were identified after sequencing 111 samples (fry, pathogen challenged fry, various developmental and adult tissues). This increased the reference miRNAome with almost one hundred genes. Prior to isomiR characterization (mature miRNA variants), the proportion of erroneous sequence variants (ESVs) arising in the analysis pipeline was assessed. The ESVs were biased towards 5' and 3' end of reads in unexpectedly high proportions indicating that measurements of ESVs rather than Phred score should be used to avoid misinterpreting ESVs as isomiRs. Forty-three isomiRs were subsequently discovered. The biological effect of the isomiRs measured as increases in target diversity was small (<3%). Five miRNA genes showed allelic variation that had a large impact on target gene diversity if present in the seed. Twenty-one miRNAs were ubiquitously expressed while 31 miRNAs showed predominant expression in one or few tissues, indicating housekeeping or tissue specific functions, respectively. The miR-10 family, known to target Hox genes, were highly expressed in the developmental stages. The proportion of miR-430 family members, participating in maternal RNA clearance, was high at the earliest developmental stage.

Identification of differentially expressed Atlantic salmon miRNAs responding to salmonid alphavirus (SAV) infection.

BACKGROUND: MicroRNAs (miRNAs) control multiple biological processes including the innate immune responses by negative post-transcriptional regulation of gene expression. As there were no studies on the role(s) of miRNAs in viral diseases in Atlantic salmon, we aimed to identify miRNAs responding to salmonid alphavirus (SAV) infection. Their expression were studied at different time points post infection with SAV isolates associated with different mortalities. Furthermore, the genome sequences of the identified miRNAs were analysed to reveal putative cis-regulatory elements, and, finally, their putative target genes were predicted. RESULTS: Twenty differentially expressed miRNAs (DE miRNAs) were identified. The expression of the majority of these increased post infection with maximum levels reached after the viral load were stabilized or decreasing. On the other hand, some miRNAs (e.g. the miRNA-21 family) showed decreased expression at the early time points post infection. There were significant differences in the temporal expression of individual miRNA associated with different SAV isolates. Target gene prediction in SAV responsive immune network genes showed that seventeen of the DE miRNAs could target 24 genes (e.g. IRF3, IRF7). Applying the Atlantic salmon transcriptome as input 28 more immune network genes were revealed as putative targets (e.g. IRF5, IRF4). The majority of the predicted target genes promote inflammatory response. The upstream sequences of the miRNA genes revealed a high density of cis-regulatory sequences known as binding sites for immune network transcription factors (TFs). A high expression in the late phase could therefore be due to increased transcription promoted by immune response activated TFs. Based on the in silico target predictions, we discuss their putative roles as early promotors or late inhibitors of inflammation. We propose that the differences in expressions associated with different SAV isolates could contribute to their differences in mortality rates. CONCLUSIONS: This study represents the first steps in exploring miRNAs important in viral-host interaction in Atlantic salmon. We identified several miRNAs responding to SAV infection. Some likely to prohibit harmful inflammation while other may promote an early immune response. Their predicted functions need to be validated and further studied in functional assays to fully understand their roles in immune homeostasis.