The Study of Myo-Inositol's Anxiolytic Activity on Zebrafish (Danio rerio).

INTRODUCTION: Myo-inositol (MI) is the most abundant inositol found in nature. To date MI supplementation is reported to be effective in the treatment of polycystic ovary syndrome, it is also suggested to alleviate the symptoms of diabetes and neurodegenerative disorders, but to date no statistically significant effects of inositol on depressive and anxiety symptoms were proven. In the study of anxiolytic effects in zebrafish, we often use the thigmotaxis index measuring the ratio of the amount of time the animal spends near the walls compared to the entire arena. AIM: The objective of this paper was to examine the effect of MI on zebrafish embryos' locomotor activity, as well as its potential anxiolytic activity in zebrafish larvae. MATERIAL AND METHODS: In the first part of the experiment, the embryos were incubated with 5, 10, 20, and 40 mg/mL MI. 1-day post fertilization, embryo mobility was evaluated and burst activity was calculated. In the next part of the study, the behavior of 5-day-old larvae was tested. RESULTS: Tests on embryo movement showed an increase in burst activity in the MI group at concentrations of 40 mg/mL (p < 0.0001) and a slight decrease in the group at concentrations of 10 mg/mL (p < 0.05). MI in the light/dark challenge had no impact on the thigmotaxis index. CONCLUSIONS: MI was shown to not affect stress reduction in zebrafish larvae. Further research on the potential of MI and other stereoisomers is needed.

Simple Coumarins from Peucedanum luxurians Fruits: Evaluation of Anxiolytic Activity and Influence on Gene Expression Related to Anxiety in Zebrafish Model.

Anxiety is one of the most common central nervous system disorders, affecting at least one-quarter of the worldwide population. The medications routinely used for the treatment of anxiety (mainly benzodiazepines) are a cause of addiction and are characterized by many undesirable side effects. Thus, there is an important and urgent need for screening and finding novel drug candidates that can be used in the prevention or treatment of anxiety. Simple coumarins usually do not show side effects, or these effects are much lower than in the case of synthetic drugs acting on the central nervous system (CNS). This study aimed to evaluate the anxiolytic activity of three simple coumarins from Peucedanum luxurians Tamamsch, namely officinalin, stenocarpin isobutyrate, and officinalin isobutyrate, in a 5 dpf larval zebrafish model. Moreover, the influence of the tested coumarins on the expression of genes involved in the neural activity (c-fos, bdnf) or dopaminergic (th1), serotoninergic (htr1Aa, htr1b, htr2b), GABA-ergic (gabarapa, gabarapb), enkephalinergic (penka, penkb), and galaninergic (galn) neurotransmission was assessed by quantitative PCR. All tested coumarins showed significant anxiolytic activity, with officinalin as the most potent compound. The presence of a free hydroxyl group at position C-7 and the lack of methoxy moiety at position C-8 might be key structural features responsible for the observed effects. In addition, officinalin and its isobutyrate upregulated the expression of genes involved in neurotransmission and decreased the expression of genes connected with neural activity. Therefore, the coumarins from P. luxurians might be considered as promising drug candidates for the therapy of anxiety and related disorders.

Antiepileptic Properties of Scyllo-Inositol on Pentylenetetrazol-Induced Seizures.

Epilepsy, with about 70 million affected people worldwide, is one of the biggest challenges of medicine today. It is estimated that about one-third of epileptic patients receive inadequate treatment. Inositols have proved effective in many disorders; hence, in the current study, we tested potential antiepileptic properties of scyllo-inositol (SCI)-one of the most common commercially available inositols-in zebrafish larvae with pentylenetetrazol-induced seizures. First, we studied the general effect of SCI on zebrafish motility, and then we tested SCI antiepileptic properties over short (1 h) and long (120 h) exposure protocols. Our results demonstrated that SCI alone does not reduce zebrafish motility regardless of the dose. We also observed that short-term exposure to SCI groups reduced PTZ-treated larva motility compared to controls (p < 0.05). In contrast, prolonged exposure did not produce similar results, likely due to the insufficient concentration of SCI given. Our results highlight the potential of SCI use in epilepsy treatment and warrant further clinical studies with inositols as potential seizure-reducing drugs.

Evaluation of the Antiseizure Activity of Endemic Plant Halfordia kendack Guillaumin and Its Main Constituent, Halfordin, on a Zebrafish Pentylenetetrazole (PTZ)-Induced Seizure Model.

Epilepsy is a neurological disease that burdens over 50 million people worldwide. Despite the considerable number of available antiseizure medications, it is estimated that around 30% of patients still do not respond to available treatment. Herbal medicines represent a promising source of new antiseizure drugs. This study aimed to identify new drug lead candidates with antiseizure activity from endemic plants of New Caledonia. The crude methanolic leaf extract of Halfordia kendack Guillaumin (Rutaceae) significantly decreased (75 µg/mL and 100 µg/mL) seizure-like behaviour compared to sodium valproate in a zebrafish pentylenetetrazole (PTZ)-induced acute seizure model. The main coumarin compound, halfordin, was subsequently isolated by liquid-liquid chromatography and subjected to locomotor, local field potential (LFP), and gene expression assays. Halfordin (20 µM) significantly decreased convulsive-like behaviour in the locomotor and LFP analysis (by 41.4% and 60%, respectively) and significantly modulated galn, and penka gene expression.

MiR92b-3p synthetic analogue impairs zebrafish embryonic development, leading to ocular defects, decreased movement and hatching rate, and increased mortality.

The aim of this study was to examine the effect of microRNA 92b-3p (MiR92b-3p) overexpression on the embryonic development of zebrafish. A synthetic MiR92b-3p analogue (mirVana™ mimic, in vivo-ready) was injected at doses up to 5 ng/embryo into the yolk sac of embryos (2-16 cell stage). At 24 h post fertilization (hpf), the locomotor activity of the embryos was measured, and after hatching (72 hpf), the rates of malformation occurrence, hatching, and mortality were determined. Next, the larvae were fixed for histological and molecular examinations. Exposure to the MiR92b-3p mimic impaired embryonic development, leading to increased occurrence of malformations (i.e., pericardial edema, spine curvature, smaller eyes), decreased locomotor activity and hatching rate, and increased mortality. Importantly, the mimic affected retinal differentiation and lens formation during zebrafish embryogenesis, which suggests that MiR92b-3p could be an important factor in the regulation of fish embryogenesis and ocular development. The expression level of MiR92b-3p was substantially higher in the exposed larvae than in the untreated larvae, indicating that the mimic was successfully delivered to the zebrafish. Although screening of potential MiR92b-3p target genes suggested some changes in their expression levels, these results were inconclusive. Together, this study indicates that MiR92b-3p mimic impairs zebrafish embryonic development, and further research is necessary to identify the MiR92b-3p-regulated cell pathways involved in the impairment of the fish's development.

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly.

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, γ-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly.

Unique Features of River Lamprey (Lampetra fluviatilis) Myogenesis.

The river lamprey (L. fluviatilis) is a representative of the ancestral jawless vertebrate group. We performed a histological analysis of trunk muscle fiber differentiation during embryonal, larval, and adult musculature development in this previously unstudied species. Investigation using light, transmission electron (TEM), and confocal microscopy revealed that embryonal and larval musculature differs from adult muscle mass. Here, we present the morphological analysis of L. fluviatilis myogenesis, from unsegmented mesoderm through somite formation, and their differentiation into multinucleated muscle lamellae. Our analysis also revealed the presence of myogenic factors LfPax3/7 and Myf5 in the dermomyotome. In the next stages of development, two types of muscle lamellae can be distinguished: central surrounded by parietal. This pattern is maintained until adulthood, when parietal muscle fibers surround the central muscles on both sides. The two types show different morphological characteristics. Although lampreys are phylogenetically distant from jawed vertebrates, somite morphology, especially dermomyotome function, shows similarity. Here we demonstrate that somitogenesis is a conservative process among all vertebrates. We conclude that river lamprey myogenesis shares features with both ancestral and higher vertebrates.

The Anti-Tumoral Potential of Phosphonate Analog of Sulforaphane in Zebrafish Xenograft Model.

Isothiocyanates (ITCs) show strong activity against numerous human tumors. Five structurally diverse ITCs were tested in vivo using the zebrafish embryos 6 and 48 h post-fertilization (hpf). The survival rate, hatching time, and gross morphological changes were assessed 24, 48, and 72 h after treatment with all compounds in various doses (1-10 µM). As a result, we selected a phosphonate analog of sulforaphane (P-ITC; 1-3 µM) as a non-toxic treatment for zebrafish embryos, both 6 and 48 hpf. Furthermore, the in vivo anti-cancerogenic studies with selected 3 µM P-ITC were performed using a set of cell lines derived from the brain (U87), cervical (HeLa), and breast (MDA-MB-231) tumors. For the experiment, cells were labeled using red fluorescence dye Dil (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine, 10 µg/mL) and injected into the hindbrain ventricle, yolk sac region and Cuvier duct of zebrafish embryos. The tumor size measurement after 48 h of treatment demonstrated the significant inhibition of cancer cell growth in all tested cases by P-ITC compared to the non-treated controls. Our studies provided evidence for P-ITC anti-cancerogenic properties with versatile activity against different cancer types. Additionally, P-ITC demonstrated the safety of use in the living organism at various stages of embryogenesis.

Study of the Potential Hepatoprotective Effect of Myo-Inositol and Its Influence on Zebrafish Development.

Inositol is a natural substance found widely in plants. It is used in therapies for many medical cases. The aim of this study was to determine the toxicity of myo-inositol (MI) and to investigate its potential hepatoprotective character. In the first part of the study, zebrafish embryos were incubated with 5, 10, 20, 40, 60, 80, and 100 mg/mL MI. Endpoints such as survivability, hatching rate, malformation, and mobility were evaluated. Our results demonstrated that the high doses of MI lead to increased mortality and malformations and reduce the hatching rate in comparison to the control group. Moreover, low doses of this compound do not produce a negative effect on zebrafish and even have the ability to increase the hatching rate and mobility. In the second part of the study, the hepatoprotective effect of MI was tested. Zebrafish larvae from the line Tg (fabp10a:DsRed) were incubated for 24 h with 1% and 2% ethanol (EtOH), 5 mg/mL of MI with 1% EtOH, and 5 mg/mL of MI with 2% EtOH. No significant differences between the groups with EtOH and the group treated with EtOH with MI were observed. Our results suggest that MI has no positive benefits on hepatocytes of zebrafish larvae.

Tilapia Lake Virus-Induced Neuroinflammation in Zebrafish: Microglia Activation and Sickness Behavior.

In mammals, the relationship between the immune system and behavior is widely studied. In fish, however, the knowledge concerning the brain immune response and behavioral changes during brain viral infection is very limited. To further investigate this subject, we used the model of tilapia lake virus (TiLV) infection of zebrafish (Danio rerio), which was previously developed in our laboratory. We demonstrated that TiLV persists in the brain of adult zebrafish for at least 90 days, even when the virus is not detectable in other peripheral organs. The virions were found in the whole brain. During TiLV infection, zebrafish displayed a clear sickness behavior: decreased locomotor activity, reduced food intake, and primarily localizes near the bottom zone of aquaria. Moreover, during swimming, individual fish exhibited also unusual spiral movement patterns. Gene expression study revealed that TiLV induces in the brain of adult fish strong antiviral and inflammatory response and upregulates expression of genes encoding microglia/macrophage markers. Finally, using zebrafish larvae, we showed that TiLV infection induces histopathological abnormalities in the brain and causes activation of the microglia which is manifested by changes in cell shape from a resting ramified state in mock-infected to a highly ameboid active state in TiLV-infected larvae. This is the first study presenting a comprehensive analysis of the brain immune response associated with microglia activation and subsequent sickness behavior during systemic viral infection in zebrafish.

The Role of Galanin during Bacterial Infection in Larval Zebrafish.

Galanin is a peptide that is conserved among different species and plays various roles in an organism, although its entire role is not completely understood. For many years, galanin has been linked mainly with the neurotransmission in the nervous system; however, recent reports underline its role in immunity. Zebrafish (Danio rerio) is an intensively developing animal model to study infectious diseases. In this study, we used larval zebrafish to determine the role of galanin in bacterial infection. We showed that knockout of galanin in zebrafish leads to a higher bacterial burden and mortality during Mycobacterium marinum and Staphylococcus aureus infection, whereas administration of a galanin analogue, NAX 5055, improves the ability of fish to control the infection caused by both pathogens. Moreover, the transcriptomics data revealed that a lower number of genes were regulated in response to mycobacterial infection in gal-/- mutants compared with their gal+/+ wild-type counterparts. We also found that galanin deficiency led to significant changes in immune-related pathways, mostly connected with cytokine and chemokine functions. The results show that galanin acts not only as a neurotransmitter but is also involved in immune response to bacterial infections, demonstrating the complexity of the neuroendocrine system and its possible connection with immunity.

Is the Activity-Based Anorexia Model a Reliable Method of Presenting Peripheral Clinical Features of Anorexia Nervosa?

Anorexia nervosa (AN) causes the highest number of deaths among all psychiatric disorders. Reduction in food intake and hyperactivity/increased anxiety observed in AN are also the core features of the activity-based anorexia animal model (ABA). Our aim was to assess how the acute ABA protocol mimics common AN complications, including gonadal and cardiovascular dysfunctions, depending on gender, age, and initial body weight, to form a comprehensive description of ABA as a reliable research tool. Wheel running, body weight, and food intake of adolescent female and male rats were monitored. Electrocardiography, heart rate variability, systolic blood pressure, and magnetic resonance imaging (MRI) measurements were performed. Immediately after euthanasia, tissue fragments and blood were collected for further analysis. Uterine weight was 2 times lower in ABA female rats, and ovarian tissue exhibited a reduced number of antral follicles and decreased expression of estrogen and progesterone receptors. Cardiovascular measurements revealed autonomic decompensation with prolongation of QRS complex and QT interval. The ABA model is a reliable research tool for presenting the breakdown of adaptation mechanisms observed in severe AN. Cardiac and hormonal features of ABA with underlying altered neuroendocrine pathways create a valid phenotype of a human disease.

Correction: Effect of partial hysterectomy on the neurons of the paracervical ganglion (PCG) of the pig.

[This corrects the article DOI: 10.1371/journal.pone.0245974.].

Cardiovascular and Respiratory Toxicity of Protamine Sulfate in Zebrafish and Rodent Models.

Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration-dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge-dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.

Effects of a Low Dose of T-2 Toxin on the Percentage of T and B Lymphocytes and Cytokine Secretion in the Porcine Ileal Wall.

Plant materials used in the production of pig feed are frequently contaminated with mycotoxins. T-2 toxin is a secondary metabolite of selected Fusarium species, and it can exert a harmful influence on living organisms. Most mycotoxins enter the body via the gastrointestinal tract, and they can modulate the gut-associated lymphoid tissue (GALT) function. However, little is known about the influence of low T-2 toxin doses on GALT. Therefore, the aim of this study was to evaluate the effect of T-2 toxin administered at 50% of the lowest-observed-adverse-effect level (LOAEL) on the percentage of CD2+ T cells, CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+CD8+ double-positive T cells, TCRγδ+ cells, CD5+CD8- B1 cells, and CD21+ B2 cells, and the secretion of proinflammatory (IFN-γ, IL-1ß, IL-2, IL-12/23p40, IL-17A), anti-inflammatory, and regulatory (IL-4, IL-10, TGF-ß) cytokines in the porcine ileal wall. The results of the study revealed that T-2 toxin disrupts the development of tolerance to food antigens by enhancing the secretion of proinflammatory and regulatory cytokines and decreasing the production of anti-inflammatory TGF-ß. T-2 toxin triggered the cellular response, which was manifested by an increase in the percentage of CD8+ T cells and a decrease in the percentage of B2 and Tγδ lymphocytes.

Protective Role of Galanin during Chemically Induced Inflammation in Zebrafish Larvae.

During a pathological condition, many different systems are involved in the response of an affected organism. Galanin is considered to be a neuropeptide that plays an important role in the central nervous system; however, it is involved in many other biological processes, including the immune response. During our studies, we showed that galanin became upregulated in zebrafish larvae when exposed to copper sulfate. Moreover, the presence of normal levels of galanin, administration of a galanin analog NAX 5055 or galanin overexpression led to lowered lateral line damage and enhanced expression of inflammatory markers compared to the knockout larvae. The results showed that the neuroendocrine system acts multifunctionally and should be considered as a part of the complex neuro-immune-endocrine axis.

Effect of partial hysterectomy on the neurons of the paracervical ganglion (PCG) of the pig.

Autonomic neurons innervating uterine horn is probably the only nerve cell population capable of periodical physiological degeneration and regeneration. One of the main sources of innervation of the uterus is paracervical ganglion (PCG). PCG is a unique structure of the autonomic nervous system. It contains components of both the sympathetic and parasympathetic nervous system. The present study examines the response of neurons of PCG innervating uterine horn to axotomy caused by partial hysterectomy in the domestic pig animal model. The study was performed using a neuronal retrograde tracing and double immunofluorescent staining for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DßH), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neuronal nictric oxide synthase (nNOS), galanin, neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin and substance P (SP). Our study showed that virtually all neurons of the porcine PCG innervating uterine horn are adrenergic and we did not confirm that PCG is the source of cholinergic fibers innervating uterine horn of the pig. After axotomy there was a decrease in expression of catecholamine-synthesizing enzymes (TH, DßH) and a strong increase in the galanin expression. The increase of the number of NPY-IR neurons in the ganglia after axotomy was observed. There were no changes in the expression of other studied substances in the PCG neurons innervating the uterine horn, what was often found in rodents studies. This indicates that neurons can respond to damage in a species-specific way.

Virulence Characterization of Listeria monocytogenes, Listeria innocua, and Listeria welshimeri Isolated from Fish and Shrimp Using In Vivo Early Zebrafish Larvae Models and Molecular Study.

Listeriosis is one of the most notable foodborne diseases and is characterized by high rates of mortality. L. monocytogenes is the main cause of human listeriosis outbreaks, however, there are isolated cases of disease caused by other species of the genus Listeria. The aim of this study was to evaluate strains of L. monocytogenes (n = 7), L. innocua (n = 6), and L. welshimeri (n = 2) isolated from fish and shrimps for their virulence based on the presence of virulence genes and the in vivo Danio rerio (zebrafish) larvae models. A total of 15 strains were analyzed. The zebrafish larvae model showed that the larvae injected with L. monocytogenes strains were characterized by the lowest survival rate (46.5%), followed by L. innocua strains (64.2%) and L. welshimeri (83.0%) strains. Multiplex PCRs were used for detection of selected virulence genes (luxS, actA2, prfA, inlB, rrn, iap, sigB, plcB, actA, hlyA), the majority of which were present in L. monocytogenes. Only a few virulence-related genes were found in L. welshimeri, however, no correlation between the occurrence of these genes and larval survival was confirmed. This research highlights the importance of the potential impact that Listeria spp. strains isolated from fish and shrimps may have on consumers.

Dietary ARA Improves COX Activity in Broodstock and Offspring Survival Fitness of a Model Organism (Medaka Oryzias latipes).

A 3-week feeding trial was conducted in medaka broodstock (age five months) to examine the effect of dietary arachidonic acid (ARA) level (range: 4-23 mg g-1 of total fatty acids (TFAs)) on fertility, cyclooxygenase (COX) activity, egg size, sperm motility parameters, hatching rate and weight of hatch, survival and growth of larvae. After spawn induction and dietary exposure to 4 mg ARA g-1 TFA, broodstock were fed a diet containing ARA in the amounts: 4 (continued, as control), 5, 14 and 23 mg g-1 TFA. COX1 activity in the liver and the number of COX2-positive cells in the ovaries was increased in females fed the diets containing the two highest amounts of ARA. The highest sperm motility parameters were observed in males fed a diet containing 23 mg ARA g-1 TFA. The hatchability rate and bodyweight of hatchlings were higher in the group obtained from broodstock fed a diet containing 23 mg ARA g-1 TFA (79% and 0.66 mg fish-1, respectively) compared with 4 mg ARA g-1 TFA (50% and 0.40 mg fish-1). The average mortality of offspring obtained from this group at 7 days post hatching was significantly higher than that of all other groups.

Viral infection-induced changes in the expression profile of non-RLR DExD/H-box RNA helicases (DDX1, DDX3, DHX9, DDX21 and DHX36) in zebrafish and common carp.

In mammals, several non-RLR DExD/H-box RNA helicases are involve in sensing of viral nucleic acids and activation of antiviral immune response, however their role in the immune defense of fish is much less known. In this study, the expression profile of non-RLR DExD/H-box RNA helicase genes: ddx1, ddx3, dhx9, ddx21 and dhx36, was studied in zebrafish (Danio rerio) and common carp (Cyprinus carpio L.) during infection with two RNA viruses: spring viremia of carp virus (SVCV) and Chum salmon reovirus (CSV). Bioinformatic analysis of the amino acid sequences of the core helicase of DDX1, DDX3, DHX9, DDX21 and DHX36 in zebrafish and common carp revealed presence of all conserved motifs found amongst all other species, with the exception of common carp DHX9 which do not possess motif V. The transcripts of studied DExD/H-box RNA helicases were found in zebrafish ZF4 cell line as well as in all studied organs from zebrafish and common carp. The expression study demonstrated the up-regulation of the expression of selected non-RLR DExD/H-box RNA helicases during viral infections in ZF4 cell line (in vitro study) and in zebrafish and common carp organs (in vivo study). DDX1 was the only DExD/H-box RNA helicase which expression was repetitively up-regulated during in vivo infections with SVCV and CSV in zebrafish and SVCV in common carp. In ZF4 cells and kidney of common carp, viral infection-induced up-regulation of DExD/H-box RNA helicases preceded the up-regulation of type I IFN gene. Our results suggest that studied non-RLR DExD/H-box RNA helicases might be involved in antiviral immune response in fish.