Flow cytometry scatter plots (A) and distribution of IgM+ cells in lymphoid organs (B).

<p><b>A)</b> Representative FSC/SSC scatter plots used to define cellular populations and composition (mean ± standard deviations, n = 9) in pooled lymphoid organs from different phenotypes. <b>P1 black</b> (13.2 ± 5.5%) and <b>P3 blue</b> (12.6 ± 4.9%), damaged cells and/or cellular debris identified using sonicated cells. <b>P2 red</b> (20.4 ± 21.2%). <b>P4 purple</b> (40.8 ± 21.5%), lymphocytes as determined before [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.ref039" target="_blank">39</a>]. <b>P10 green</b> (4.3 ± 2.7%). <b>B)</b> Cells from lymphoid organs were pooled from 3 NI (open bars), VHSVS (hatched bars) or bacterial-survivor (black bars) zebrafish phenotypes. Cells were stained with no MAb (in the absence of any MAb), an irrelevant MAb, FITC-phytohemaglutinin (PHA) and zebrafish crossreacting anti-trout IgM MAb 6B7 (aIgMt). The percentage of cells above the threshold fluorescence in the P4 population was calculated by the formula: 100 x number of cells in P4 with fluorescence above the threshold / number of P2+P4+P5 cells. Mean and standard deviations were represented (n = 2). <b>+</b>, significantly different from the staining with the irrelevant MAb. *, significantly different from irrelevant MAb or NI stained with anti-IgM.</p

Cumulative percent survival of zebrafish after <i>VHSV</i> infections (A) and levels of anti-<i>VHSV</i> neutralizing antibodies (NAbs) in plasma (B).

<p><b>A)</b> primary infected VHSV+ and vaccinated plus booster VHSVS were obtained as described in detail in methods and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s001" target="_blank">S1 Fig</a> After infection-by-immersion in 10⁷ ffu of <i>VHSV</i> per ml, daily cumulative survival at 14°C was calculated for each experiment using the formula, 100 x (1- daily cumulative mortality / total mortality after 30 days) (n = 15 to 35 zebrafish per experiment). Different symbols (circles, squares, triangles and stars) correspond to independent experiments. <b>Open circles and squares</b>, primary infection-by-immersion at 14°C (VHSV+). <b>Solid symbols</b>, vaccination- by-injection at 18°C plus booster-by-immersion at 14°C 3-months later. <b>Blue stars</b>, infection-by-immersion at 14°C after booster of the VHSVS fish (VHSVS+). <b>B)</b> Levels of neutralizing Abs (NAbs) in plasma from: vaccination plus booster VHSVS (6 months after the first infection-by-injection); infection after booster VHSVS+ (VHSVS 2-days after a third infection); chronically infected with bacteria (bacteria); non-infected (NI) zebrafish and no added plasma (no plasma). The percentage of infected cells was calculated by the formula, 100 x number of cells with fluorescences above the threshold / total number of cells gated per well. <i>VHSV</i>-infected cell controls in the absence of added zebrafish plasma showed that 55% of the EPC cells were infected (fluorescent). The results were then expressed in percentage of neutralization by the formula, 100–100 x % of infected cells / 55. Each open circle corresponds to an individual zebrafish. <b>Red horizontal lines</b>, mean neutralization percentage values. <b>Dash line</b>, mean + 2 standard deviations of neutralization percentage of non-infected plasma (n = 8). <b>*,</b> mean percentage values significantly higher than non-infected mean at the p<0.05 level (Student t-test).</p

Comparison of gene ES from the Leading Edge novel GSs with significant NES.

<p>The novel GSs with significant NES are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.t002" target="_blank">Table 2</a> (gene compositions in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s009" target="_blank">S5 Table</a>). <b>Open circles</b>, novel GSs containing 12 <i>mapks</i> and 5 <i>pirp</i> (phosphoinositide-related proteins). <b>Red circles</b>, novel GSs containing 8 <i>tlr</i>, 5 <i>ifn</i> and 5 <i>mx</i> genes. <b>Red triangles</b>, novel GSs containing 5 <i>ifn</i> and 4 <i>mx</i>. <b>Blue circles</b>, novel GS containing 5 <i>caspases</i>. <b>Green circles</b>, novel GS containing 9 chemokines (<i>9cxc</i>). The complete list of genes for each of the novel 14 GSs are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s009" target="_blank">S5 Table</a>.</p

Heat map of Modulated MultiPath Genes (mMPG).

<p>The mMPG with folds >2 or <0.5 in at least one of the phenotypes, were ordered by the expression levels in VHSVS. Relative differential expressions were calculated versus NI fish. <b>Bright gree</b>n, <0.2. <b>Light green</b>, <0.66 and >0.2. <b>Yellow</b>, folds <1.5 and >0.66. <b>Light red</b>, >1.5 and <2. <b>Red</b>, >2 and <3. <b>Intense red</b>, folds >3. <b>P,</b> number of pathways in which the mMPG were present. <b>1–12</b>, biological replicates. <b>Blue M,</b> mMPG genes present in the “Mitogen activated protein kinase pathway” (MAPK). <b>Blue E</b>, mMPG genes present in the "EGFR1 signaling pathway". <b>Blue MP,</b> mMPG genes present in the novel 12MAPKS+5PIRP GS defined after Leading Edge Analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s009" target="_blank">S5 Table</a>, red).</p

Comparison of Enrichment Scores (ES) for individual genes from GSs with significant NES.

<p>Some of the most enriched human/zebrafish orthologous GSs of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.t001" target="_blank">Table 1</a> were compared by their corresponding individual gene enrichment plots (ES per gene in the Y axis <i>versus</i> ranked list of genes ordered from their highest to lowest differential expression folds in the X axis). Those genes ranked first in the X axis correlated with the first phenotype of the comparison (VHSV+, VHSVS, VHSVS+) while those at the end correlated with NI. <b>Black circles</b>, <i>crp</i> (c-reactive protein) keyword-selected GSs with added complement components (genes listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Red circles</b>, <i>mx</i> (myxovirus-induced protein) keyword-selected GSs with added interferon genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Green circles</b>, <i>nitr</i> (novel immune-type receptor) keyword-selected GSs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Blue circles</b>, “Proteasome degradation” WIKI pathway (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Open circles</b>, “complement and coagulation cascades” KEGG pathway (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Red squares</b>, “Type II interferon signaling (<i>ifng</i>)” WIKI pathway (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). <b>Red triangles</b>, <i>ifn</i> (interferon) keyword-selected GSs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s008" target="_blank">S4 Table</a>). The complete list of zebrafish GS genes in the in-house microarray and their corresponding probe sequences can be found at GEO’s GPL17670.</p

Comparison of NES from GSEA of novel GSs derived from Leading Edge Gene Analysis (gene symbols described in S5 Table) of the data summarized in Table 1

<p>The results show the significant NES among the 14 novel GSs proposed by the Leading Edge Gene Analysis. The differential expressions were calculated versus NI zebrafish. The numbers before the gene names indicate the total number of these genes in each novel GS. The novel GS names indicate some of the majoritary genes which form part of the novel GSs (The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s009" target="_blank">S5 Table</a> shows the corresponding gene symbols of all novel GSs).</p><p><b>Bold Novel GSs</b>, novel GSs proposed by Leading Edge analysis of GSEA summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.t001" target="_blank">Table 1</a>. <b>MAPKS</b>, mitogen-activated protein kinases.</p><p><b>PIRP</b>, phosphatidyl-inositol related gene proteins.</p><p><b>CASPS</b>, caspases.</p><p><b>TLR</b>, Toll-like receptors.</p><p><b>IFN</b>, interferons.</p><p><b>MX</b>, myxovirus-induced proteins.</p><p><b>+</b>, NES correlating with the first phenotype in the comparison.</p><p><b>-</b>, NES correlating with NI in the comparison.</p><p><b>** (bold)</b>, FDR q value < 0.05.</p><p><b>*</b>, FDR q value < 0.25.</p><p>Comparison of NES from GSEA of novel GSs derived from Leading Edge Gene Analysis (gene symbols described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s009" target="_blank">S5 Table</a>) of the data summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.t001" target="_blank">Table 1</a></p

Differential expression profiles of individual <i>crp</i>, <i>mx</i>, <i>nitr</i> and <i>psm</i> genes from multigene family GSs.

<p>After normalization and reduction to a list of unique genes, folds were calculated for each gene using the formula: fluorescence of each <i>VHSV</i>-infected replicate / mean fluorescences from the NI replicates (n = 4). Means and standard deviations were then obtained for each gene and outliers were removed to obtain final folds (n = 4). <b>A,</b><i>crp</i>, <i>mx</i> and <i>nitr</i> multigene families. <b>B</b>, <i>psm</i> multigene family. <b>Open bars</b>, VHSV+. <b>Hatched bars</b>, VHSVS. <b>Black bars</b>, VHSVS+. <b>Red horizontal bars</b>, 1.5- and 0.66-fold thresholds. <b>*,</b> significantly >1.5 or <0.66 at the p = 0.05 level (Student t-test).</p

Comparison of NES from GSEA of GS defining distinct immune cell types (gene symbols described in S6 Table)

<p>To estimate the different immune cell activities, new Gene Sets (GSs) were defined (gene compositions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s010" target="_blank">S6 Table</a>). To define genes for each cellular type, activating, membrane and secreted genes were selected and added to the GS from data obtained from various sources. The resulting GSs shown by their symbols in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135483#pone.0135483.s010" target="_blank">S6 Table</a>, were used as inputs for GSEA analysis. The NES values of each cellular type ordered by those in VHSVS are shown. The differential expressions were calculated versus <b>NI</b>, non-infected zebrafish.</p><p><b>Th1</b>, T helper 1 cells.</p><p><b>Th2</b>, T helper 2 cells.</p><p><b>Th17</b>, T helper 17 cells.</p><p><b>Treg,</b> T regulatory cells.</p><p><b>B cells,</b> IgM-producing cells.</p><p><b>BZ</b> cells, IgZ-producing cells.</p><p><b>Dendritic</b>, dendritic cells.</p><p><b>Cytotoxic,</b> antigen-specific cytotoxic cells.</p><p><b>NK cells</b>, natural killer cells.</p><p><b>Macrophages</b>, monocyte and macrophages.</p><p><b>Neutrophil,</b> neutrophil and granulocyte cells.</p><p><b>+</b>, NES correlating with the first phenotype in the comparison.</p><p><b>-</b>, NES correlating with NI in the comparison.</p><p><b>** (bold)</b>, FDR q value<0.05.</p><p><b>*</b>, FDR q value <0.25.</p><p>Comparison of NES from GSEA of GS defining distinct immune cell types (gene symbols described in S6 Table)</p

Microarray hybridization and RTqPCR fold comparison of differentially expressed multipath genes.

<p>Microarray folds of the differentially expressed multipath genes from Table 1 were compared with the corresponding folds obtained by RTqPCR as described in Methods. To increase clarity the genes were distributed in three groups: <b>A</b>, <b>B</b> and <b>C</b>. Points inside the shadowed ellipses include most of the data. Logarithmic scales were used to best compare all data because of the wide relative differences between microarray and RTqPCR fold values. ○, Mean folds from head kidney and spleen from 2-day infected zebrafish. ●, Mean folds from head kidney and spleen from 30-day survivor zebrafish. Arrows indicate the direction of the fold changes from 2- to 30-days by both microarray and RTqPCR estimations. The <i>mapk10</i>, a multipath gene whose fold did not changed from 2- to 30-days, was included as a control (Figure 4C).</p

VENN diagram between genome-wide and targeted microarrays and comparison of fluorescence intensities obtained by hybridization to targeted microarrays of transcripts from 2- and 30-days after SVCV infection with those obtained from non-infected zebrafish controls.

<p><b>A</b>) A VENN diagram was constructed by comparing unique accession numbers between genome-wide zebrafish vs2 ID019161 microarray of Agilent (43803 probes, 37464 accession numbers) and targeted microarray zfin ID041401 (11586 probes, 8636 unique accession numbers: 2286 and 6350 pathway and keyword sections, respectively). The online software from BioInfoRx (<a href="http://apps.bioinforx.com" target="_blank">http://apps.bioinforx.com</a>) was used. <b>A1</b>, wide-genome zebrafish vs 2 ID019161. <b>A2</b>, pathway section of the targeted microarray zfin ID041401. A3, keyword section of the targeted microarray zfin ID041401. <b>B</b>, <b>C</b>) The zfin ID041401 was used to estimate transcript levels in pooled head kidney and spleen from SVCV-infected and non-infected zebrafish after 2-days (<b>B</b>) or 30-days (<b>C</b>). The Figure shows the range of mean fluorescences obtained from different experiments (6 fish per experiment, n=3 for non-infected zebrafish and for SVCV infected zebrafish after 2-days and n=2 for SVCV infected zebrafish after 30-days). A white straight line shows fold = 1.</p