RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad spectrum of PIM kinases in zebrafish kidney that promote viral entry
Patricia Pereiro, Margarita Álvarez-Rodríguez, Valentina Valenzuela-Muñoz, Cristian Gallardo-Escárate, Antonio Figueras, Beatriz Novoa
PII: S1050-4648(20)30063-2
DOI: https://doi.org/10.1016/j.fsi.2020.01.055
Reference: YFSIM 6791
To appear in: Fish and Shellfish Immunology
Received Date: 21 November 2019 Revised Date: 24 January 2020 Accepted Date: 27 January 2020
Please cite this article as: Pereiro P, Álvarez-Rodríguez M, Valenzuela-Muñoz V, Gallardo-Escárate C, Figueras A, Novoa B, RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad spectrum of PIM kinases in zebrafish kidney that promote viral entry, Fish and Shellfish Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.01.055.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.
1 RNA-Seq analysis reveals that spring viraemia of carp virus induces a broad
2
3
spectrum of Pim kinases in zebrafish kidney that promote viral entry
4 Patricia Pereiro1,2,#, Margarita Álvarez-Rodríguez1,#, Valentina Valenzuela-Muñoz2,
5 Cristian Gallardo-Escárate2, Antonio Figueras1, Beatriz Novoa1,*
6 1 Institute of Marine Research (IIM), National Research Council (CSIC), Eduardo
7 Cabello, 6, 36208, Vigo, Spain
8 2 Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for
9 Aquaculture Research (INCAR), University of Concepción, P.O. Box 160-C,
10
11
Concepción, Chile
12*Corresponding author:
13Dr. Beatriz Novoa
14Email: [email protected]
15
16
Tel: +34 986231930
17
18
19
#These authors equally contributed to this work
20 Abstract
21 PIM kinases are a family of serine/threonine protein kinases that potentiate the
22progression of the cell cycle and inhibit apoptosis. Because of this, they are considered
23to be proto-oncogenes, and they represent an interesting target for the development of
24anticancer drugs. In mammals, three PIM kinases exist (PIM-1, PIM-2 and PIM-3), and
25different inhibitors have been developed to block their activity. In addition to their
26involvement in cancer, some publications have reported that the PIM kinases have pro-
27viral activity, and different mechanisms where PIM kinases favour viral infections have
28been proposed. Zebrafish possess more than 300 Pim kinase members in their genome,
29and by using RNA-Seq analysis, we found a high number of Pim kinase genes that were
30significantly induced after infection with spring viraemia of carp virus (SVCV).
31Moreover, analysis of the miRNAs modulated by this infection revealed that some of
32them could be involved in the post-transcriptional regulation of Pim kinase abundance.
33To elucidate the potential role of the 16 overexpressed Pim kinases in the infectivity of
34SVCV, we used three different pan-PIM kinase inhibitors (SGI-1776, INCB053914 and
35AZD1208), and different experiments were conducted both in vitro and in vivo. We
36observed that the PIM kinase inhibitors had a protective effect against SVCV, indicating
37that, similar to what is observed in mammals, PIM kinases are beneficial for the virus in
38zebrafish. Moreover, zebrafish Pim kinases seem to facilitate viral entry into the host
39cells because when ZF4 cells were pre-incubated with the virus and then were treated
40with the inhibitors, the protective effect of the inhibitors was abrogated. Although more
41investigation is necessary, these results show that pan-PIM kinase inhibitors could serve
42
43
as a useful treatment for preventing the spread of viral diseases.
44Keywords: zebrafish, SVCV, Pim kinases, pan-PIM kinase inhibitors, viral entry,
45antiviral
46Introduction
47 The proviral insertion site in Moloney murine leukaemia virus (PIM) kinases are
48a family of serine/threonine protein kinases involved in the regulation of different
49cellular processes [1]. In mammals, three PIM kinase members exist (PIM-1, PIM-2 and
50PIM-3). They are considered proto-oncogenes due to their ability to promote cell
51survival and proliferation by inhibiting apoptosis and positively regulating cell cycle
52progression, among other functions [1]. Because of these functions, PIM kinases are
53involved in tumourigenesis and represent an attractive target for pharmacological
54anticancer therapy [2]. PIM protein expression is induced via the JAK/STAT signalling
55pathway, which is activated by several cytokines after cytokine-receptor interaction [3].
56Therefore, these kinases are induced after a variety of immune stimuli, indicating their
57role in the immune response [4].
58 PIM kinases lack a regulatory domain [5]; therefore, it is thought that these
59proteins are constitutively active when expressed in cells and, consequently, that their
60activity is directly correlated with their transcription level [3]. Due to their involvement
61in cancer progression, many efforts have been made to develop efficient PIM kinase
62inhibitors. Most of them block the activity of all three PIM isoforms, so they serve as
63pan-PIM kinase inhibitors [1]. Moreover, although only a few inhibitors have been
64tested in human clinical trials, the inhibition of all the PIM isoforms in murine models
65revealed very modest side effects, maintaining animal viability and fertility [6].
66 PIM kinases have also been linked to the progression of certain viral pathogens.
67A few publications reported that PIM kinase inhibition ameliorated the resolution of a
68variety of viral diseases, although this effect was tested exclusively in vitro, and the
69mechanisms by which PIM kinases promote viral replication seem to be different [7-
7011].
71 Whereas mammals possess three highly evolutionarily conserved PIM kinase
72isoforms, more than 300 Pim kinases have been identified in zebrafish by computational
73analysis [12]. The expansion of gene families is frequent in teleost species [13, 14],
74which sometimes makes the establishment of functional equivalences between
75mammalian and fish proteins difficult. Nevertheless, despite this great diversity of Pim
76kinases in zebrafish, a high conservation of functionally important residues has been
77observed between human and zebrafish PIM kinases [12].
78 As a model species, the zebrafish is a very useful organism to study a multitude
79of biological processes, including infectious diseases [15]. Among viruses, one of the
80most commonly used to challenge zebrafish is the spring viraemia of carp virus (SVCV)
81[16]. SVCV is an enveloped, bullet-shaped, negative-sense, single-stranded RNA virus
82belonging to the Rhabdoviridae family [17]. This family also includes other viruses
83causing relevant economic losses in the fish aquaculture industry [18-20]. Moreover,
84humans and mammals are also affected by rhabdoviruses, such as the rabies virus and
85the vesicular stomatitis virus [17]. Therefore, advances in the knowledge of the antiviral
86immune response in zebrafish could help to gain a better understanding of the defence
87mechanisms against rhabdoviruses or viruses in general in other species.
88 High-throughput sequencing technologies have emerged as a powerful tool to
89thoroughly analyse the transcriptome response to a specific stimulus or condition. In
90this work, we conducted RNA-Seq analysis of kidney samples from SVSV-infected and
91uninfected zebrafish to evaluate the gene modulations (mRNA changes) induced after
92infection. The differential expression analysis between infected and control individuals
93showed that in addition to a multitude of immune-related genes, a broad spectrum of
94Pim kinases is induced after SVSV challenge. Additionally, the microRNA (miRNA)
95profile was also obtained. We found that at least five different miRNAs affected by the
96infection have Pim kinase mRNA as a potential target, revealing that the level of these
97kinases could be regulated by miRNAs after viral challenge. To better understand the
98involvement of Pim kinases in SVCV progression, we tested three different pan-Pim
99kinase inhibitors both in vivo and in vitro. Our results clearly showed that blocking Pim
100kinase activity reduces SVCV entry into the cells and consequently ameliorates the
101
102
survival of infected zebrafish larvae.
103Material and methods
104Animals, virus and cell lines
105 Six-month-old wild-type zebrafish were obtained from the facilities at the
106Instituto de Investigaciones Marinas (Vigo, Spain), where zebrafish are maintained
107following established protocols [21-22]. Zebrafish were euthanized using a tricaine
108methanesulfonate (MS-222) overdose (500 mg/l). Fish care and challenge experiments
109were conducted according to the guidelines of the CSIC National Committee on
110Bioethics under approval number ES360570202001/16/FUN01/PAT.05/tipoE/BNG.
111Wild-type zebrafish larvae were also obtained in the same facilities.
112 SVCV isolate 56/70 was propagated in epithelioma papulosum cyprini (EPC)
113carp cells (ATCC CRL-2872) that were maintained in MEM (Gibco) supplemented with
1142 % FBS (Gibco) and 1 % penicillin/streptomycin solution (Gibco), and the cells were
115titrated in 96-well plates. The TCID50/ml was calculated according to the Reed and
116Muench method [23].
117 For in vitro assays, the zebrafish fibroblastic cell line ZF4 (ATCC CRL-2050)
118was maintained in DMEM (Gibco) supplemented with 10 or 2 % FBS (Gibco) and 1 %
119penicillin/streptomycin solution (Gibco), and the cells were kept at 27°C.
120Experimental design and samples for sequencing
121 Twelve adult zebrafish were injected intraperitoneally (i.p.) with 20 µl of SVCV
122(3 x 102 TCID50/ml), and as a control group, the same number of fish were inoculated
123with an equivalent volume of MEM + 2 % FBS + penicillin/streptomycin. That viral
124concentration was previously tested and resulted in a survival rate of 20 % [24]. Kidney
125samples were collected at 24 h post-infection (hpi), and the same quantity of tissue from
1264 animals was pooled, obtaining 3 biological replicates (4 fish/replicate) per condition.
127Samples were stored at -80°C until RNA extraction.
128High-throughput transcriptome sequencing (mRNA and miRNA)
129 Total RNA from the different samples was extracted using a Maxwell 16 LEV
130simplyRNA Tissue kit (Promega) with an automated Maxwell 16 Instrument in
131accordance with the instructions provided by the manufacturer. The quantity of RNA
132was measured in a NanoDrop ND-1000 (NanoDrop Technologies, Inc.), and RNA
133integrity was analysed in an Agilent 2100 Bioanalyzer (Agilent Technologies Inc.,
134Santa Clara, CA, USA) according to the manufacturer’s instructions. All the samples
135passed the quality control tests and were used for Illumina library preparation.
136 For mRNA sequencing, double-stranded cDNA libraries were constructed using
137the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA), and
138sequencing was performed using Illumina HiSeq 4000 technology. For miRNA-Seq, a
139TruSeq small RNA Library Preparation Kit (Illumina, San Diego, CA, USA) was used,
140and sequencing was conducted with HiSeq 2500 technology. Both types of sequencing
141were conducted by Macrogen Inc. (Seoul, Republic of Korea).
142 The read sequences obtained with both methodologies were deposited in the
143Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra) under the BioProject
144accession number PRJNA532380.
145Trimming, mapping, RNA-Seq and differential expression analysis of mRNAs
146 CLC Genomics Workbench, v. 11.0.2 (CLC Bio, Aarhus, Denmark) was used to
147filter and trim reads, map the high-quality reads against the last version of the zebrafish
148genome (GRCz11) and perform the RNA-Seq statistical analyses. Raw reads were
149trimmed to remove adaptor sequences and low-quality reads (quality score limit 0.05 on
150the Phred scale). RNA-Seq analyses were performed using the zebrafish genome with
151the following parameters: length fraction = 0.8, similarity fraction = 0.8, mismatch cost
152= 2, insertion cost = 3 and deletion cost = 3. The expression values were set as
153transcripts per million (TPM). Finally, a differential expression analysis test was used to
154compare gene expression levels and to identify differentially expressed genes (DEGs).
155Transcripts with absolute fold change (FC) values > 2 and p-values < 0.05 were retained
156for further analyses. A heat map showing hierarchical clustering of gene expression
157(TPM values) was constructed using the complete linkage method with Euclidean
158distance.
159Gene Ontology (GO) enrichment, KEGG pathways and domain analyses
160 For the up- and downregulated DEGs between SVCV-infected and uninfected
161zebrafish, we conducted GO enrichment analysis of biological processes, KEGG
162pathway analysis and domain enrichment using DAVID software [25, 26]. The
163significance level was set at 0.05 (p ˂0.05) in all cases. For domain enrichment, the
164Protein Information Resource (PIR) database [27] was used. The representation of the
165different categories was based on the fold-enrichment value.
166Sequence alignment and identity/similarity matrix
167 Protein sequences for the Pim kinase genes modulated after infection were
168obtained from the zebrafish genome via Ensembl
169(http://www.ensembl.org/Danio_rerio/Info/Index) [28]. The region corresponding to the
170Pim kinase domain was selected, and alignment was conducted using the ClustalW
171server [29]. Sequence similarity and identity scores were calculated with the software
172MatGAT [30] using the BLOSUM62 matrix.
173Analysis of the zebrafish miRNome and target prediction
174 miRNAs are small non-coding RNAs that are evolutionarily conserved, and they
175regulate gene expression at the post-transcriptional level by interacting with the 3’UTR
176of mRNAs and recruiting molecular machinery that degrades the target mRNAs [31,
17732]. Therefore, miRNAs could serve as key mechanisms of post-transcriptional gene
178silencing.
179 The CLC Genomics Workbench, v. 11.0.2 (CLC Bio, Aarhus, Denmark) was
180also used for small RNA analysis. The raw reads were also filtered (quality score limit
1810.05 on the Phred scale) and trimmed to delete adaptor sequences. High-quality reads
182with lengths ranging from 15 to 30 nucleotides were retained as small RNAs. RNA-Seq
183analyses were conducted using the zebrafish mature miRNAs database downloaded
184from the miRBase 22.1 (http://www.mirbase.org/cgi-bin/mirna_summary.pl?org=dre)
185[33] as reference sequences. For the analysis, the following settings were used:
186mismatches = 2, length fraction = 0.6, similarity fraction = 0.5. The expression values
187were set as transcripts per million (TPM). Finally, a differential expression analysis test
188was used to compare gene expression levels. Those miRNAs with an FC > 2 were
189selected for further analyses (statistical restriction was not applied due to the presence
190of only one biological replicate per condition).
191 Based on the probability of interaction between the different mature miRNAs
192and the 3’UTR of the zebrafish genes, the prediction of the potential gene targets for the
193differentially expressed miRNAs was conducted using TargetScanFish v6.2 [34] and
194mirMAP [35]. For the TargetScanFish, those potential targets with a total context+
195score < -0.3 were considered, and for the mirMAP, those with a mirMAP score > 90
196were considered.
197Quantitative PCR (qPCR) validation of RNA-Seq and miRNA data
198 For DEG validation, cDNA synthesis of the samples was performed with an
199NZY First-Strand cDNA Synthesis kit (NZYTech) using 0.5 µg of total RNA. A total of
2004 genes were used to validate the RNA-Seq results. Specific qPCR primers were
201designed using Primer 3 software [36], and their amplification efficiency was calculated
202with the threshold cycle (CT) slope method [37]. Primer sequences are listed in
203Supplementary Table S1. Individual qPCR reactions were carried out in a 25 µl reaction
204volume that contained 12.5 µl of SYBR GREEN PCR Master Mix (Applied
205Biosystems), 10.5 µl of ultrapure water, 0.5 µl of each specific primer (10 µM) and 1 µl
206of two-fold diluted cDNA template; reactions were performed in MicroAmp optical 96-
207well reaction plates (Applied Biosystems). Reactions were conducted using technical
208triplicates in a 7300 Real-Time PCR System thermocycler (Applied Biosystems). qPCR
209conditions consisted of an initial denaturation step (95°C, 10 min), which was followed
210by 40 cycles of a denaturation step (95°C, 15 s) and one hybridization-elongation step
211(60°C, 1 min). The relative expression levels of the different genes were normalized
212following the Pfaffl method [37]; 18s ribosomal RNA (18s) was used as a reference
213gene. Fold-change units were calculated by dividing the normalized expression values
214in SVCV-infected zebrafish by the normalized expression values of the controls.
215 For miRNA validation, RNA samples (0.25 µg) were reverse transcribed with a
216miScript II RT kit (Qiagen). Primers for 4 miRNAs were purchased based on the exact
217sequence of the zebrafish mature miRNA deposited in the mirBASE [33]. We selected
218the U6 snRNA (5’-ATGACACGCAAATCCGTGAAG-3’) as a reference sequence for
219normalization. qPCR reactions were conducted with a miScript SYBR Green PCR Kit
220(Qiagen) following the manufacturer’s recommendations. Reactions were conducted
221using technical triplicates in a 7300 Real-Time PCR System thermocycler (Applied
222Biosystems). qPCR conditions consisted of an initial denaturation step (95°C, 15 min),
223which was followed by 40 cycles of denaturation (94°C, 15 s), annealing (55°C, 30 s)
224and extension (70°C, 34 s). Fold-change units were calculated by dividing the
225normalized expression values in SVCV-infected zebrafish by the normalized expression
226values of the controls.
227Pimr106 expression after SVCV or Poly I:C challenge
228 Adult (9 month) zebrafish were i.p. injected with 10 µl of an SVCV suspension
229(3 × 106 TCID50/ml), and the corresponding controls were injected with the same
230volume of culture medium (MEM + 2 % FBS + penicillin/streptomycin). The same
231experiment was conducted using polyinosinic:polycytidylic acid (Poly I:C) (1 mg/ml in
232PBS; Sigma–P1530), and the corresponding controls were injected with PBS. Both the
233SVCV and Poly I:C concentrations were previously tested for the induction of a
234significant immune response [38,39]. To analyse the induction of the pim proto-
235oncogene, serine/threonine kinase, related 106 (pimr106) gene by qPCR, kidney
236samples were taken from anaesthetized fish at 3, 6 and 24 h post-stimulation, and 4
237biological replicates (4 fish/replicate) per time point were obtained. Additionally, the
238expression of two pivotal genes involved in the type I interferon response was analysed
239in the Poly I:C-stimulated fish and the corresponding controls to confirm the activation
240of the typical antiviral response: interferon phi 1 (ifnphi1) and interferon-stimulated
241gene 15 (isg15). The primers used for gene amplification are listed in Supplementary
242Table S1.
243Pan-PIM kinase inhibitors
244 PIM kinase inhibitors used in this work were SGI-1776 (Calbiochem; Ref.
245526528), INCB053914 (Selleckchem; Ref. S8800) and AZD1208 (Sigma-Aldrich; Ref.
246SML2595). The compounds were resuspended in DMSO.
247Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in ZF4 cells
248 Based on the literature [40-42], we selected the following concentrations of PIM
249kinase inhibitors to conduct the functional assays: 5 µM SGI-1776, 2 µM INCB053914
250and 10 µM AZD1208. ZF4 cells were seeded in 96-well plates and treated for 24 h with
251these concentrations of inhibitors; as a control condition, cells were treated with the
252vehicle alone (0.002 % DMSO). A total of 12 wells per treatment were included. To test
253cell viability, an MTT assay was conducted with a Vybrant MTT Cell Proliferation
254Assay Kit (Life Technologies). Briefly, the cell medium was replaced with 100 µl of
255fresh medium, and then 10 µl of MTT stock solution (12 mM) was added to each well
256and incubated for 4 h. After this period, a 25 µl volume was removed from each well,
257and 50 µL of DMSO was added. After 10 min, the absorbance was measured at 540 nm
258with a spectrophotometer microplate reader (iEMS reader MF; Labsystems).
259Effect of PIM kinase inhibitors on the expression of cell cycle-related genes
260 ZF4 cells were seeded in 24-well plates, and on the next day, they were treated
261for 24 h with the pan-PIM kinase inhibitors SGI-1776 (5 µM), INCB053914 (2 µM),
262AZD1208 (10 µM) or vehicle alone (0.002 % DMSO). Total RNA was isolated (3
263biological replicates/treatment), and qPCRs were conducted to detect the expression of
264the cell cycle-related genes cellular tumour antigen p53 (tp53), cyclin-dependent kinase
265inhibitor 1a (p21) and e3 ubiquitin-protein ligase mdm2 (mdm2). Moreover, we also
266analysed whether the inhibition of Pim kinase activity is compensated for by higher
267gene expression of these proteins. For this, we selected pimr106 as a prototypical gene.
268The primer pairs used are listed in Supplementary Table S1.
269Treatment of ZF4 cells with pan-PIM kinase inhibitors and infection with SVCV
270 ZF4 cells were seeded in 96-well plates, and on the next day, the media was
271removed and was replaced by new media (DMEM + 2 % FBS + P/S) containing 5 µM
272SGI-1776, 2 µM INCB053914, 10 µM AZD1208 or 0.002 % vehicle (DMSO). These
273concentrations were previously reported to inhibit the activity of the PIM kinases in a
274variety of cell lines [40-42]. After incubation for 24 h at 27°C, new treatments were
275added that contained seven 10-fold serial dilutions of SVCV (highest concentration: 3 ×
276107 TCID50/ml) for viral titration, which was performed in triplicate according to the
277Reed and Muench method [23]. Non-infected controls were also included. This
278experiment was conducted five times. In parallel, 24-well plates were also seeded with
279ZF4 cells and treated with the different pan-PIM kinase inhibitors or the vehicle alone.
280After 24 h, new treatments were also added together with the SVCV (3 × 105
281TCID50/ml). At 24 h post-infection (hpi), the media was removed, the cells were washed
282with PBS, total RNA was isolated (4 biological replicates/treatment) and qPCR was
283conducted to detect the SVCV N gene (the primers used are listed in Supplementary
284Table S1).
285 To elucidate if the pan-PIM kinase inhibitors could affect viral entry, ZF4
286seeded onto 96-well plates were infected with seven 10-fold serial dilutions of SVCV
287(highest concentration: 3 × 109 TCID50/ml) in triplicate; after 5 h, the media was
288removed, the wells were washed twice with PBS and then cells were treated with the
289pan-PIM inhibitors or the vehicle alone at the same concentrations mentioned above.
290The viral titer for the different conditions was estimated based on the visualization of
291cytopathic effect (CPE) according to the Reed and Muench method [23]. This
292experiment was replicated three times. As in the previous experiment, 24-well plates
293were also seeded with ZF4, and in this case they were infected with the virus (3 × 107
294TCID50/ml) for 5 h, washed twice with PBS and then treated with the different pan-PIM
295kinases. At 24 h post-infection (hpi), the media was removed, the cells were washed
296with PBS, total RNA was isolated (4 biological replicates/treatment) and qPCR was
297conducted to detect the SVCV N gene.
298In vivo treatment of zebrafish larvae with pan-PIM kinase inhibitors
299 Zebrafish larvae (2 days post-fertilization–dpf) were placed in 6-well plates (10
300larvae per well) in a volume of 6 ml. Larvae from 6 wells were pre-treated with SGI-
3011776 (5 µM), INCB053914 (2 µM), AZD1208 (10 µM) or vehicle alone (0.002 %
302DMSO). After 24 h (3 dpf larvae), half of the larvae from each treatment were infected
303via the duct of Cuvier with 2 nl of an SVCV suspension (5 × 104 TCID50/ml; 10 %
304phenol red), and the other half were inoculated with the same volume of PBS with 10 %
305phenol red, as previously described [39]. For microinjections, we used glass capillaries
306coupled to a micromanipulator (MN-151, Narishige, Japan) and a FemtoJet 4x
307microinjector (Eppendorf, Germany). Mortality was assessed through 6 dpi using three
308biological replicates comprised of 10 larvae each. This experiment was replicated three
309times. In parallel, samples were also taken after 24 h (3 biological replicates, 4-5 larvae
310
311
replicate) to analyse the viral replication in infected larvae by qPCR.
312 Statistical analyses
313 For qPCR experiments, the results are represented graphically as the mean ±
314standard error of the biological replicates. Significant differences were determined with
315the computer software package IBM SPSS Statistics v25 using Student’s t-tests.
316Kaplan-Meier survival curves were analysed with a log-Rank (Mantel-Cox) test.
317Significant differences are displayed as *** (0.0001 < p < 0.001), ** (0.001 < p < 0.01)
318
319
or * (0.01 < p < 0.05).
320Results
321Sequencing and mapping information of the coding RNA
322 A summary of the reads per sample, trimming results, and mapping information
323is included in Table 1. A total of 543.596.316 million reads were obtained from the
324different samples of zebrafish, with an average of 90 million per sample, and over 99 %
325of raw reads passed the quality control. From these high-quality reads, 97.28 %
326successfully mapped to the zebrafish genome, with an average value of 97.27 % per
327sample. Therefore, only 2.72 % of the reads remained unmapped, with an average value
328of 2.73 % per sample.
329Differentially modulated genes, GO enrichment, KEGG pathways and domain
330enrichment analysis
331 When we analysed the expression of the different zebrafish genes in those
332individuals infected with SVCV compared to the uninfected fish, a total of 714 DEGs
333were observed (Supplementary Table S2; Figure 1). A heat map representing the TPM
334values of the DEGs across the different samples showed well-differentiated clusters of
335genes (Figure 1A) one of the clusters corresponded to those genes overexpressed in the
336control samples and another to those overexpressed in infected fish. The three biological
337replicates of each condition clustered together (Figure 1A), indicating a good
338consistency of the results.
339 Whereas 343 DEGs were significantly upregulated after viral infection, a total
340of 371 were inhibited following the viral challenge (Supplementary Table S2; Figure
3411B). These RNA-Seq results were validated by qPCR of 4 genes. The qPCR results for
342the tested genes exhibited the same modulation pattern that was observed in the RNA-
343Seq data (Supplementary Table S3A).
344 For the genes that were downregulated after SVCV infection, GO biological
345process enrichment showed a variety of terms, but a large number were related to the
346synthesis of corticosteroids, muscle contraction/formation, cytoskeleton organization
347and calcium transport (Figure 2A). This was also reflected in the KEGG pathways
348analysis (Figure 2B). Domain enrichment analysis showed the “Kelch-like protein,
349gigaxonin type” and “ATP-gated ion channel P2X4 receptor” as the domain families
350enriched for the downregulated genes (Figure 2C).
351 As expected, GO enrichment analysis of the upregulated genes revealed a high
352representation of biological processes related to immunity, especially to the antiviral
353response (Figure 3A). These significantly enriched immune terms were “response to the
354virus”, “cell chemotaxis”, “neutrophil chemotaxis”, “defence response to virus”,
355“negative regulation of apoptotic process”, “inflammatory response”, “immune
356response” and “innate immune response”. This elevated representation in immune terms
357was also reflected in the KEGG pathways, where all the pathways significantly enriched
358for the genes induced after SVCV were related to the antiviral response: “Cytosolic
359DNA-sensing pathway”, “NOD-like receptor signalling pathway“, “Toll-like receptor
360signalling pathway”, “Cytokine-cytokine receptor interaction”, “Jak-STAT signalling
361pathway” and “Herpes simplex infection” (Figure 3B). Interestingly, when the domain
362enrichment analysis was conducted, only one domain family was represented for the
363overexpressed genes, and it corresponded to “Proto-oncogene serine/threonine-protein
364kinase Pim-1” (Figure 3C).
365A variety of Pim kinases are induced after SVCV infection
366 Because the “Proto-oncogene serine/threonine-protein kinase Pim-1” domain
367was the only domain overrepresented among the genes induced after viral challenge, we
368wanted to analyse in a more detailed way this family of proteins identified in our RNA-
369Seq results. We found a total of 16 Pim kinases upregulated in zebrafish kidneys 24 h
370after SVCV challenge (Table 2). The fold-change values ranged from 3.94 to 89.15. The
371TPM values of the different replicates are represented in Figure 4.
372 To further understand the potential implication of the 16 Pim kinases modulated
373in response to the virus, we first analysed whether they effectively correspond to the
374PIM kinase family. We searched the Pim kinase domain and the characteristic
375adenosine triphosphate (ATP)-binding site in all of these sequences. We conducted an
376alignment of the Pim kinase domain of these 16 Pim kinases, although the domain was
377incomplete for some partial sequences due to genome sequencing ambiguities (Figure
3785A). In general, the Pim kinase domain was relatively well conserved across the
379different zebrafish Pim kinases induced upon SVCV challenge. Indeed, the similarity
380percentage was always above 40 % for the different comparisons (Figure 5B).
381Therefore, we confirmed that these proteins corresponded to the Pim kinase family.
382Pimr106 is induced early after SVSV infection, and its increase is not mediated by viral
383nucleic acids
384 We selected one of the most overexpressed Pim kinases, pimr106 (pimr106, FC
385= 60), to analyse its expression pattern after viral challenge. We analysed its
386transcription in adult zebrafish infected with SVCV or stimulated with Poly I:C for 3, 6
387and 24 h. Poly I:C, as a synthetic analogue of viral dsRNA, was also inoculated to
388determine if viral nucleic acids affected the expression of Pim kinases. The gene
389pimr106 was already overexpressed in SVCV-infected fish at 3 hpi, and its expression
390remained higher than the uninfected control until 24 hpi (Figure 6). Poly I:C did not
391induce significant differences in the expression of pimr106 at the tested sampling points
392(Figure 6), although this compound significantly increased the expression of the
393antiviral genes ifnphi1 and isg15 (Supplementary Figure S1). Based on this, viral
394nucleic acids do not seem to induce Pim kinases during viral infection.
395miRNAs as potential modulators of Pim kinase expression
396 In addition to mRNA, we wanted to test the miRNA profile after infection. Due
397to the presence of only one biological replicate, these results should be carefully
398considered. Nevertheless, we validated 4 different miRNAs in three independent
399biological replicates, and a very comparable expression pattern was observed between
400the RNA-Seq and qPCR results (Supplementary Table S3B). For the control zebrafish,
401more than 27 million raw reads were obtained, and 59.18 % passed the filter parameters
402(Table 3A). For the infected fish, we obtained more than 25 million raw reads, and
40382.46 % passed the filters (Table 3A). Because we annotated our results using the
404mature miRNA database of zebrafish, only 597 and 546 reads from the control and
405infected samples, respectively, were successfully annotated to one of the 355 mature
406miRNAs (Table 3A).
407 We found 47 mature miRNAs modulated (FC >2) in the kidney after SVCV
408infection (Figure 7; Supplementary Table S4); 24 of them were particularly
409overexpressed, whereas 23 were downregulated. By analysing the potential 3’
410untranslated region (3’UTR) targets of these miRNAs, we found that 5 of the modulated
411miRNAs could interact with Pim kinases (Table 3B). Although only one of the
412predicted targets corresponded to a significantly modulated Pim kinase, this opens the
413door to further studies of whether zebrafish Pim kinase expression is regulated by
414miRNAs.
415Assay to determine ZF4 cell viability after treatment with pan-PIM kinase inhibitors
416 To confirm the non-cytotoxic effect of the concentrations of pan-PIM kinase
417inhibitors used in this work, we conducted an MTT assay in ZF4 cells treated for 24 h
418with the three inhibitors. The tested concentrations seemed not to be cytotoxic to ZF4
419cells, and even a slight increase in the formazan precipitation was observed for the cells
420treated with the inhibitors compared to the control cells (Supplementary Figure S2).
421Therefore, the inhibitors were not cytotoxic to ZF4 cells at the tested concentrations.
422PIM kinase inhibitors do not alter the expression of pimr106, but they do affect the
423expression of genes related to the cell cycle
424 We analysed whether the use of pan-PIM kinase inhibitors could affect the
425expression of zebrafish Pim kinases. When we analysed the expression of pimr106 in
426ZF4 cells treated with the inhibitors for 24 h, we did not observe significant differences
427in the expression of this gene (Supplementary Figure S3). This could indicate that the
428use of the inhibitors is not compensated for by increased transcription of the Pim kinase
429genes.
430 Due to the involvement of PIM kinases in the progression of the cell cycle, we
431wanted to confirm the alteration of this process by pan-PIM kinase inhibitors in
432zebrafish cells. To do this, we analysed the expression of three genes directly involved
433in the cell cycle; two of them act as tumour suppressors (tp53 and p21), and another one
434has oncogenic activity (mdm2), similar to that of the PIM kinases. Independent of
435whether their impact on the cell cycle is positive or negative, the three genes were
436inhibited by the three pan-PIM kinase inhibitors (Supplementary Figure S4).
437Pim kinase inhibition reduces the SVCV titer in ZF4 cells
438 We wanted to study the potential effect of the repertoire of zebrafish Pim kinases
439in SVCV infection. For this, we conducted two different assays in ZF4 cells. In one of
440them, we pre-treated the cells with three pan-PIM kinase inhibitors (SGI-1776,
441INCB053914 or AZD1208). Then, after 24 h, we infected the cells with seven 10-fold
442dilutions of SVCV in the presence of the inhibitors. We observed a significant reduction
443in the viral titer in the presence of the three inhibitors, especially SGI-1776 (Figure 8A).
444For this compound, the reduction was 3-log compared to the untreated cells, whereas for
445INCB053914 and AZD1208, the viral titer was reduced by 2-log or more than 2-log,
446respectively (Figure 8A). When the expression of the N gene from SVCV was analysed
447by qPCR at 24 h post-infection, a significant reduction in the viral nucleoprotein gene
448levels was also observed following treatment with the three drugs (Figure 8B).
449 Interestingly, when the cells were infected with serial dilutions of SVCV for 5 h,
450washed and then treated with the inhibitors, these differences in the viral titer almost
451disappeared; differences were less than 1-log, or there was no difference (Figure 8C).
452This was also confirmed by qPCR at 24 h post-infection (Figure 8D). Therefore, it
453seems that Pim kinases mainly mediate SVCV entry.
454Pan-PIM kinase inhibitors protect zebrafish larvae from SVCV infection
455 To better understand the implication of the Pim kinases in the death caused by
456SVCV, we pre-treated 2 dpf larvae with the PIM kinase inhibitors for 24 h, and they
457were infected by microinjection into the duct of Cuvier and returned to the water
458containing the different inhibitors. Kaplan-Meier survival curves showed that the three
459drugs increased the survival of the larvae (Figure 9A). Whereas the untreated larvae
460showed a 33.3 % survival, this percentage increased to 70.4 % with SGI-1776, 55.6 %
461with INCB053914 and 53.6 % with AZD1208. Although the differences in survival
462were significantly different only between the control and SGI-1776 groups, as assessed
463by a log-Rank (Mantel-Cox) test, if we analyse the survival at the end of the experiment
464with a Student’s t-test, the three inhibitors significantly protected larvae from SVCV.
465For the uninfected larvae, a mean survival of 95 % was achieved. qPCR analysis of the
466SVCV N gene at 24 h post-challenge showed a significant reduction in the viral
467
468
detection in the groups treated with the three inhibitors (Figure 9B).
469 Discussion
470 SVCV is a Rhabdovirus predominantly affecting cyprinid fish, and it is a cause
471of death and, consequently, economic losses in the aquaculture industry [43]. Moreover,
472due to the high susceptibility of the model species zebrafish to this virus, the SVCV-
473zebrafish interaction could be a useful tool for studying antiviral mechanisms or
474potential treatments for Rhabdovirus infecting mammals.
475 Some previous publications reported the transcriptome of zebrafish in response
476to SVCV. This is the case for the microarrays conducted for kidney samples [44-46] or
477an RNA-Seq analysis of the brain and spleen [47]. However, these transcriptome studies
478were mainly focused on the typical immune response and on the effect of certain
479mutations or immunostimulants in the response to SVCV. Some publications also
480reported the modulation of non-coding RNAs after SVCV challenge, as seen in long
481non-coding RNAs (lncRNAs) [24] or the miRNA profile, which was analysed in vitro
482using the carp cell line EPC [48].
483 In this work, we conducted RNA-Seq analysis of kidney samples from adult
484zebrafish infected or not infected with SVCV for 24 h. Both the mRNA and miRNA
485profiles were analysed. A total of 714 DEGs were significantly modulated (343
486upregulated and 371 downregulated) following the infection. Whereas the
487downregulated genes were mainly involved in the synthesis of steroid hormones and
488muscle and cytoskeleton organization, the genes overexpressed following virus
489treatment were directly related to the antiviral immune response. Interestingly, GO
490terms related to the negative regulation of apoptosis were also enriched, and these were
491mainly conformed by several Pim kinase proteins. As mentioned in the introduction,
492PIM kinases are inhibitors of apoptosis and are positive regulators of cell cycle
493progression [1]. Moreover, the Pim kinase domain was the only domain significantly
494enriched in the upregulated genes. In addition, four miRNAs affected by the SVCV
495challenge are potential modulators of different members of the PIM kinase family.
496Although further functional studies would help us to determine if the interaction of
497these miRNAs and the 3’UTR of certain zebrafish Pim kinases exist, this observation
498could shed some light on the mechanisms regulating the mRNA levels of PIM kinases.
499 The involvement of PIM kinases in the context of viral infections has hardly
500been studied. To the best of our knowledge, only a few publications have investigated
501the potential role of PIM kinases in the progression of viral diseases. The first
502publication reporting the pro-viral effect of PIM kinases was published by Rainio et al.
503[7], and it was based on the role that PIM kinases played in the ability of Epstein-Barr
504virus to immortalize B-cells and predispose them to malignant growth. PIM-1 and PIM-
5053 also induce reactivation of a herpes virus, Kaposi’s sarcoma herpesvirus (KSHV),
506from its latency due to the phosphorylation of the KSHV latency-associated nuclear
507antigen (LANA) on specific serine residues [8]. After that, another study revealed that
508the inhibition of PIM-1 reduced viral replication in primary bronchial epithelial cells,
509and this was attributed to enhanced apoptosis upon viral infection, limiting viral
510replication and spread [9]. In the same year, Park et al. [10] found that the hepatitis C
511virus (HCV) nonstructural 5A protein interacts with PIM kinases and stabilizes them,
512and PIM kinases regulate HCV entry via unknown mechanisms without affecting the
513other steps of the HCV life cycle. Finally, it has been shown that PIM kinases
514phosphorylate the human immunodeficiency virus (HIV) protein Vpx, which in turn
515promotes the ubiquitin-mediated proteolysis of Sterile alpha motif and histidine-
516aspartate domain-containing protein 1 (SAMHD1), an inhibitor of the transcription of
517several lentiviruses, including HIV [11]. Therefore, inhibition of the PIM kinases by
518treatment with the inhibitor AZD1208 allowed increased SAMHD1 activity and, as a
519consequence, decreased lentivirus replication [11].
520 Although the potential mode of action for PIM kinases in favouring viral
521progression varies enormously among the different publications, all of them reported a
522beneficial effect of blocking the PIM kinases to reduce viral progression. For that
523reason, pan-PIM kinase inhibitors are promising drugs not only for cancer therapy but
524also as new treatments against certain viral infections. Many of these inhibitors are PIM
525kinase ATP-competitive inhibitors. Because the Pim kinases induced in zebrafish
526conserved the characteristic ATP-binding site at the beginning of the PIM kinase
527domain, we tested its effectiveness in zebrafish cells. This is the first time that PIM
528kinase inhibitors have been used in zebrafish. Due to the existence of more than 300
529Pim kinases in this species, we first confirmed that SGI-1776, INCB053914 and
530AZD1208 were not cytotoxic to ZF4 cells at the concentrations used in this work, and
531we showed that they were able to downregulate the expression of three pivotal genes
532involved in the cell cycle (p53, p21 and mdm2). The MDM2/p53/p21 axis is a core
533pathway controlling the cell cycle [49], and it is known that mammalian PIM kinases
534interact with this axis [50-52]. Although PIM kinases do not directly affect the
535expression of these genes, modulations in their mRNA levels are indicative of
536alterations in the cell cycle. Therefore, we can assume that pan-PIM kinase inhibitors
537effectively affect the functionality of zebrafish Pim kinases.
538 Based on the high number of Pim kinases induced in zebrafish after SVCV
539infection, we wanted to evaluate whether this family of proteins in this model organism
540is also involved in SVCV infectivity. We first tested the effect of the pan-PIM kinase
541inhibitors in vitro using a zebrafish fibroblast cell line, ZF4. When the cells were pre-
542incubated for 24 h with the inhibitors and then infected with SVCV in the presence of
543the inhibitors, we observed a large reduction in the viral titer and viral replication in
544these cells. The results showed a pro-viral effect of the PIM kinases and an antiviral
545activity of the pan-PIM kinase inhibitors. Interestingly, when the cells were pre-
546incubated with the virus for 5 h and then treated with the inhibitors, these differences
547were dramatically reduced and even abrogated, which is consistent with an effect of the
548PIM kinases in the SVCV entry and is in agreement what was observed with HCV [10].
549 Due to the potential use of these inhibitors in prophylaxis and/or treatment of
550viral infections, we also wanted to analyse their effect in vivo using zebrafish larvae.
551Larvae were pre-treated for 24 h with SGI-1776, INCB053914 or AZD1208 diluted in
552the water, and then they were microinjected with SVCV and returned to the water
553containing the inhibitors. These treated larvae showed an increase in survival after
554infection compared to the untreated and infected larvae. Moreover, larvae treated with
555the drugs showed significantly lower SVCV detection. As observed in the in vitro
556experiments, SGI-1776 was the most protective pan-PIM kinase inhibitor. Therefore,
557although larvae were microinjected into the duct of Cuvier, these results could indicate
558that PIM kinase inhibitors penetrate into the larvae and avoid the entry of the SVCV
559into the host cells for efficient replication. However, due to the effects that PIM kinases
560can exert on mammalian immune cells, we cannot rule out the activation of additional
561antiviral mechanisms in the whole organism. Nevertheless, the activity of the PIM
562kinases on immune cells was mainly investigated in T-lymphocytes [53-55], which are
563absent in zebrafish larvae [56].
564 The different bioactivity of the inhibitors observed both in vitro and in vivo cold
565be conditioned by the concentrations used, but also by the different inhibition constant
566(Ki) values. The Ki is the concentration of an inhibitor that gives half maximal rate of
567inhibition and therefore, it is an indicative of how potent an inhibitor is. Indeed, pan-
568PIM kinase inhibitors possess different Ki values against the three PIM isoforms
569described in mammals [57]. In zebrafish, this could be much more complex due to the
570expansion of this gene family.
571 In conclusion, in this work, we conducted RNA-Seq analysis of kidneys from
572adult zebrafish that were i.p. infected or not with SVCV for 24 h. We observed a high
573induction of typical antiviral immune genes (type I interferon-related genes,
574chemokines, pro-inflammatory cytokines, etc.) However, one of the observations that
575drawn our attention was the high representation of Pim kinases overexpressed during
576SVCV infection. In vitro and in vivo assays with three different pan-PIM kinase
577inhibitors allowed us to corroborate previous observations with mammalian viruses [7,
5789-11], showing that Pim kinase activity is beneficial for SVCV. Moreover, in the
579particular case of this virus, the zebrafish Pim kinases seem to facilitate the entry of the
580SVCV into the cells. For that reason, PIM kinase inhibitors deserve certain attention due
581to their antiviral effect, and their use could help to control the spread of different viral
582diseases. However, future investigations will be needed to test the potential pernicious
583collateral effects of these drugs.
584Acknowledgements
585 This work was funded by the BIO2017-82851-C3-1R project of the Spanish
586Ministerio de Economía y Competitividad and the IN607B 2019/01 from Consellería de
587Economía, Emprego e Industria (GAIN), Xunta de Galicia. Patricia Pereiro wishes to
588thank the Axencia Galega de Innovación (GAIN, Xunta de Galicia) for her postdoctoral
589contract (IN606B-2018/010). Margarita Álvarez-Rodríguez was the recipient of an FPU
590fellowship from the Ministerio de Educación (FPU014/05517).
591References
592[1] M. Narlik-Grassow, C. Blanco-Aparicio, A. Carnero, The PIM family of
593serine/threonine kinases in cancer, Med. Res. Rev. 34 (2014) 136–159.
594[2] R. Swords, K. Kelly, J. Carew, S. Nawrocki, D. Mahalingam, J. Sarantopoulos, D.
595Bearss, F. Giles, The Pim kinases: new targets for drug development, Curr. Drug
596Targets. 12 (2011) 2059–2066.
597[3] N.A. Warfel, AS Kraft, PIM kinase (and Akt) biology and signaling in tumors,
598Pharmacol. Ther. 151 (2015) 41-49.
599[4] G.G. Jinesh, S. Mokkapati, K. Zhu, E.E. Morales, Pim kinase isoforms: devils
600defending cancer cells from therapeutic and immune attacks, Apoptosis. 21 (2016)
6011203–1213.
602[5] K.C. Qian, J. Studts, L. Wang, K. Barringer, A. Kronkaitis, C. Peng, A. Baptiste, R.
603LaFrance, S. Mische, B. Farmer, Expression, purification, crystallization and
604preliminary crystallographic analysis of human Pim-1 kinase, Acta Crystallogr. Sect F:
605Struct. Biol. Cryst. Commun.61 (2005) 96–99.
606[6] H. Mikkers, M. Nawijn, J. Allen, C. Brouwers, E. Verhoeven, J. Jonkers, A. Berns,
607Mice deficient for all PIM kinases display reduced body size and impaired responses to
608hematopoietic growth factors, Mol. Cell. Biol. 24 (2004) 6104–6115.
609[7] E.M. Rainio, H. Ahlfors, K.L. Carter, M. Ruuska, S. Matikainen, E. Kieff, P.J.
610Koskinen, Pim kinases are upregulated during Epstein-Barr virus infection and enhance
611EBNA2 activity, Virology. 15 (2005) 201–206.
612[8] F. Cheng, M. Weidner-Glunde, M. Varjosalo, E.M. Rainio, A. Lehtonen, T.F.
613Schulz, P.J. Koskinen, J. Taipale, P.M. Ojala, KSHV reactivation from latency requires
614Pim-1 and Pim-3 kinases to inactivate the latency-associated nuclear antigen LANA,
615PLoS Pathog. 5 (2009) e1000324.
616[9] M. de Vries, NP Smithers, PH Howarth, MC Nawijn, DE Davies, Inhibition of Pim1
617kinase reduces viral replication in primary bronchial epithelial cells, Eur. Respir. J. 45
618(2015) 1745–1748.
619[10] C. Park, S. Min, E.M. Park, Y.S. Lim, S. Kang, T. Suzuki, E.C. Shin, S.B. Hwang,
620Pim Kinase Interacts with nonstructural 5A protein and regulates hepatitis c virus entry,
621J. Virol. 89 (2015) 10073–10086.
622[11] K. Miyakawa, S. Matsunaga, M. Yokoyama, M. Nomaguchi, Y. Kimura, M. Nishi,
623H. Kimura, H. Sato, H. Hirano, T. Tamura, H. Akari, T. Miura, A. Adachi, T. Sawasaki,
624N. Yamamoto, A. Ryo, PIM kinases facilitate lentiviral evasion from SAMHD1
625restriction via Vpx phosphorylation, Nat. Commun. 10 (2019) 1844.
626[12] R. Rakshambikai, N. Srinivasan, R.A. Gadkari, Repertoire of protein kinases
627encoded in the genome of zebrafish shows remarkably large population of PIM kinases,
628J. Bioinform. Comput. Biol. 12 (2014) 1350014.
629[13] J. Wittbrodt, A. Meyer, M. Schartl, More genes in fish?, BioEssays 20 (1998) 511–
630515.
631[14] I.G.Woods, P.D. Kelly, F. Chu, P. Ngo-Hazelett, Y.L. Yan, H. Huang, J.H.
632Postlethwait, W.S. Talbot, A comparative map of the zebrafish genome, Genome Res.
63310 (2000) 1903–1914.
634[15] M.C. Gomes, S. Mostowy, The case for modeling human infection in zebrafish,
635Trends Microbiol. In Press (2019). https://doi.org/10.1016/j.tim.2019.08.005
636[16] G.E. Sanders, W.N. Batts, J.R. Winton, Susceptibility of zebrafish (Danio rerio) to
637a model pathogen, spring viremia of carp virus, Comp. Med. 53 (2003) 514–521.
638[17] P.J. Walker, A. Benmansour, R. Dietzgen, R.X. Fang, A.O. Jackson, G. Kurath,
639J.C. Leong, S. Nadin-Davies, R.B. Tesh RB, N. Tordo, Family Rhabdoviridae, in:
640M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes,
641S.M. Lemon, J. Maniloff, M.A. Mayo, D.J. McGeoch, C.R. Pringle, R.B. Wickner
642(Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Seventh Report
643of the International Committee on Taxonomy of Viruses, Academic Press, San Diego,
6442000, pp. 563–583.
645[18] M.K. Purcell, K.J. Laing, J.R. Winton, Immunity to fish rhabdoviruses, Viruses 4
646(2012) 140–166.
647[19] P. Dixon, R. Paley, R. Alegria-Moran, B. Oidtmann, Epidemiological
648characteristics of infectious hematopoietic necrosis virus (IHNV): a review, Vet. Res.
64947 (2016) 63.
650[20] P. Pereiro, A. Figueras, B. Novoa, Turbot (Scophthalmus maximus) vs. VHSV
651(Viral Hemorrhagic Septicemia Virus): A Review, Front. Physiol. 7 (2016) 192.
652[21] M. Westerfield, The zebrafish book: A guide for the laboratory use of zebrafish,
653University of Oregon Press, 2000.
654[22] C. Nusslein-Volhard, R. Dahm, Zebrafish: a practical approach, Oxford University
655Press, 2002.
656[23] L.J. Reed, H. Muench, A simple method of estimating fifty percent endpoints, Am.
657J. Epidemiol. 27 (1938) 493–497.
658[24] V. Valenzuela-Muñoz, P. Pereiro, M. Álvarez-Rodríguez, C. Gallardo-Escárate, A.
659Figueras, B. Novoa, Comparative modulation of lncRNAs in wild-type and rag1-
660heterozygous mutant zebrafish exposed to immune challenge with spring viraemia of
661carp virus (SVCV), Sci. Rep. 9 (2019) 14174.
662[25] D.W. Huang, B.T. Sherman, R.A. Lempicki, Systematic and integrative analysis of
663large gene lists using DAVID Bioinformatics Resources, Nature Protoc. 4 (2019a) 44–
66457.
665[26] D.W. Huang, B.T. Sherman, R.A. Lempicki, Bioinformatics enrichment tools:
666paths toward the comprehensive functional analysis of large gene lists, Nucleic Acids
667Res. 37 (2009b) 1–13.
668[27] C.H. Wu, L.S.L. Yeh, H. Huang, L. Arminski, J. Castro-Alvear, Y. Chen, Z. Hu, P.
669Kourtesis, R.S. Ledley, B.E. Suzek, C.R. Vinayaka, J. Zhang, W.C. Barker, The
670Protein Information Resource, Nucleic Acids Res. 31(2003) 345–347.
671[28] D.R. Zerbino, P. Achuthan, W. Akanni, M.R. Amode, D. Barrell, J. Bhai, K. Billis,
672C. Cummins, A. Gall, C. García Girón, L. Gil, L. Gordon, L. Haggerty, E. Haskell, T.
673Hourlier, O.G. Izuogu, S.H. Janacek, T. Juettemann, J. Kiang To, M.R. Laird, I.
674Lavidas, Z. Liu, J.E. Loveland, T. Maurel, W. McLaren, B. Moore, J. Mudge, D.N.
675Murphy, V. Newman, M. Nuhn, D. Ogeh, C. Kee Ong, A. Parker, M. Patricio, H. Singh
676Riat, H. Schuilenburg, D. Sheppard, H. Sparrow, K. Taylor, A. Thormann, A. Vullo, B.
677Walts, A. Zadissa, A. Frankish, S.E. Hunt, M. Kostadima, N. Langridge, F.J. Martin, M.
678Muffato, E. Perry, M. Ruffier, D.M. Staines, S.J. Trevanion, B.L. Aken, F.
679Cunningham, A.Yates, P. Flicek, Ensembl 2018, Nucleic Acids Res. 46 (2018) D754–
680D761.
681[29] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the
682sensitivity of progressive multiple sequence alignments through sequence weighting,
683position specific gap penalties and weight matrix choice, Nucleic Acid Res. 22 (1994)
6844673–4680.
685[30] J.J. Campanella, L. Bitincka, J. Smalley, MatGAT: an application that generates
686similarity/identity matrices using protein or DNA sequences, BMC Bioinform. 4 (2003),
68729.
688[31] M. Chekulaeva, W. Filipowicz, Mechanisms of miRNA-mediated post-
689transcriptional regulation in animal cells, Curr. Opin. Cell Biol. 21 (2009) 452–460.
690[32] M. Hafner, M.Jr. Ascano, T. Tuschl, New insights in the mechanism of
691microRNA-mediated target repression, Nat. Struct. Mol. Biol. 18 (2011) 1181–1182.
692[33] S. Griffiths-Jones, R.J. Grocock, S. van Dongen, A. Bateman, A.J. Enright,
693miRBase: microRNA sequences, targets and gene nomenclature, Nucleic Acids Res. 34
694(2006) D140–D144.
695[34] D.M. Garcia, D. Baek, C. Shin, G.W. Bell, A. Grimson, D.P. Bartel, Weak seed-
696pairing stability and high target-site abundance decrease the proficiency of lsy-6 and
697other microRNAs, Nat. Struct. Mol. Biol. 18 (2011) 1139–1146.
698[35] C.E. Vejnar, M. Blum, E.M. Zdobnov, miRmap web: comprehensive microRNA
699target prediction online, Nucleic Acids Res. 41 (2013) W165–W168.
700[36] S. Rozen, H.J. Skaletsky, Primer3 on the WWW for general users and for biologist
701programmers, in: S. Krawetz, S. Misener (Eds.), Bioinformatics Methods and Protocols:
702Methods in Molecular Biology, Humana Press, Totowa, 2000, pp. 365–386.
703[37] M.W. Pfaffl, A new mathematical model for relative quantification in real-time
704RT-PCR, Nucleic Acids Res. 29 (2001) e45.
705[38] P. Pereiro, M. Varela, P. Diaz-Rosales, A. Romero, S. Dios, A. Figueras, B.
706Novoa, Zebrafish Nk-lysins: First insights about their cellular and functional
707diversification, Dev. Comp. Immunol. 51 (2015) 148–159.
708[39] P. Pereiro, G. Forn-Cuni, S. Dios, J. Coll, A. Figueras, B. Novoa, Interferon-
709independent antiviral activity of 25-hydroxycholesterol in a teleost fish, Antiviral Res.
710145 (2017) 146–159.
711[40] J. Li, M. Favata, J.A. Kelley, E. Caulder, B. Thomas, X. Wen, R.B. Sparks, A.
712Arvanitis, J.D. Rogers, A.P. Combs, K. Vaddi, K.A. Solomon, P.A. Scherle, R. Newton,
713J.S. Fridman, INCB16562, a JAK1/2 selective inhibitor, is efficacious against multiple
714myeloma cells and reverses the protective effects of cytokine and stromal cell support,
715Neoplasia. 12 (2010) 28–38.
716[41] Q. Yang, L.S. Chen, S.S. Neelapu, V. Gandhi, Combination of Pim kinase inhibitor
717SGI-1776 and bendamustine in B-cell lymphoma, Clin. Lymphoma Myeloma Leuk. 13
718(2013) S355–S362.
719[42] S. Kreuz, K.B. Holmes, R.M. Tooze, P.F. Lefevre, Loss of PIM2 enhances the anti-
720proliferative effect of the pan-PIM kinase inhibitor AZD1208 in non-Hodgkin
721lymphomas, Mol. Cancer. 14 (2015) 205.
722[43] W. Ahne, H.V. Bjorklund, S. Essbauer, N. Fijan, G. Kurath, J.R. Winton, Spring
723viraemia of carp (SVC), Dis. Aquat. Organ. 52 (2002) 261–272.
724[44] P. Encinas, P. Garcia-Valtanen, B. Chinchilla, E. Gomez-Casado, A. Estepa, J.
725Coll, Identification of multipath genes differentially expressed in pathway-targeted
726microarrays in zebrafish infected and surviving spring viremia carp virus (SVCV)
727suggest preventive drug candidates, PLoS One. 8 (2013) e73553.
728[45] P. García-Valtanen, A. Martínez-López, A. López-Muñoz, M. Bello-Perez, R.M.
729Medina-Gali, M.D. Ortega-Villaizán, M. Varela, A. Figueras, V. Mulero, B. Novoa, A.
730Estepa, J. Coll, Zebra fish lacking adaptive immunity acquire an antiviral alert state
731characterized by upregulated gene expression of apoptosis, multigene families, and
732interferon-related genes, Front. Immunol. 8 (2017) 121.
733[46] M. Álvarez-Rodríguez, P. Pereiro, F.E. Reyes-López, L. Tort, A. Figueras, B.
734Novoa, Analysis of the long-lived responses induced by immunostimulants and their
735effects on a viral infection in zebrafish (Danio rerio), Front. Immunol. 9 (2018) 1575.
736[47] Y. Wang, H. Zhang, Y. Lu, F. Wang, L. Liu, J. Liu, X. Liu, Comparative
737transcriptome analysis of zebrafish (Danio rerio) brain and spleen infected with spring
738viremia of carp virus (SVCV), Fish Shellfish Immunol. 69 (2017) 35–45.
739[48] S. Wu, L. Liu, A. Zohaib, L. Lin, J. Yuan, M. Wang, X. Liu, MicroRNA profile
740analysis of Epithelioma papulosum cyprini cell line before and after SVCV infection,
741Dev. Comp. Immunol. 48 (2015) 124–128.
742[49] S. Nag, J. Qin, K.S. Srivenugopal, M. Wang, R. Zhang, The MDM2-p53 pathway
743revisited, J. Biomed. Res. 27 (2013) 254–271.
744[50] C. Hogan, C. Hutchison, L. Marcar, D. Milne, M. Saville, J. Goodlad, N.
745Kernohan, D. Meek, Elevated levels of oncogenic protein kinase Pim-1 induce the p53
746pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma,
747J. Biol. Chem. 283 (2008) 18012–18023.
748[51] Z. Wang, N. Bhattacharya, P.F. Mixter, W. Wei, J. Sedivy, N.S. Magnuson,
749Phosphorylation of the cell cycle inhibitor p21Cip1/WAF1 by Pim-1 kinase, Biochim.
750Biophys. Acta. 1593 (2002) 45–55.
751[52] Z. Wang, Y. Zhang, J.J. Gu, C. Davitt, R. Reeves, N.S. Magnuson, Pim-2
752phosphorylation of p21(Cip1/WAF1) enhances its stability and inhibits cell proliferation
753in HCT116 cells, Int. J. Biochem. Cell Biol. 42 (2010) 1030–1038.
754[53] X.P. Chen, J.A. Losman, S. Cowan, E. Donahue, S. Fay, B.Q. Vuong, M.C.
755Nawijn, D. Capece, V.L. Cohan, P. Rothman, Pim serine/threonine kinases regulate the
756stability of Socs-1 protein, Proc. Natl. Acad. Sci. USA. 99 (2002) 2175–2180.
757[54] C.J. Fox, P.S. Hammerman, C.B. Thompson, The Pim kinases control rapamycin-
758resistant T cell survival and activation, J. Exp. Med. 201(2005) 259–266.
759[55] G. Deng, Y. Nagai, Y. Xiao, Z. Li, S. Dai, T. Ohtani, A. Banham, B. Li, S.L. Wu,
760W. Hancock, A. Samanta, H. Zhang, M.I. Greene, Pim-2 kinase influences regulatory T
761cell function and stability by mediating Foxp3 protein N-terminal phosphorylation, J.
762Biol. Chem. 290 (2005) 20211–20220.
763[56] C.E. Willett, A. Cortes, A. Zuasti, A.G. Zapata, Early hematopoiesis and
764developing lymphoid organs in the zebrafish, Dev. Dyn. 214 (1999) 323–336.
765[57] M.T. Burger, W. Han, J. Lan, G. Nishiguchi, C. Bellamacina, M. Lindval, G.
766Atallah, Y. Ding, M. Mathur, C. McBride, E.L. Beans, K. Muller, V. Tamez, Y. Zhang,
767K. Huh, P. Feucht, T. Zavorotinskaya, Y. Dai, J. Holash, J. Castillo, J. Langowski, Y.
768Wang, M.Y. Chen, P.D. Garcia, Structure guided optimization, in vitro activity, and in
769vivo activity of pan-PIM kinase inhibitors, ACS Med. Chem. Lett. 4 (2013) 1193–1197.
770Tables
771Table 1. Summary of the mRNA Illumina sequencing, trimming and genome
772mapping.
mRNA
Sample Raw reads Reads after trim Mapped to genome (%) Unmapped (%)
WT C1 94,617,822 93,976,916 97.21 2.79
WT C2 91,411,306 90,745,708 97.41 2.59
WT C3 89,359,334 88,664,158 97.39 2.61
WT-SVCV 1 90,571,676 90,011,432 97.17 2.83
WT-SVCV 2 95,483,786 94,975,756 97.60 2.40
WT-SVCV 3 82,152,392 81,664,334 96.84 3.16
Average value 90,599,386 90,006,384 97.27 2.73
Total value 543,596,316 540,038,304 97.28 2.72
773
774Table 2. Summary of the Pim kinases significantly induced in zebrafish kidney at
77524 h after SVCV infection.
ENSEMBL ID Gene Symbol Description FC p-value
ENSDARG00000055056 ENSDARG00000094651 ENSDARG00000104164 ENSDARG00000100992 ENSDARG00000069851 ENSDARG00000037246 ENSDARG00000070015 ENSDARG00000052677 ENSDARG00000100216 ENSDARG00000092339 ENSDARG00000095386 ENSDARG00000103910 ENSDARG00000057265 ENSDARG00000102396 ENSDARG00000093631 ENSDARG00000098638 si:ch73-129a22.11_3 pimr106 CABZ01028711.1 pimr101_2 pimr152_8
pimr212 pimr173
si:ch211-138g9.2 pimr117
pimr61 pimr179 pimr202_2 pimr52 pimr66 pimr20 pimr65 PREDICTED: Serine/threonine-protein kinase pim-1-like Pim proto-oncogene, serine/threonine kinase, related 106 PREDICTED: Serine/threonine-protein kinase pim-3-like Pim proto-oncogene, serine/threonine kinase, related 101 Pim proto-oncogene, serine/threonine kinase, related 152 Pim proto-oncogene, serine/threonine kinase, related 212 Pim proto-oncogene, serine/threonine kinase, related 173 PREDICTED: Serine/threonine-protein kinase pim-3-like Pim proto-oncogene, serine/threonine kinase, related 117 Pim proto-oncogene, serine/threonine kinase, related 61 Pim proto-oncogene, serine/threonine kinase, related 179 Pim proto-oncogene, serine/threonine kinase, related 202 Pim proto-oncogene, serine/threonine kinase, related 52 Pim proto-oncogene, serine/threonine kinase, related 66 Pim proto-oncogene, serine/threonine kinase, related 20 Pim proto-oncogene, serine/threonine kinase, related 65 89.15
60.36
34.95
17.56
15.31
13.25
10.23
10.00
7.91
6.97
6.83
5.94
5.40
5.00
4.72
3.94 0.01192 0.019852 0.045781 0.005791 0.012636 0.012636 0.031065 0.002023 0.017364 0.012071 0.009817 0.036673 0.022613 0.006272 0.037309 0.011634
776
777Table 3. Summary of the small RNA sequencing and miRNA annotation to the
778mature miRNAs present in miRBase (A), and the representation of those miRNAs
779that were modulated by the infection with SVCV and have a Pim kinase as a
780
781
potential target (B).
A
TargetScanFish
B miRNA Target score mirMAP score
dre-miR-2188 ENSDARG00000059001 -0.50 94.77
dre-miR-199 ENSDARG00000059001 -0.65 -
dre-miR-210-5p ENSDARG00000055129 - 95.94
dre-miR-124 ENSDARG00000055129 - 90.99
dre-miR-181b ENSDARG00000070015 -0.54 98.59
782
783Figure Legends
784Figure 1. Differentially expressed genes in zebrafish kidneys after infection with
785SVCV (FC >2, p-value < 0.05). A) Heat map representing the expression level of those
786genes differentially expressed and hierarchical clustering of the different samples
787constructed based on TPM values. Two well-differentiated clusters are observed: one
788for those genes inhibited after viral challenge and another for the genes overexpressed
789after infection. B) Stacked column chart reflecting the distribution (up- or
790downregulated) and intensity (FC value) of regulated genes.
791Figure 2. GO enrichment of biological processes (A), KEGG pathways (B) and
792domain enrichment (C) analyses of the downregulated genes in SVCV-infected
793fish.
794Figure 3. GO enrichment of biological processes (A), KEGG pathways (B) and
795domain enrichment (C) analyses of the upregulated genes in SVCV-infected fish.
796Figure 4. Representation of the TPM values of the zebrafish Pim kinases
797overexpressed in kidney after SVCV infection. Three biological replicates per
798condition are shown.
799Figure 5. Analysis of the PIM kinase domain of the zebrafish Pim kinases induced
800by SVCV. A) Alignment of the PIM kinase domain of 16 Pim kinases. With the
801exception of two Pim kinases where the predicted domain was incomplete in the
802genome, all the Pim kinases contained a typical ATP binding site (highlighted in pink).
803B) Identity/similarity matrix of the zebrafish Pim kinase domains.
804Figure 6. Expression of the zebrafish pimr106 (ENSDARG00000094651) gene in
805kidney samples at 3, 6 and 24 h post-challenge with SVCV or Poly I:C. Whereas
806SVCV increased the expression of this gene from the first few hours post-infection,
807Poly I:C does not significantly affect the expression of pimr106.
808Figure 7. Representation of mature zebrafish miRNAs modulated (FC > 2) by
809SVCV. In green colour are represented the overexpressed miRNAs and in red colour the
810down-regulated miRNAs.
811Figure 8. Pan-PIM kinase inhibitors reduce the entry of SVCV into ZF4 cells. A)
812ZF4 cells were pre-treated with the different PIM kinase inhibitors and were then
813infected with 1:10 serial dilutions of SVCV in the presence of the inhibitors. Changes in
814the CPE were checked every day, and the viral titer was calculated following the Reed
815and Muench method. A 3-log reduction in the viral titer was detected for SGI-1776, 2-
816log for INCB053914 and more than 2-log for AZD1208. B) When ZF4 cells were
817infected in the presence of the inhibitors, there was a significant reduction in the qPCR
818detection of the SVCV nucleoprotein (N) gene. C) ZF4 cells were pre-incubated for 5 h
819with 1:10 serial dilutions of SVCV, washed twice with PBS and then treated with the
820pan-PIM kinase inhibitors. Viral titer was also calculated according to the Reed and
821Muench method. The antiviral effects mediated by the inhibitors almost disappeared
822when the cells were first infected and were then treated with the drugs, indicating that
823Pim kinases are probably involved in the entry of the virus into the cells. A lower titer
824was obtained for SGI-1776, but the difference compared to the control was less than 1-
825log. D) qPCR detection of the SVCV N gene was performed after ZF4 cells were pre-
826incubated with the virus for 5 h and then treated with the inhibitors and sampled after 24
827h. No significant differences were observed between the control cells and those treated
828with the different PIM kinase inhibitors.
829Figure 9. Pan-PIM kinase inhibitors protect zebrafish larvae against SVCV
830infection. A) Kaplan-Meier survival curves are shown for zebrafish larvae infected with
831SVCV in the presence of the different inhibitors. Groups of 2 dpf larvae were pre-
832treated with the inhibitors or the vehicle alone (0.002 % DMSO) diluted in the water.
833After 24 h, larvae were microinjected via the duct of Cuvier with an SVCV suspension.
834Control larvae were inoculated with the same volume of viral medium diluted in PBS.
835Then, larvae were returned to the water containing the PIM kinase inhibitors. Mortality
836was registered during the next 6 dpi. The three drugs reduced the mortality caused by
837SVCV, but statistically significant differences were only obtained for SGI-1776. The
838survival of the uninfected larvae was: 93 % (control), 97 % (SGI-1776), 97 %
839(AZD1208) and 93 % (INCB053914). B) Detection of the SVCV N gene was
840performed after 24 h in zebrafish larvae treated with the different inhibitors or with the
841vehicle alone. A lower detection of the viral gene was observed in the zebrafish larvae
842
843
infected in the presence of the inhibitors.
Highlights:
- Numerous genes encoding for Pim kinases were induced in zebrafish after SVCV challenge
- Zebrafish Pim kinases seem to be involved in the SVCV entry step
- Pan-PIM kinase inhibitors reduce SVCV entry into ZF4 cells
- Pan-PIM kinase inhibitors protect zebrafish larvae from SVCV