Wild-type AB family founder zebra fish (D. rerio) were originally obtained from David Raible’s fish facility at the University of Washington (USA). Mutant adult zebra fish recombinant activation gene (rag1^−/−) and wild type (rag1^+/+) were reproduced and characterized at the University of Murcia (Dr. Víctoriano Mulero). Adult zebra fish rag1^−/− and rag1^+/+ were raised and genotyped when they reached 0.5–1 g (~6 months of age). Figure S1 in Supplementary Material shows the smaller size and apparent accelerated aging in rag1^−/− fish, compared to rag1^+/+ fish. As we have experienced in three different laboratories (CSIC, UM, and UMH), rag1^−/− zebra fish mutants are difficult to raise and even more to reproduce compared to similar mutants in mice (46). These difficulties may explain why few people could make experiments with them and why only heterozygous rag1^± rather than homozygous rag1^−/− have been used for microarray analysis (49). Zebra fish were maintained at 28°C in 30 l aquaria with tap-dechlorinated carbon-filtered water with 1 g of CaCl₂, 1 g of NaHCO₃, and 0.5 g of Instant Ocean sea salts added to water resulting in a conductivity of 200–300 μS and pH of 7.8–8.2. The aquaria were provided with biological filters and fish fed daily with a commercial feed diet. Previously to the infection experiments, all fish were acclimatized to 22°C for 2 weeks.

The zebra fish embryonic fibroblast (ZF4) cell line (58) was purchased from the American Type Culture Collection (number CRL-2050). ZF4 cells were maintained at 28°C in a 5% CO₂ atmosphere in RPMI 1640-Dutch modified culture medium (Gibco, Invitrogen Co., UK) supplemented with 20 mM HEPES, 10% fetal calf serum (Sigma, St. Louis, MO, USA), 1 mM piruvate, 2 mM glutamine, 50 μg/ml of gentamicin, and 2.5 μg/ml of fungizone.

The SVCV isolate 56/70 (59, 60) was grown in ZF4 cells at 22°C in the presence of 2% fetal calf serum. Supernatants from ZF4 cell monolayers infected with SVCV were clarified by centrifugation at 4,000 × g for 30 min and kept in aliquots at −70°C until used as described before (22, 61, 62). Viral titers of SVCV were determined by methylcellulose plaque assays (56). Briefly, ZF4 cell monolayers were infected with different dilutions of SVCV in 24-well plates for 90 min. Then, the cell culture media were removed, wells covered with 2% methyl cellulose (Sigma, St. Louis, USA) in cell culture media and plates incubated at 22°C. After 5 days, the media were removed and cell monolayers stained with 1% crystal violet-formalin to count plaque forming units (pfu). Please note that SVCV was recently renamed Carp Sprivivirus (53). However, to avoid confusion we have kept the traditional name for this publication.

Spring viremia carp virus infections were conducted as in previous studies (22, 61, 62). Briefly, zebra fish were exposed to SVCV (10³, 10⁴, or 10⁵ pfu/ ml) by bath immersion for 90 min at 22°C (optimal temperature for SVCV replication). Mock-infected zebra fish were incubated with cell culture medium in parallel experiments. After SVCV infection, zebra fish were transferred to tanks with clean water and kept at 22°C to allow the progress of SVCV infection to until tissues were harvested or challenges ended.

Transcript expression folds were evaluated at 2 days after infection. At this time point, higher percentages of genes are differentially transcribed in virally infected fish (63–71), no new viruses are yet released into the water and external SVCV infection symptoms start to appear (52).

To evaluate mortalities, SVCV infections were allowed to proceed during 33 days. From days 2 to 33, infected and non-infected zebra fish were monitored daily to remove those fish that presented external hemorrhages.

During SVCV-induced mortalities, zebra fish were monitored 2–4 times per day and those with external hemorrhages killed by an overdose of anesthetics (methanesulfonate 3-aminobenzoic acid ethyl ester, MS-222) (Sigma-Aldrich) to minimize their suffering (72). Zebra fish were killed by an overdose of MS-222 to extract their tissues. To date, there is no evidence that short exposure to MS-222 has measurable effects on gene expression (since maximum changes are found >2 days of infection) and the use of differential expression fold calculations should eliminate any small differences; however, this cannot be completely rule out since it may affect different genes differentially. Fish were handled in accordance with the National and European guidelines and regulations on laboratory animal care. All the experiments were performed using protocols approved by the European Union Council Guidelines (86/609/EU). Animal work was approved by the UMH, CSIC, UMU, or INIA corresponding Ethic Committees.

Viral titers in zebra fish were determined as described previously (61). Briefly, pooled internal organs, skin, and fins from four fish culled by exposure to MS-222 (see above) were disrupted and homogenized using a pestle, homogenized using a sterile nylon cell strainer (BD Falcon, MA, USA), resuspended in 3 ml of cell culture medium and passed through 0.2 μm sterile filters to remove bacterial contamination. Virus containing suspensions were assessed using the same methylcellulose method as above.

To evaluated transcript expression in zebra fish organs by reverse transcriptase and quantitative polymerase chain reaction (RTqPCR), caudal and pectoral fins and adjacent skin were excised from four to six adult zebra fish per group before and after SVCV infection. Additionally, whole larvae and embryo eggs were pooled (n = 10–20 fish) in each group. RNA was extracted using the E.Z.N.A HP Tissue RNA kit (Omega Bio-tek, Norcross, GA, USA) following manufacturer’s instructions. Isolated RNAs were stored at −80°C until used.

For microarray analysis of rag^−/− and rag^+/+ zebra fish 2 (SVCV infection) or 4 [poly(I:C) injection] experiments were carried out. In each group, head kidney and spleen from individual zebra fish were pooled to obtain enough RNA for hybridization. Pooled head kidneys and spleens (n = 3 fish) were immediately immersed in RNAlater (Ambion, Austin, TX, USA) at 4°C overnight before being frozen at −70°C until processed. RNA was extracted from sonicated (1 min × 3 times at 40 W in ice) organs using a commercial RNA isolation kit (RNeasy kit, Qiagen, Hilden, Germany). RNA concentrations were estimated with Nanodrop and the presence of 18 and 28 S bands confirmed by denatured RNA agar electrophoresis (Sigma, Che. Co, MS, USA).

To study innate/adaptive immune responses by RTqPCR in embryo eggs, larvae, and adult zebra fish in rag1^−/− versus rag1^+/+ groups, a selected gene set (sGS) was chosen as representative genes of innate and adaptive responses based in our own and also other studies. The sGS contained the following groups of immune-related genes: (i) pro-inflammatory cytokines interleukin-1β (il1b) and tumor necrosis α (tnfa), (ii) innate immunity-related genes such as the virus-induced transcription factor interferon regulatory factor-3 (irf3), tank-binding kinase-1 (tbk1), which induces IRF3 in mammalian models, tripartite-motif-21 (trim21), which directs virions to the proteasome for proteolysis, interferon PHI3 (ifnphi3) implicated in antiviral alert innate/adaptive responses in zebra fish, and interferon-induced myxovirus resistance isoforms A–B (mxab) and C (mxc) important in the zebra fish response to rhabdoviral infections (26), (iii) effector proteins such as the antimicrobial peptide defensin 2-like (defbl2), which exhibits antiviral properties against SVCV (73), and the antibacterial protein released from NK-cells NK-LYSIN (nklysin) and, (iv) adaptive immunity-related genes such as the markers of helper and cytotoxic T lymphocytes cluster of differentiation 4 and 8 (cd4 and cd8a, respectively), NK cell- and T cell-generated interferon gamma (ifng) produced after viral infections and finally immunoglobulin M (igm) a marker of mature B-cells. Their corresponding primers are listed in Table S1 in Supplementary Material.

To perform RTqPCR assays 1 μg of RNA was used to obtain cDNA using reverse transcriptase (Moloney murine leukemia virus, Invitrogen) as previously described (73). Quantitative PCR was performed using ABI PRISM 7300 (Applied Biosystems, NJ, USA) and SYBR Green PCR master mix (Life Technologies, UK). Reactions were prepared in 20 μl volume with 2 μl of cDNA, 900 nM of each primer (Table S1 in Supplementary Material) and 10 μl of SYBR Green PCR master mix. Non-template controls were included for each gene analysis. The cycling conditions were 95°C for 10 min, followed by 40 cycles at 65°C 1 min, 95°C for 1 min, and extension of 10 min. The results were analyzed using the 2^−δδCt method (74). Each gene expression value was normalized by the formula, expression of each gene/expression of ef1a. Similar results were obtained using the sum of all gene expression values for normalization (not shown). Differential folds were then calculated by the formula, normalized expression of each rag1^−/− gene/normalized expression of each rag1^+/+ gene.

To target a larger amount of immune-related genes even those which are found in the lowest concentrations, we employed two formats of zebra fish 60-mer oligo microarrays: (i) immune-targeted in-house-designed microarray, Agilent’s ID47562 in a 8 × 15 K format-containing 14,541 fully annotated sequences, previously described, validated (26, 52), and deposited in Gene Expression Omnibus GEO’s GPL17670 (SVCV-infected zebra fish) and (ii) genome-wide commercial microarray Agilent’s ID019161 in a 4 × 44 K format vs2-containing 43,803 partially annotated sequences.

To account for the biological effects arising from small changes in several related genes, we used our previously designed 104 gene set (GS) collection (26). The GS collection was designed from immune-related sequences obtained from key-word searches at the GeneBank, KEGG, and WIKI human/zebra fish pathways (as accessed in 2012) plus new fish GS resulting from leading edge gene analysis (LEGA) of previously studied microarray results in VHSV (a rhabdovirus related to SVCV) survivors (26). Our GS collection was used here for analysis of rag1^−/− and rag1^+/+ by gene set enrichment analysis (GSEA) (http://www.broad.mit.edu/GSEA) (75–77). GSEA assigns a normalized enrichment score (NES) to each GS calculating its corresponding false discovery rates (FDR) for significance evaluation of differential expression folds. The most stringent cut-off value of <0.05 (**) or <0.25 FDR (*) were used in this work for NES to determine statistical significance.

Samples labeled from 2 μg of high quality RNA (50 μg/ml) were hybridized to the microarrays by Nimgenetics (Cantoblanco, Madrid, Spain). Raw and normalized data were deposited in the GEO bank at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc, at GSE54096 (SVCV-infected zebra fish) for the experiments performed at the INIA laboratory and at GSE91397 [poly(I:C)-injected zebra fish] for the experiments performed at the CSIC laboratory. Biological replicates were obtained from head kidney/spleens from 3 pooled zebra fish per replica. Normalized data from two (SVCV-infection) or four [poly(I:C) injection] biological replicas were downsized to about 2,000 unique annotated genes after removing repeated and non-annotated genes and/or genes having outlier fold values. The resulting gene lists were compared and ranked by the t-test statistic metric to calculate GSEA NES. The different genotype or phenotypes comparisons were labeled as A, rag1^−/− versus rag1^+/+ genotypes; B, SVCV-infected or poly(I:C) injected rag1^−/− phenotypes versus rag1^−/− genotypes; or C, SVCV-infected or poly(I:C)-injected rag1^+/+ phenotypes versus rag1^+/+ genotype.

To search for modulated MultiPath Genes (mMPG), those genes present in >6 pathways were extracted as described before using the software Origin pro vs8.6 (Northampton, MA, USA) (52). After normalization, fluorescence outliers defined as values outside the means ± SDs, were first masked from mean calculations (n = 2). Folds were then calculated by applying the formula, normalized gene fluorescence value for each biological replica/non-infected mean. Fold outliers were then eliminated and their mean and SDs recalculated. Those MPG with folds >1.5 or <0.66, significant at the p > 0.05 level (n = 2 or 4) using the two-tail independent t-test, were considered modulated (mMPG).

We used the cellular gene sets (cGS) described before (26), for Th1, T helper 1 cells; Th2, T helper 2 cells; Th17, T helper 17 cells; Treg, T regulatory cells; BZ cells, mucosal IgZ producing cells; B-cells, IgM-producing cells; Dendritic, dendritic cells; CTL, antigen-specific cytotoxic cells; NK-cells, NK cells; macrophages, monocyte and macrophages; and Neutrophil, neutrophil and granulocyte cells. To define the cGS, activating, membrane, and secreted genes were searched for each cellular type from different sources. The resulting cGS were used as inputs for GSEA.

Zebra fish were euthanized with MS-222 (as described above) and a small incision made on the ventral body from the anus to the anterior part of the body cavity. They were then fixed in 10% buffered formalin, embedded in paraffin and cut in sections of 3 μm. Sections were stained with anti-human/mouse active CASPASE-3 antibody (R&D Systems, MN, USA), which recognizes a fully conserved epitope of zebra fish CASPASE-3 (78) or anti-human l-plastin antibody (Gene Tex, CA, USA). All sections were slightly counterstained with hematoxylin.

In non-sterile aquaria both rag1^−/− and rag1^+/+ zebra fish survive early stages of development (egg/larvae) and reach adulthood. However, adult fish in each group present differences in their physical appearance. Commonly, rag1^−/− are smaller and present features that are indicative of accelerated physical deterioration or aging (Figure S1 in Supplementary Material), in contrast to mice rag1^−/− mutants. The contrast between the cleanliness of mouse rooms and that of fish tanks may explain such difficulties. Based on the above mentioned observations, we asked the question, is the innate immune function at early stages of development different in rag1^−/− zebra fish?

To answer this question, we selected some immune genes that, based on our own and other publications, are regulated when zebra fish are exposed to pathogens. Our resulting selected gene set (sGS) included the genes of inflammatory cytokines (il1b, tnfa), innate immune transcription factors and effector molecules (irf3, tbk1, trim21, ifnphi3, mxab, mxc), antimicrobial peptides (defbl2, nklysin), and molecules involved in adaptive immune responses (cd4, cd8, ifng, igm).

Except for ifnphi3, and mxab, the all of the genes were downregulated (folds < 1) in rag1^−/− egg embryos at 24 h post-fertilization (hpf), compared to rag1^+/+ eggs (Figure 1A). This indicated that immune factors directly inherited from the mother were less efficiently passed or absent in rag1^−/− egg embryos, compared to embryos in the rag1^+/+group. Similarly, we evaluated the transcript expression folds (rag1^−/− versus rag1^+/+) in hatched larvae at 72 hpf. The differential expression in rag1^−/− larvae had increased relative to egg embryos, reaching levels similar to those of rag1^+/+ larvae (compare results from Figure 1A to those of Figure 1B). Next, we tested the resistance of zebra fish larvae to SVCV infection. In the conditions used for infection (10⁴ pfu of SVCV per ml, 22°C), both rag1^−/− and rag1^+/+ larvae were susceptible. However, survival rates were modest, 4.4% in rag1^−/− and 16.6% in rag1^+/+ (Figure 1C), similar to those previously reported for rag1^+/+ larvae (51).

The fact that neither egg embryos nor larvae in the rag1^−/− group showed upregulation in the expression of immune-related genes prior to SVCV infection or increased survival after SVCV infection, supports the idea that zebra fish need more time to develop an innate immune system to survive prolonged exposure to microorganisms in the water. To test this hypothesis, we then studied the innate immune response to SVCV infection in adult zebra fish.

When naïve rag1^−/− or rag1^+/+ zebra fish of 6 months of age were infected by bath immersion with 10⁴ pfu/ml of SVCV, there were neither infection symptoms nor deaths for the first 2 days after infection. At 3 days post-infection, zebra fish began to show external hemorrhagic symptoms in mouth, gills, lateral skin, and/or fin bases. Later on, 40–45% of adult rag1^−/− zebra fish survived infection, while 100% of rag1^+/+ died in the first 12 days after infection (Figure 2A). Lower viral titers (~100-fold lower) observed in rag1^−/− fish organs correlated with their delayed and lower mortality, compared to rag1^+/+ zebra fish (Figure 2B). Together, the mortality rates and viral loads observed in the viral challenge experiments suggest that rag1^−/− individuals were capable of mounting an enhanced innate immune response in the absence of adaptive immunity, compared to fully immunocompetent rag1^+/+ zebra fish. Alternatively, the absence of antibody responses in rag1^−/− could have caused less non-specific inflammation and less organ damage resulting in lower the mortality in this group. On the other hand, it is also possible that limitations of the antiviral innate response imposed by the adaptive immune system (i.e., antibodies) could explain higher mortalities in the rag1^+/+.

Because previous reports suggested the importance of the skin barrier in protecting zebra fish against viral infection and skin and fins are principal entry sites for SVCV (17, 47, 51), we studied the selected transcript differential expression in these organs. Before infection, all of the genes in the sGS except irf3 were upregulated in skin/fins of adult rag1^−/− (Figure 3A) with nklysin and cd8 being the most upregulated. After SVCV infection, however, most of the individual gene differential expressions were reduced except for irf3 and nklysin (Figure 3B). To note that cd8 and nklysin, a marker for T cytotoxic cells and a gene highly expressed in both cytotoxic and NK-cells, respectively (79), were both upregulated before and after SVCV infection in rag1^−/− zebra fish, suggesting that the participation of these cellular types are important in the antiviral response in these fish. These results indicate that transcription of the sGS genes is more active in naïve rag1^−/− skin/fins fish than in rag1^+/+, an advantage for resisting SVCV infection. It is also worth noting that transcript expression folds for the genes upregulated in adult rag1^−/− skin/fins correlated with folds of rag1^−/− larvae with a Pearson’s coefficient of 0.63 (using a polynomial fit) but not with those from embryo eggs (data not shown). Presently, whether the mechanisms that lead to the acquired overexpression of these genes in rag1^−/− larvae involve epigenetic changes or other mechanisms is being evaluated but, for now, this is beyond the scope of this study.

To further investigate the transcriptomic profile behind the enhanced survival of naïve adult rag1^−/− after SVCV infection, transcript expression was estimated using both immune-targeted, in-house-designed, and commercially available genome wide microarrays. Analysis was performed in transcripts from pooled head kidneys (the analog of mammalian bone marrow) and spleens before and after SVCV infection or after poly(I:C) injection (to mimic viral replication intermediates). A total of 104 immune-related GSs and/or pathways obtained from keyword searches, KEGG, WIKI, and LEGA (see methods) were used to perform GS enrichment analysis (GSEA) and modulated multi-pathway gene (mMPG) analysis.

Table 1 shows that 25 out of the 104 GS were significantly modulated in at least one of the genotype/SVCV-infected phenotypes analyzed by GSEA using data obtained from the immune-targeted, in-house-designed microarrays. Of those 25 modulated GS, 18 (72%) were upregulated in uninfected rag1^−/− fish, including 3 GS containing genes related to apoptosis. Upregulation of apoptosis-related genes in adult rag1^−/− zebra fish is described here for the first time (Table 1, gray rows, columns A and 2). In addition to apoptosis, the multigene families “NITR” and “MX” (upregulated) or “CRP” and protein degradation psm genes (downregulated), which were previously associated to transcriptional changes in VHSV-survivor zebra fish phenotypes before and after reinfection (26), were also modulated in rag1^−/− fish (Table 1, columns A and 4). Additionally, six upregulated GS contained well known interferon-related genes such as Toll-like receptors (tlr), interferons (ifn), and myxovirus-induced (mx) (Table 1, columns A and 1) or complement-related genes (Table 1, columns A and 3). Upregulation of interferon-related genes confirmed some of the data obtained in skin/fins in uninfected rag1^−/− fish (Figure 3A), strongly suggesting that a systemic upregulation of many genes traditionally implicated in the response to viral infection occurred in the absence of virus. Other GS described here for the first time in relation to uninfected rag1^−/− innate immunity were two GS related to chemokines (“CXCS” and “CHK”) (Table 1, column A).

We then compared the modulated GS after SVCV infection in both genotypes (rag1^−/− and rag1^+/+) using genotype-matched uninfected zebra fish to calculate differential expression folds. In rag1^−/− fish, all of the GS that were upregulated in naïve fish were downregulated after SVCV infection (compare the 18 GS with upregulated NES in Table 1, column A with those in Table 1, column B). One possible explanation for such infection-induced downregulation could be that cell migration from internal lymphoid organs to peripheral tissues (i.e., viral entry tissues), reduces immune gene transcript levels in internal organs. On the contrary, GS that were downregulated in uninfected rag1^−/− became upregulated after SVCV infection. For example, “Proteosome degradation” and “CRP” (Table 1, columns B and 4), complement (Table 1, columns B and 3), and others (Table 1, columns B) indicating an infection-induced response. On the other hand, SVCV infection of rag1^+/+ upregulated complement (Table 1, columns C and 4), 7TLR7CASP and AMP antimicrobial peptides (Table 1, columns C) GS.

Because some SVCV proteins have immunosuppressive effects (52, 80), we decided to study the differential expression fold profiles before and after injection of poly(I:C), a model for viral double-stranded RNA short-lived replication intermediates, in the absence of viral protein and their associated immunosuppressive effects. Thirty GS were modulated in at least one of the genotype/phenotypes when tested by GSEA using genome-wide microarrays (Table 2). In rag1^−/− fish, 10 GS were upregulated, 4 of them containing genes related to apoptosis, coinciding with some of the results obtained in the same fish infected with SVCV (Table 1 gray rows, columns A and 2). In addition, 3 GS contained interferon-related genes such as tlr, ifn, and/or mx genes (Table 2, columns A and 1). Poly (I:C) exclusively upregulated the GS “EGFR1 signaling pathway”, “P13K-AKT signaling pathway” and “TGFB signaling wikipathway” (Table 2, columns A). It is worth noting that after being injected with poly(I:C), rag1^−/− fish maintained similarly modulated GS than naïve fish (Table 2, columns B, 1, and 3), as opposed to what occurred in the same fish when infected with SVCV (Table 1, columns B). Interestingly, after injection with Poly (I:C), rag1^−/− fish upregulated 5 GS participating in protein degradation pathways including, “Heat shock proteins,” “Ubiquitin mediated proteolysis,” “Protein processing in endoplasmic reticulum,”, “Proteosome degradation,” and “Protein export” (Table 2, columns B and 3). This suggests that there is a relationship between viral RNA intermediates and protein degradation pathways. In sharper contrast with SVCV infection, poly(I:C) injection of rag1^+/+ zebra fish, downregulated GS related to complement (Table 2, columns C and 4), suggesting opposite effects of SVCV and poly(I:C) regarding this pathway.

Gene set enrichment analysis of the data obtained in the SVCV infection and poly(I:C) injection experiments revealed important contributions of immune-related multigene families to the rapid defense mechanisms observed in rag1^−/− fish. To some extent these results are in agreement with those from a previous study carried out with zebra fish which survived lethal VHSV infections. When reinfected, VHSV-survivor zebra fish responded more rapidly to infection due to a preceding modulation of specific innate multigene families, rather than to gene regulation in response to the virus (26). These multigene families containing 7–15 different genes each, included c-reactive proteins (crp), myxovirus-induced proteins (mx), novel immunoglobulin-type receptors (nitr), and proteasome subunit macropain proteins (psm). The regulation of these innate multigene families was indicative of responses similar to fish adaptive secondary responses, characterized by shorter lag times (1), rather than by both increased speed and magnitude of the response, as it ensues in mammal species. Because these multigene families present these similarities with adaptive memory responses and abundant gene polymorphisms (which allow for a higher variability), they are candidates for mediators of trained immunity or its equivalent in fish. Based on all the above commented results, it could be argued that in the absence of adaptive immunity, the continuous exposure to microorganisms present in the water activates compensatory innate immune mechanisms that lead to an antiviral alert state characterized by the modulation of apoptosis, multigene family and interferon-related transcripts that facilitate a more rapid and fish “adaptive”/memory-like response to pathogens, in this case a rhabdovirus.

To identify modulated genes with high impact on network regulation in rag1^−/− fish before and after viral infection, we performed a modulated MultiPath Gene (mMPG) analysis. In order to do this, fold data were extracted for genes that (i) were common to > 6 pathways, (ii) had a fold >1.5 or <0.66, and (iii) were significantly different (p < 0.05) from one of the 1.5/0.66-fold thresholds. The results showed that in uninfected rag1^−/− fish several apoptosis-related mMPG were regulated, being fas, faslg, hsp90, casp7, and hspb the most upregulated (Table 3 gray rows, column A, genes labeled with +), which confirms the importance of apoptosis for the survival of this genotype. Other important genes upregulated in the same fish included pro-inflammatory cytokine il1b, and the highly pleiotropic transcription factors stat1a and stat3. After infection with SVCV most of these mMPG were downregulated in rag1^−/− fish (Table 1, column B), while only il1b, irf6, and hspb were upregulated in SVCV-infected rag1^+/+ fish (Table 3, column C).

As expected, the level of immunoglobulin igm (probes designed against its heavy chain constant domain) was downregulated in uninfected rag1^−/−, compared to rag1^+/+ fish. However, the most intriguing data was the upregulation of igm in rag1^−/− fish after infection with SVCV. This might reflect a host desperate attempt to activate igm constant domains in the presence of a pathogen, despite the absence of recombination of the variable domains. Thus, it is also possible that although there is no V(D)J recombination in rag1^−/− mutants, some igm sterile transcription occurs, as previously suggested by others (81).

Because apoptosis was likely to be important for the survival of rag1^−/− fish based on the transcript upregulations of apoptosis-related GS (Tables 1 and 2) and mMPG (Table 3), we tested whether apoptosis was activated also at the protein level. To that end, we stained zebra fish head kidney, gut, skin, and liver with an anti-apoptosis protein antibody. For those experiments, we chose active CASPASE-3 because of its amplifying role in CASPASE-8-mediated mammalian/fish apoptosis (82, 83). Staining of uninfected histological sections revealed augmented positive staining in rag1^−/− tissues compared to rag1^+/+ (Figure 4A). When tissues from SVCV-infected zebra fish were stained, active CASPASE-3 positive staining increased in both rag1^−/− and rag1^+/+ fish (Figure 4B). Therefore, it could be argued that as part of the rag1^−/− antiviral alert state both transcripts and proteins facilitate the rapid induction of apoptosis, which would help to eliminate virus-infected cells and increase survival.

Because of the lack of molecular markers for zebra fish immune cells, cGS including immune cell type-representative genes were designed as alternative markers in a previous study. The same analysis was applied here to evaluate the relative importance of each particular cell type in the rag1^−/− genotype before and after SVCV infection.

Transcripts from NK-cells and macrophages were the most upregulated cGS in uninfected rag1^−/− fish (Table 4). Both NK-cells and macrophages have been identified in higher vertebrate models, as mediators of trained immunity (9, 11, 16, 18). In partial agreement with our results, rag1^−/− zebra fish developed immune memory when immunized to Edwardsiella ictaluri (a pathogenic bacterium) through the participation of NK-cells (25). NK-cells are traditionally defined as cells of the innate immune system because they lack RAG recombinase-dependent antigen receptors. The comparison of candidate mechanisms for mediating mammalian antigen-specific NK cell memory with other examples of RAG-independent pathways that generate antigen receptor diversity in non-mammalian species such as zebra fish, suggests that specific subsets of NK cells can develop long-lived and specific memory to a variety of antigens independent of B cells and T cells (13, 84). Most recently, antigen-specific NK cell responses to influenza and HIV viruses were induced in primates after infection and/or vaccination, an important opening for human vaccine improvement (13–15). T helper cell 17 (TH17) and TH2 were also upregulated in uninfected rag1^−/− fish, contrary to other cellular types such as B-cells and neutrophils. Interestingly, all head kidney/spleen cellular types studied were downregulated in rag1^−/− fish after SVCV infection, suggesting again that cellular migration to peripheral tissues ensues after infection. In contrast, in rag1^+/+ fish, B-cells and neutrophils were upregulated (Table 4).

Since the transcript data suggested that NK-cells and/or macrophages might infiltrate rag1^−/− tissues, we stained the corresponding zebra fish tissue sections with an anti-l-plastin polyclonal antibody, a pan-leukocyte marker (85, 86), to explore for the presence of leukocytes in peripheral tissues. Leukocytes were abundant in significant amounts in zebra fish muscle and skin from uninfected rag1^−/− fish (Figure 5). In contrast, equivalent tissue sections from rag1^+/+ fish showed limited leukocyte presence (Figure 5), confirming previous reports (45).

After recognizing different pathogen-associated molecular patterns, mammalian NK-cells and macrophages undergo epigenetic changes to become effector cells for trained immunity (9, 18). Our results showed that cell-specific gene expression and immunohistology data from rag1^−/− zebra fish confirmed the increased function and/or activation of NK-cells/macrophages and leukocytes, respectively. Therefore, it could be argued that in rag1^−/− zebra fish these cellular types mediate a shorter lag time responses that resemble adaptive responses in fish, consistent with trained immunity responses mediated by similar cells in other vertebrate models.