Comparison of Atlantic salmon individuals with different outcomes of cardiomyopathy syndrome (CMS)

Abstract

Background: Cardiomyopathy syndrome (CMS) is a severe disease of Atlantic salmon (Salmo salar L.) associated with significant economic losses in the aquaculture industry. CMS is diagnosed with a severe inflammation and degradation of myocardial tissue caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV), with structural similarities to the Totiviridae family. In the present study we characterized individual host responses and genomic determinants of different disease outcomes.

Results: From time course studies of experimentally infected Atlantic salmon post-smolts, fish exhibited different outcomes of infection and disease. High responder (HR) fish were characterized with sustained and increased viral load and pathology in heart tissue. Low responder (LR) fish showed declining viral load from 6-10 weeks post infection (wpi) and absence of pathology. Global gene expression (SIQ2.0 oligonucleotide microarray) in HR and LR hearts during infection was compared, in order to characterize differences in the host response and to identify genes with expression patterns that could explain or predict the different outcomes of disease. Virus-responsive genes involved in early antiviral and innate immune responses were upregulated equally in LR and HR at the first stage (2-4 wpi), reflecting the initial increase in virus replication. Repression of heart muscle development was identified by gene ontology enrichment analyses, indicating the early onset of pathology. By six weeks both responder groups had comparable viral load, while increased pathology was observed in HR fish. This was reflected by induced expression of genes implicated in apoptosis and cell death mechanisms, presumably related to lymphocyte regulation and survival. In contrast, LR fish showed earlier activation of NK cell-mediated cytotoxicity and NOD-like receptor signaling pathways. At the late stage of infection, increased pathology and viral load in HR was accompanied by a broad activation of genes involved in adaptive immunity and particularly T cell responses, probably reflecting the increased infiltration and homing of virus-specific T cells to the infected heart. This was in sharp contrast to LR fish, where recovery and reduced viral load was associated with a significantly reduced transcription of adaptive immunity genes and activation of genes involved in energy metabolism.

Conclusions: In contrast to LR, a stronger and sustained expression of genes involved in adaptive immune responses in heart tissue of HR at the late stage of disease probably reflected the increased lymphocyte infiltration and pathological outcome. In addition to controlled adaptive immunity and activation of genes involved in cardiac energy metabolism in LR at the late stage, recovery of this group could also be related to an earlier activation of NOD-like receptor signaling and NK cell-mediated cytotoxicity pathways.

Figure 1

Virus load and pathology of disease responders. PMCV RNA level and pathology score in heart tissue of challenged fish over the time course of infection. The three stages of disease (early = 2–4 wpi, mid = 6 wpi and late = 8–10 wpi) are indicated on top of the figure, as explained in Results. Relative quantification of PMCV by qPCR is given as fold change of viral transcripts relative to the median levels of 0 wpi samples set to 1. Individual values are shown as dots and colored according to the level of CMS pathology (histopathology score level based on atrium; green = score 0, yellow = 1, orange = 2, red = 3). Significant differences in viral load between high responder fish (HR, orange and red dots) and low responder fish (LR, green and yellow dots) from mid and late stages are marked with asterisks (t-test on log-transformed values, p < 0.01). Overlapping dots were plotted beside each other.

Figure 2

Enrichment analysis of gene ontology classes and pathways. Gene enrichment analysis of regulated gene ontology (GO) classes and KEGG pathways in all fish at the early disease stage, and in high (HR) and low responder (LR) groups at the mid and late stage. Categories highlighted in red indicate upregulated genes and blue indicate downregulated genes. Categories that were only regulated in one group (HR or LR) in mid and late stages are indicated with bold text. The different bioinformatics sources used for analyses are indicated (column “source”); ST:GO/KEGG: STARS platform, B2GO: BLAST2GO/Cytoscape. Column “#” shows the number of regulated genes behind the respective GO term (see Additional file 2 for primary data).

Figure 3

Gene markers of early antiviral response. Heat map of 20 selected genes with expression profile showing upregulation at the early stage and gradually decreased expression during mid and late stages of infection (complete data in Additional file 3). Left columns show the median log₂ expression ratios of genes in the different stages and responder groups (“E” = early, “M:HR” = mid stage HR, “M:LR” = mid stage LR, “L:HR” = late stage HR, “L:LR” = late stage LR). Graded levels from white to red and blue indicate respectively upregulation and downregulation, according to the color scale (row “L”). Significant differences in log₂–ER between control and infected samples (t-test, p < 0.05) are shown in bold. Column “cat” indicates the category of gene annotation according to [10]: V- early antiviral and IFN response, P- MHC antigen presentation.

Figure 4

Early pathology and outcome predictor genes. Heat map of all genes with expression profile showing upregulation in HR (A) and LR (B) fish at the mid stage of infection. Further explanations are given in Figure 3. Column “cat” indicates the category of gene annotation according to [10]: V- early antiviral and IFN response, T- T cell response, M- muscle cytoskeleton development, L- lymphocyte regulation, A- apoptosis, I- implicated in immune response, E- mitochondrial electron chain/energy metabolism, O- other/unknown. Genes may have identical name and Refseq ID match, but represent different cDNA sequences on the array (see Additional file 3.)

Figure 5

Gene markers of pathology and outcome. Heat map of selected genes with expression profile showing strongest upregulation in HR (A) or LR (B) fish at the late stage of infection. Further explanations are given in Figure 3. Column “cat” indicates the category of gene annotation according to [10]: T- T cell response, P- MHC antigen presentation, B- B cell response, A- apoptosis, M- muscle cytoskeleton development, E- mitochondrial electron chain/energy metabolism, I- implicated in immune response, O- other/unknown.

Figure 6

Gene expression of selected markers of late pathology. Real-time qPCR expression of four genes identified as pathology markers from microarrays in an extended number of individuals eight weeks post-infection. Gene abbreviations are; tnf-11b: tumor necrosis factor receptor superfamily member 11b, cd274: programmed cell death ligand 1, gzmA: granzyme A, tnf-d: tumor necrosis factor decoy receptor. The gene expression ratios are shown as boxplots based on nine HR fish (red) and ten LR fish (green) against the average of control fish. Values of the fish used in the microarray experiments are highlighted with dots and colored according to their histopathology score level (see Figure 1). Boxes represent 50% of the values, while black bars mark the median log-ER. Whiskers indicate the maximum length of 1.5 times the box length. Significance levels between all HR and LR fish are indicated on top of the plot (t-test on log-transformed values, ** = p < 0.01).

Figure 7

Gene ontology classes correlated to viral load at late stage. Gene ontology classes correlated to viral load in responder groups at the late stage of infection. Significantly enriched GO classes (FDR correction, p < 0.05) with positive or negative correlation to viral load (Pearson’s r > 0.6) in HR and LR groups at 8–10 wpi are shown. Column “#” shows the number of genes behind the respective GO term/class, and only the ten GOs with lowest p-values are shown (for completed data, see Additional file 4).

Figure 8

Confirmation of microarray results by qPCR. Comparison of gene expression results obtained with qPCR and microarrays. Log₂-ER of four genes were compared (n = 8, plotted as letters; a = radical s-adenosyl methionine domain-containing protein 2 (rsad2/viperin), b = interferon-induced protein with tetratricopeptide repeats 5 (ifit 5), c = retinoic acid inducible gene I (rigI), d = barrier to autointegration factor (baf)). The dashed black line represents the regression function of the measured values. The regression model, correlation coefficient and p-value are shown in the graph.

Figure 9

Summary of pathogenesis in responder groups. Summary of host-virus responses in high and low responder fish based on findings from a previous CMS study [10] and the present work, as indicated with superscript letters a and b, respectively. Colored circles in fish illustrate histopathology (atrium score level), analogous to Figure 1.