Cultured Fish Cells Research Articles

Tumor necrosis factor α1 decreases mucosal immune and antioxidant response in the midgut of hybrid fish (white crucian carp ♀ × red crucian carp ♂).

Tumor necrosis factor α1 (TNFα) is a pleiotropic cytokine involved in immune regulation and cellular homeostasis, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in a novel hybrid fish (WR), which was produced by crossing white crucian carp (Carassius cuvieri, ♀) with red crucian carp (Carassius auratus red var, ♂). In this study, WR-tnfα1 sequence was identified, and a high-level expression was detected in the intestine. Elevated levels of WR-tnfα1 expressions were detected in immune-related tissues and cultured fish cells on stimulation. The appearance of vacuolization and submucosal rupture was observed in TNFα1-treated midgut of WR, along with elevated levels of goblet cell atrophy, whereas no significant changes were detected in most expressions of tight-junction genes and mucin genes. In contrast, WR receiving gut perfusion with WR-TNFα1 showed a remarkable decrease in antioxidant status in midgut, whereas the expression levels of apoptotic genes and redox responsive genes increased sharply. These results suggested that TNFα1 could exhibit a detrimental effect on antioxidant defense and immune regulation in the midgut of WR.

Tumor necrosis factor α1 (TNFα1) administration can disrupt barrier function and attenuate redox defense in midgut of red crucian carp (Carassius auratus red var)

TNFα belongs to superfamily of tumor necrosis factor that can exert the pleiotropic effort in a large quantity of biological processes, including immune homeostasis, intracellular modulation and etiopathogenesis, but its regulatory role in mucosal immune regulation of fish is unclear. Currently, this study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in teleost fish. In this study, TNFα1 sequences were identified from red crucian carp (RCC, Carassius auratus red var). The high-level expression of RCC-TNFα1 mRNA was detected in gill among all the isolated samples. Then, RCC-TNFα1 expression increased dramatically in immune-related tissues after A. hydrophila infection and in cultured fish cells after lipopolysaccharide (LPS) treatment. RCC-TNFα1 fusion protein was generated and purified in vitro. RCC receiving RCC-TNFα1 perfusion showed an increased levels of villi fusion and edema in injured midgut with the fuzzy appearance. In addition, The mRNA expressions of tight junction (TJ) genes, mucin genes and redox sensitive genes decreased sharply in TNFα1-treated midgut in comparison with those of the control (P < 0.05), whereas the expression levels of apoptotic genes involved in caspase signals and unfolded protein response (UPR) attained the dramatic increase. These results demonstrated that RCC-TNFα1 stimulation could impair midgut structural integrity and immune function by induction of antioxidant collapse and apoptotic activation.

Cloning, expression and functional characterization of recombinant tumor necrosis factor α1 (TNFα1) from white crucian carp in gut immune regulation

TNFα is one of important cytokines belonging to TNF superfamily, which can exhibit a pleiotropic effect in immune modulation, homeostasis as well as pathogenesis. However, its immunoregulatory function on mucosal immunity in fish gut are still unclear. In this study, we aimed to investigated the immunoregulatory role of TNFα1 in midgut of white crucian carp (WCC). WCC-TNFα1 sequence and its deduced structure were firstly identified in WCC. Then, tissue-specific analysis revealed that high-level WCC-TNFα1 expression was detected in gill. After Aeromonas hydrophila and lipopolysaccharide (LPS) stimulated, increased trends of WCC-TNFα1 expressions were detected in immune-related tissues and cultured fish cells, respectively. WCC anal-intubated with WCC-TNFα1 fusion protein showed the increased levels of edema and fuzzy appearance in impaired villi, along with atrophy and reduction of goblet cells (GC). Moreover, the expression levels of tight junction (TJ) genes and mucin genes were consistently lower than those of the control (P < 0.05). WCC-TNFα1 treatment could sharply decrease antioxidant status in midgut, while the expression levels of caspase (CASP) genes, unfolded protein response (UPR) genes and redox response genes increased dramatically. Our results suggested that WCC-TNFα1 could exhibit a detrimental effect on antioxidant and mucosal immune regulation in midgut of WCC.

Tumor necrosis factor α2 (TNFα2) facilitates gut barrier breach by Aeromonas hydrophila and exacerbates liver injury in hybrid fish

TNFα is a multifunctional cytokine that can modulate immune homeostasis and promote cellular regulation in mammals, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα2 on gut barrier breach by A. hydrophila and systemic pathogenesis in gut and liver of WR (White crucian carp ♀ × red crucian carp ♂). WR-TNFα2 encoded 232 amino acid residues and showed a high-level of WR-TNFα2 expression in spleen. After A. hydrophila and lipopolysaccharide (LPS) stimulated, elevated levels of WR-TNFα2 expressions were detected in immune-related tissues and cultured fish cells, respectively. Gut perfusion with WR-TNFα2 alone could alter antioxidant status, increase extrinsic apoptosis as well as promote inflammatory response in midgut, causing severe injury of epithelial barrier. Moreover, WR-TNFα2-treated midgut exhibited a decline of mucosal protection against invasive A. hydrophila, which could elevate bacterial burden in tissues, aggravate tissue injury as well as attenuate immune activation. Our results suggested that WR-TNFα2 could exhibit a detrimental effect on gut barrier defense against A. hydrophila infection and dysregulate immune response in midgut and liver of hybrid crucian carp.

Effect of Biokos, a natural lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on infection of Cryptocaryon irritans.

Cryptocaryoniasis (marine white spot disease), caused by Cryptocaryon irritans, is a major threat to marine fish cultures in tropical and subtropical waters, and a serious nuisance to hobbyists with saltwater fish tanks. With only classical treatment schedules such as copper salts or hyposaline baths being available, control of the disease remains a challenge. In this study, we investigated the effect of Biokos, a viscosin-like lipopeptide surfactant extracted from a bacterium of the Pseudomonas genus, on the external life stages of C. irritans, including theronts, protomonts and tomonts. The present study demonstrated that the compound has an antiparasitic effect on all tested external stages of the parasite. In particular, when Biokos was used at 48 mg/L, it was able to kill almost all theronts and protomonts within 1 h in in vitro experiments, and using the same concentration in an in vivo challenge experiment, the parasitic load was reduced by more than 95% compared to the control group with no Biokos. Additionally, cultured fish cells were able to proliferate, and fish showed no adverse signs at Biokos concentrations that were effective in killing the parasite. Thus, Biokos may be a promising way for preventing or reducing the burden of this parasitic disease in the future.

Comparative analysis on the immunoregulatory roles of ferritin M in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) and its parental species after bacterial infection

Ferritin M is involved in the regulation of fish immunity. In this study, open reading frame (ORF) sequences of ferritin M from hybrid fish and its parental species were 534 bp. Tissue-specific analysis indicated that the highest level of ferritin M from red crucian carp was observed in kidney, while peaked expressions of ferritin M from white crucian carp and hybrid carp were observed in gill. Elevated levels of ferritin M from hybrid carp and its parental species were detected in immune-related tissues following Aeromonas hydrophila infection or in cultured fish cell lines after lipopolysaccharide (LPS) challenge. Ferritin M overexpression could attenuate NF-κB and TNFα promoter activity in their respective fish cells. Purified ferritin M fusion proteins elicited in vitro binding activity to A. hydrophila and Edwardsiella tarda, lowered bacterial dissemination to tissues and alleviated inflammatory response. Furthermore, treatment with ferritin M fusion proteins could mitigate bacteria-induced liver damage and rescue antioxidant activity. These results suggested that ferritin M in hybrid fish showed a similar immune defense against bacteria infection in comparison with those of its parental species.

Effect of Lipopolysaccharide (LPS) stimulation on apoptotic process and oxidative stress in fibroblast cell of hybrid crucian carp compared with those of Carassius cuvieri and Carassius auratus red var

Bacterial LPS is a heat-stable endotoxin and wall components of gram negative bacteria, which can exhibit a toxicological effect on physiology and biochemical activities of fish. In this study, we investigated the effect of LPS exposure on cell viability, oxidative stress, caspase activity and immune-related gene expressions in cultured fin cell lines of red crucian carp, white crucian carp and their hybrid offspring. LPS stimulation could reduce fish cell viability, whereas gene expression levels and promoter activities in inflammatory signals increased dramatically. Moreover, enhanced levels of intracellular oxidative stress and decreased levels of mitochondrial membrane potential (MMP) were observed in LPS-induced fish cells. N-Acetyl-L-cysteine (NAC) could alleviate LPS-stimulated reactive oxygen species (ROS) generation and caspase-3 activity in fish cells. These results suggested that ROS-mediated cytotoxic stress was involved in LPS-induced inflammation and mitochondrial damage in cultured fish cells.

A New Method for Chromosomes Preparation by ATP-Competitive Inhibitor SP600125 via Enhancement of Endomitosis in Fish.

Previous studies have suggested that 1,9-Pyrazoloanthrone, known as SP600125, can induce cell polyploidization. However, what is the phase of cell cycle arrest caused by SP600125 and the underlying regulation is still an interesting issue to be further addressed. Research in this article shows that SP600125 can block cell cycle progression at the prometaphase of mitosis and cause endomitosis. It is suggested that enhancement of the p53 signaling pathway and weakening of the spindle assembly checkpoint are associated with the SP600125-induced cell cycle arrest. Using preliminary SP600125 treatment, the samples of the cultured fish cells and the fish tissues display a great number of chromosome splitting phases. Summarily, SP600125 can provide a new protocol of chromosomes preparation for karyotype analysis owing to its interference with prometaphase of mitosis.

Efficient genome editing using CRISPR/Cas9 ribonucleoprotein approach in cultured Medaka fish cells.

ABSTRACTGene editing with CRISPR/Cas9 is a powerful tool to study the function of target genes. Although this technology has demonstrated wide efficiency in many species, including fertilized zebrafish and medaka fish embryos when microinjected, its application to achieve efficient gene editing in cultured fish cells have met some difficulty. Here, we report an efficient and reliable approach to edit genes in cultured medaka (Oryzias latipes) fish cells using pre-formed gRNA-Cas9 ribonucleoprotein (RNP) complex. Both medaka fish haploid and diploid cells were transfected with the RNP complex by electroporation. Efficient gene editing was demonstrated by polymerase chain reaction (PCR) amplification of the target gene from genomic DNA and heteroduplex mobility assay carried out with polyacrylamide gel electrophoresis (PAGE). The heteroduplex bands caused by RNP cleavage and non-homologous end joining could be readily detected by PAGE. DNA sequencing confirmed that these heteroduplex bands contains the mutated target gene sequence. The average gene editing efficiency in haploid cells reached 50%, enabling us to generate a clonal cell line with ntrk3b gene mutation for further study. This RNP transfection method also works efficiently in diploid medaka cells, with the highest mutation efficiency of 61.5%. The specificity of this synthetic RNP CRISPR/Cas9 approach was verified by candidate off-target gene sequencing. Our result indicated that transfection of pre-formed gRNA-Cas9 RNP into fish cells is efficient and reliable to edit target genes in cultured medaka fish cells. This method will be very useful for gene function studies using cultured fish cells.

A portable chemical toxicity biochip based on electronic enzymatic monitoring of cytotoxic effects on fish cells

Herein we describe a miniaturised chemical toxicity analyser with capacity to assess water quality without intervention of laboratory equipment. The device, as presented, integrates living cells and electrode-based sensors to monitor cell viability upon exposure to toxic chemicals. The methodology involved cultured fish cells Poeciliopsis lucidia hepatocellular carcinoma (PLHC-1) and Oncorhynchus mykiss rainbow trout gonad (RTG-2) cells, exposed to toxic chemicals (CdCl2 and pentachlorophenol (PCP)) after which the activity of a cellular enzyme, acid phosphatase (AP) was measured. Reduced AP activity was quantified electrochemically, allowing IC50 values (50% reduction in AP activity) to be determined. Fish cells are a relevant model of the toxicity risks posed by contaminated water to aquatic health. Such cells are relatively slow growing and do not require a source of CO2 for incubation, thus ensuring portability for single-use point of site applications. The 24hour IC50 values determined for CdCl2 in the fish cell lines (16.2±0.7 and 91±11μM for PLHC and RTG-2, respectively) were in agreement with those found using the MTT assay (16.3±1.6 and 164±96μM). In the case of PCP, the IC50 value in PHLC cells (127±22μM) suggested enhanced sensitivity towards PCP compared with the MTT assay (IC50>160μM). The optimised electronic assay was transferred to a prototype integrated assay cartridge with associated fluidic and transducer elements. This device (TOXOR) monitored changes in the metabolic state of fish cells, realising high-value toxicity information as a potential early warning on-site screening system.

Singapore grouper iridovirus (SGIV) encoded SGIV-miR-13 attenuates viral infection via modulating major capsid protein expression

Singapore grouper iridovirus (SGIV) encodes a number of microRNAs (miRNAs) during infection. Among these, SGIV-miR-13 has robust expression at early stage after SGIV inoculation, raising a huge possibility that it participates in the viral infection. In the present study, we found that SGIV-miR-13 overexpression led to a significant reduction in viral load in cultured fish cells with SGIV infection, as demonstrated by less level of viral transcripts, viral-induced cytopathic effect (CPE) and assembled viral particles. In silico analysis showed that SGIV-miR-13 maps antisense to the coding region of SGIV major capsid protein (SGIV-MCP), suggesting it to be a potential target of SGIV-miR-13. Coincidently, SGIV-miR-13 showed an inverted expression profile with SGIV-MCP during SGIV infection, and luciferase reporter assay further demonstrated SGIV-MCP as the direct target of SGIV-miR-13. Functionally, overexpression of SGIV-miR-13 inhibited, whereas knockdown of SGIV-miR-13 restored the expression of SGIV-MCP during viral infection, resulting in altered viral progeny emergences. In conclusion, our data suggest that SGIV-miR-13 functions in a negative regulatory mechanism to restrict early viral replication, and thus prevents excessive cellular antiviral responses during SGIV infection. The detailed investigation of SGIV encoded miRNAs may provide new insights into the mechanism of iridovirus pathogenesis.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

Quantification of malondialdehyde by HPLC-FL - application to various biological samples.

Malondialdehyde (MDA) is stabile product of lipid peroxidation (LPO), and therefore MDA is frequently used as a biomarker of LPO. To determine MDA level in various biological samples (human plasma, fish liver tissue and cells in culture), we used an HPLC method with fluorescent detection based on 2-thiobarbituric acid (TBA) assay. The method was validated by the use of spiked pooled plasma samples. In tested concentration range (0.15-3.0 µmol/L) the method was linear (R(2) = 0.9963), the between-day variability (coefficient of variations, CVs) was between 4.7 and 7.6%, the within-day variability CVs was between 2.6 and 6.4% and recovery was between 91.2 and 107.6%. The level of MDA in human plasma (healthy male, non-smokers, 46.3 ± 4.7 years; N = 38) was 2.2 ± 1.4 µmol/L; that in liver tissue of common carp (Cyprinus carpio; N = 12) was 0.02 ± 0.004 µmol/g tissue, and in cultured cells (human laryngeal carcinoma cells; N = 10) it was 0.18 ± 0.02 nmol/mg proteins. The HPLC-FL method is rapid, accurate and reliable to follow the extent of LPO in various biological samples, particularly in samples in which a low level of MDA is expected, such as cells in culture. Owing to the rapid analytical process and run time, it can be used for routine analysis of MDA in clinical laboratory.

Cell-penetrating peptide delivery of biologically active oct4 protein into cultured Takifugu rubripes spermary cells.

Continuous cell culture of a puffer fish Takifugu rubripes has been established for efficient delivery of exogenous genes or proteins to cultured fish cells. Transcription factor oct4 was chosen for transduction into cultured fish cells because of its conserved structure and function between fish and mammals. In this work, the T. rubripes oct4 gene was cloned and expressed in Escherichia coli as a recombinant protein by introducing cell-penetrating peptide (CPP) poly-arginine (11R) and 6His-tag at the C-terminus. After purification, recombinant proteins were added to the growth medium and incubated with T. rubripes spermary cells. Recombinant proteins that crossed the cell membrane were detected in the cytoplasm and nucleus by western blot and immunofluorescent observation. The function of transduced oct4 as a transcription factor in fish cells was confirmed by driving green fluorescent protein expression in the pEGFP-1 reporter construct with the conserved specific oct4-binding sequence from mouse Mus musculus. Taken together, 11R can be an efficient CPP in delivering fusion proteins to cultured fish cells.

Fish viperin exerts a conserved antiviral function through RLR-triggered IFN signaling pathway

Mammalian viperin is a typical interferon (IFN)-induced antiviral protein. Fish have viperin homologs; however, little is known about the expression regulation of fish viperins. In this study, we report the expression regulation and antiviral function of a fish viperin from crucian carp Carassius auratus during IFN response. Crucian carp viperin is induced at mRNA and protein levels by fish IFNs and IFN stimuli such as poly(I:C). Consistently, this gene promoter contains multiple transcription factor binding sites including IFN-stimulated response elements (ISRE) and IFN gamma activation sequences (GAS), and is activated by two types of fish IFNs and also by the intracellular and extracellular poly(I:C). Activation of crucian carp viperin promoter by the intracellular poly(I:C) is mediated by retinoic acid-inducing gene I (RIG-I)-like receptors (RLR)-triggered IFN signaling pathway, which is further verified by the findings that each signaling molecule of RLR pathway is able to induce the expression of crucian carp viperin at mRNA and protein levels. Finally, overexpression of crucian carp viperin in cultured fish cells confers significant protection against infection of grass carp reovirus (GCRV). These data suggest that similar to mammalian homologs, crucian carp viperin exerts a conserved function through RLR-triggered IFN signaling pathway.

Isolation and characterization of a new class of DNA aptamers specific binding to Singapore grouper iridovirus (SGIV) with antiviral activities

The Singapore grouper iridovirus (SGIV), a member of the genus Ranavirus, is a major viral pathogen that has caused heavy economic losses to the grouper aquaculture industry in China and Southeast Asia. No efficient method of controlling SGIV outbreaks is currently available. Systematic evolution of ligands by exponential enrichment (SELEX) is now widely used for the in vitro selection of artificial ssDNA or RNA ligands, known as aptamers, which bind to targets through their stable three-dimensional structures. In our current study, we generated ssDNA aptamers against the SGIV, and evaluated their ability to block SGIV infection in cultured fish cells and cultured fish in vivo. The anti-SGIV DNA aptamers, LMB-761, LMB-764, LMB-748, LMB-439, LMB-755, and LMB-767, were selected from a pool of oligonucleotides randomly generated using a SELEX iterative method. The analysis of the secondary structure of the aptamers revealed that they all formed similar stem-loop structures. Electrophoretic mobility shift assays showed that the aptamers bound SGIV specifically, as evidenced by a lack cross-reactivity with the soft shell turtle iridovirus. The aptamers produced no cytotoxic effects in cultured grouper spleen cells (GS). Assessment of cytopathic effects (CPE) and viral titer assays showed that LMB-761, LMB-764, LMB-748, LMB-755, and LMB-767 significantly inhibited SGIV infection in GS cells. The in vivo experiments showed that LMB-761 and LMB-764 reduced SGIV-related mortality, and no negative effects were observed in orange-spotted grouper, Epinephelus coioides, indicating that these DNA aptamers may be suitable antiviral candidates for controlling SGIV infections in fish reared in marine aquaculture facilities.

Measured and modeled toxicokinetics in cultured fish cells and application to in vitro-in vivo toxicity extrapolation.

Effect concentrations in the toxicity assessment of chemicals with fish and fish cells are generally based on external exposure concentrations. External concentrations as dose metrics, may, however, hamper interpretation and extrapolation of toxicological effects because it is the internal concentration that gives rise to the biological effective dose. Thus, we need to understand the relationship between the external and internal concentrations of chemicals. The objectives of this study were to: (i) elucidate the time-course of the concentration of chemicals with a wide range of physicochemical properties in the compartments of an in vitro test system, (ii) derive a predictive model for toxicokinetics in the in vitro test system, (iii) test the hypothesis that internal effect concentrations in fish (in vivo) and fish cell lines (in vitro) correlate, and (iv) develop a quantitative in vitro to in vivo toxicity extrapolation method for fish acute toxicity. To achieve these goals, time-dependent amounts of organic chemicals were measured in medium, cells (RTgill-W1) and the plastic of exposure wells. Then, the relation between uptake, elimination rate constants, and log KOW was investigated for cells in order to develop a toxicokinetic model. This model was used to predict internal effect concentrations in cells, which were compared with internal effect concentrations in fish gills predicted by a Physiologically Based Toxicokinetic model. Our model could predict concentrations of non-volatile organic chemicals with log KOW between 0.5 and 7 in cells. The correlation of the log ratio of internal effect concentrations in fish gills and the fish gill cell line with the log KOW was significant (r>0.85, p = 0.0008, F-test). This ratio can be predicted from the log KOW of the chemical (77% of variance explained), comprising a promising model to predict lethal effects on fish based on in vitro data.

Turbot (Scophthalmus maximus) hepcidin-1 and hepcidin-2 possess antimicrobial activity and promote resistance against bacterial and viral infection

Hepcidin is an antimicrobial peptide and a regulator of iron homeostasis. In turbot (Scophthalmus maximus), two types of hepcidins have been identified, which share approximately 50% sequence identity. In this study, we examined the antimicrobial potentials of the two hepcidins in the form of synthesized peptides, SmHep1P and SmHep2P. We found that SmHep1P and SmHep2P exhibited apparent bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner. The bactericidal effect of SmHep1P was stronger against Gram-positive bacteria, while the bactericidal effect of SmHep2P was stronger against Gram-negative bacteria. Fluorescence and electron microscopy showed that both peptides were able to bind to the target bacterial cells and alter the surface structure of the cells. In vitro studies showed that SmHep1P and SmHep2P reduced bacterial invasion into cultured fish cells. In vivo studies showed that turbot administered with SmHep1P and SmHep2P exhibited significantly enhanced resistance against bacterial and viral infection. In both in vivo and in vitro studies, the antimicrobial activities of SmHep2P were in most cases significantly stronger than those of SmHep1P. Together these results indicate that the two hepcidins of turbot most likely possess antimicrobial properties and play a role in the innate immune defense against bacterial and viral pathogens.

Gill cell culture systems as models for aquatic environmental monitoring

A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples.

Rock bream (Oplegnathus fasciatus) viperin is a virus-responsive protein that modulates innate immunity and promotes resistance against megalocytivirus infection

Viperin in mammals is known to be an antiviral protein that inhibits the replication of diverse DNA and RNA viruses. In teleost, viperin homologues have been identified in a large number of species and, in some cases, are stimulated in transcription by viruses. However, the biological significance of fish viperin protein in antiviral immunity has not been investigated. In this study, we identified a viperin homologue from rock bream (Oplegnathus fasciatus) (named OfVip) and examined its expression pattern, subcellular localization, and immune effect. We found that OfVip expression occurred in eight tissues, and experimental challenge of rock bream with the viral fish pathogen megalocytivirus upregulated OfVip expression in kidney, liver, and spleen. OfVip was localized in the endoplasmic reticulum under normal physiological conditions, and viral infection induced subcellular redistribution of OfVip. Transient transfection of cultured fish cells with an OfVip-expressing plasmid caused enhanced cellular resistance against megalocytivirus challenge. Consistently, in vivo study showed that rock bream overexpressing OfVip exhibited significantly reduced viral loads in tissues following experimental infection with megalocytivirus. Furthermore, OfVip upregulated the expression of a wide range of immune genes, including those that are known to participate in antiviral immunity. Taken together, these results indicate for the first time that a teleost viperin is a virus-responsive protein that is modulated in subcellular localization by viral infection, and that viperin regulates the immune reactions of the host fish in a manner that augments resistance against viral infection.