ABSTRACT
Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC50 of 100 µg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 µg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1ß, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed.
Subject(s)
Antiviral Agents , Novirhabdovirus , Umbelliferones , Animals , Umbelliferones/pharmacology , Antiviral Agents/pharmacology , Novirhabdovirus/physiology , Novirhabdovirus/drug effects , Fish Diseases/drug therapy , Fish Diseases/immunology , Fish Diseases/virology , Flounder/immunology , Hemorrhagic Septicemia, Viral/virology , Hemorrhagic Septicemia, Viral/immunology , Cell LineABSTRACT
The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.
Subject(s)
Amino Acid Sequence , Extracellular Matrix Proteins , Fish Proteins , Gene Expression Regulation , Hemorrhagic Septicemia, Viral , Immunity, Innate , Novirhabdovirus , Phylogeny , Animals , Novirhabdovirus/physiology , Fish Proteins/genetics , Fish Proteins/immunology , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/immunology , Hemorrhagic Septicemia, Viral/immunology , Hemorrhagic Septicemia, Viral/genetics , Immunity, Innate/genetics , Gene Expression Regulation/immunology , Sequence Alignment/veterinary , Fish Diseases/immunology , Fish Diseases/virology , Gene Expression Profiling/veterinary , Flatfishes/immunology , Flatfishes/geneticsABSTRACT
Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.
Subject(s)
CRISPR-Cas Systems , Gene Knockout Techniques , Hemorrhagic Septicemia, Viral , Interferon Regulatory Factor-3 , Novirhabdovirus , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/immunology , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Novirhabdovirus/physiology , Novirhabdovirus/immunology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Hemorrhagic Septicemia, Viral/immunology , Hemorrhagic Septicemia, Viral/genetics , Hemorrhagic Septicemia, Viral/virology , Animals, Genetically Modified , Fish Diseases/immunology , Fish Diseases/virology , Fish Diseases/genetics , Immunity, Innate/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Rhabdoviridae Infections/immunology , Rhabdoviridae Infections/virology , Disease Models, Animal , InterferonsABSTRACT
Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.
Subject(s)
Gastropoda , Immunity, Innate , Metallothionein , Novirhabdovirus , Oxidative Stress , Vibrio parahaemolyticus , Animals , Metallothionein/genetics , Metallothionein/immunology , Gastropoda/immunology , Gastropoda/genetics , Gastropoda/microbiology , Oxidative Stress/drug effects , Vibrio parahaemolyticus/physiology , Immunity, Innate/genetics , Novirhabdovirus/physiology , Gene Expression Regulation/immunology , Amino Acid Sequence , Phylogeny , Sequence Alignment/veterinary , Listeria monocytogenes/physiology , Listeria monocytogenes/immunology , Mice , Gene Expression Profiling/veterinary , RAW 264.7 Cells , Metals, Heavy/toxicity , Water Pollutants, ChemicalABSTRACT
Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to ß-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 µg/mL or 100 µg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H2O2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet.
Subject(s)
Antioxidants , Fish Proteins , Galectins , Smegmamorpha , Animals , Fish Proteins/genetics , Fish Proteins/metabolism , Fish Proteins/immunology , Smegmamorpha/immunology , Smegmamorpha/genetics , Galectins/metabolism , Galectins/genetics , Antioxidants/metabolism , Fish Diseases/immunology , Cytokines/metabolism , Immunity, Innate , Poly I-C/immunology , Lactococcus/physiology , Lipopolysaccharides/immunology , Chemokines/metabolism , Chemokines/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Novirhabdovirus/physiology , Novirhabdovirus/immunology , Antiviral Agents/metabolismABSTRACT
The influence of colour background on the regulation of behavioural and physiological responses in zebrafish is widely recognized. However, its specific effect on virus infection in zebrafish remains unclear. This study aimed to explore the susceptibility of zebrafish to viral haemorrhagic septicaemia virus (VHSV) infection in relation to background colour, investigate the underlying mechanisms, and elucidate the involvement of key molecules, using proteomic and gene expression analyses. The results revealed that zebrafish housed in a blue tank exhibited higher survival rates and considerably reduced VHSV replication compared to those housed in a yellow tank. Further, up-regulation of apolipoprotein 1 (APOA1) was identified as a crucial shared mechanism associated with survival in zebrafish exposed to VHSV infection and reared in a blue background. The mRNA expression level of bmal1a, a core gene involved in the circadian rhythm, was consistently downregulated in fish from the blue tank compared to fish from the yellow tank, regardless of infection status. Subsequently, zebrafish in the blue tank were exposed to daylight conditions to stimulate per2 and pgc1a expression, aiming to investigate their potential impact on VHSV infection. The validity of these interconnected events, triggered by background colour, involving APOA1 up-regulation, circadian rhythm modulation, and antiviral responses, was confirmed by treatments with hesperetin and cyclosporine A, an activator and inhibitor of apoa1 respectively. Our findings revealed the influence of background colour on the apoa1 expression level, thus establishing the involvement of a novel network through circadian rhythm signalling.
Subject(s)
Fish Diseases , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , Zebrafish , Color , Proteomics , Antiviral Agents/pharmacology , Novirhabdovirus/physiologyABSTRACT
Viral haemorrhagic septicaemia virus (VHSV) is one of the highly pathogenic virus, which causes viral haemorrhagic septicaemia disease in both marine and freshwater fish. Micro RNA-155 (miRNA-155) is a multifunctional small non-coding RNA and it involves regulation of immune responses during viral infection. In this study, dre-miR-155 mimics were encapsulated into chitosan nanoparticles (CNPs). Resulted encapsulated product (miR-155-CNPs) was investigated for its immunomodulation role in zebrafish during experimentally challenged VHSV infection. Successful encapsulation of dre-miR-155 mimics into CNPs was confirmed through average nanoparticle (NPs) size (341.45 ± 10.00 nm), increased encapsulation efficiency percentage (98.80%), bound dre-miR-155 with chitosan, sustained release in vitro (up to 40%), and the integrity of RNA. Overexpressed miR-155 was observed in gills, muscle, and kidney tissues (5.42, 19.62, and 140.72-folds, respectively) after intraperitoneal delivery of miR-155-CNPs into zebrafish upon VHSV infection (miR-155-CNPs + VHSV). The miR-155-CNPs + VHSV infected fish had the highest cumulative survival (85%), which was associated with low viral copy numbers. The miR-155-overexpressing fish showed significantly decreased expression of ifnγ, irf2bpl, irf9, socs1a, il10, and caspase3, compared to that of the miR-155 inhibitor + VHSV infected fish group. In contrast, il1ß, tnfα, il6, cd8a, and p53 expressions were upregulated in miR-155-overexpressed zebrafish compared to that of the control. The overall findings indicate the successful delivery of dre-miR-155 through miR-155-CNPs that enabled restriction of VHSV infection in zebrafish presumably by modulating immune gene expression.
Subject(s)
Chitosan , Fish Diseases , Hemorrhagic Septicemia, Viral , MicroRNAs , Nanoparticles , Novirhabdovirus , Animals , Zebrafish , Immunity , Novirhabdovirus/physiology , MicroRNAs/geneticsABSTRACT
Viral hemorrhagic septicemia causes considerable economic losses for Korea's olive flounder (Paralichthys olivaceus) aquaculture farms; therefore, effective antiviral agents for controlling viral hemorrhagic septicemia virus (VHSV) infection are imperative. The present study implemented a Box-Behnken design and cytopathic reduction assay to derive an optimized extract of Sanguisorba officinalis L. roots (OE-SOR) with maximum antiviral activity against VHSV. OE-SOR prepared under optimized extraction conditions (55% ethanol concentration at 50 °C for 5 h) exhibited potent antiviral activity against VHSV, with a 50% effective 0.21 µg/mL concentration and a 340 selective index. OE-SOR also showed direct virucidal activity in the plaque reduction assay. Administering OE-SOR to olive flounder exhibited substantial efficacies against VHSV infection. Fish receiving 100 mg/kg body weight/day of OE-SOR as a preventive (40.0%; p < 0.05) or therapeutic (44.4%; p < 0.05) exhibited a higher relative survival than the untreated VHSV-infected control group (mortalities of 100% and 90%, respectively). In addition, fish fed with OE-SOR (100 mg/kg body weight/day) for two weeks conveyed a significantly higher inflammatory cytokine expression (nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB], interleukin-1 beta [IL-1ß], and tumor necrosis factor-alpha [TNF-α]) than the control group one to two days post-administration. Moreover, no hematological or histological changes were observed in olive flounder treated with OE-SOR over four weeks. Liquid chromatography-quadrupole-time of flight tandem mass spectrometry and -triple quadrupole tandem mass spectrometry analyses identified ziyuglycoside I as a prominent OE-SOR constituent and marker compound (content: 14.5%). This study verifies that OE-SOR is an effective alternative for controlling viral hemorrhagic septicemia in olive flounder farms as it exhibits efficient in vivo anti-VHSV activity and increases innate immune responses.
Subject(s)
Fish Diseases , Flounder , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Sanguisorba , Animals , Hemorrhagic Septicemia, Viral/prevention & control , Antiviral Agents/pharmacology , Novirhabdovirus/physiology , Body Weight , Fish Diseases/prevention & controlABSTRACT
MicroRNAs (miRNAs) are small non-coding RNAs known to play a significant role in the regulation of gene expression in various living organisms including fish. MiR-155 is known to enhance immunity in cells and several reports have demonstrated the antiviral properties of miR-155 in mammals. In this study, we investigated the antiviral role of miR-155 in Epithelioma papulosum cyprini (EPC) cells with viral hemorrhagic septicemia virus (VHSV) infection. EPC cells were transfected with miR-155 mimic and then infected with VHSV at different MOIs (0.01 and 0.001). The cytopathogenic effect (CPE) was observed at 0, 24, 48, and 72 h post infection (h.p.i). CPE progression appeared at 48 h.p.i in mock groups (VHSV only infected groups) and the VHSV infection group transfected with miR-155 inhibitors. On the other hand, the groups transfected with the miR-155 mimic did not show any CPE formation after infection with VHSV. The supernatant was collected at 24, 48 and 72 h.p.i., and the viral titers were measured by plaque assay. The viral titers increased at 48 and 72 h.p.i in groups infected only with VHSV. In contrast, the groups transfected with miR-155 did not show any increase in the virus titer and had a similar titer to 0 h.p.i. Furthermore, the real-time RT-PCR of immune gene expression showed upregulation of Mx1 and ISG15 at 0, 24, and 48 h.p.i in groups transfected with miR-155, while the genes were upregulated at 48 h.p.i in groups infected only with VHSV. Based on these results, miR-155 can induce the overexpression of type I interferon-related immune genes in EPCs and inhibit the viral replication of VHSV. Therefore, these results suggest that miR-155 could possess an antiviral effect against VHSV.
Subject(s)
Carcinoma , Fish Diseases , Hemorrhagic Septicemia, Viral , MicroRNAs , Novirhabdovirus , Animals , Antiviral Agents , Cell Line, Tumor , MicroRNAs/genetics , MicroRNAs/metabolism , Novirhabdovirus/physiology , Mammals/metabolismABSTRACT
As filter-feeding bivalves, mussels have been traditionally studied as possible vectors of different bacterial or viral pathogens. The absence of a known viral pathogen in these bivalves makes it particularly interesting to study the interaction of the mussel innate immune system with a virus of interest. In the present work, mussels were challenged with viral haemorrhagic septicaemia virus (VHSV), which is a pathogen in several fish species. The viral load was eliminated after 24 h and mussels evidenced antiviral activity towards VHSV, demonstrating that the virus was recognized and eliminated by the immune system of the host and confirming that mussels are not VHSV vectors in the marine environment. The transcriptome activating the antiviral response was studied, revealing the involvement of cytoplasmic viral sensors with the subsequent activation of the JAK-STAT pathway and several downstream antiviral effectors. The inflammatory response was inhibited with the profound downregulation of MyD88, shifting the immune balance towards antiviral functions. High modulation of retrotransposon activity was observed, revealing a mechanism that facilitates the antiviral response and that had not been previously observed in these species. The expression of several inhibitors of apoptosis and apoptosis-promoting genes was modulated, although clear inhibition of apoptosis in bivalves after severe viral infection and subsequent disease was not observed in this study. Finally, the modulated expression of several long noncoding RNAs that were correlated with genes involved in the immune response was detected.
Subject(s)
Fish Diseases , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , Transcriptome , Janus Kinases , STAT Transcription Factors , Signal Transduction , Novirhabdovirus/physiology , Antiviral Agents/pharmacologyABSTRACT
The replication of viral hemorrhagic septicemia virus (VHSV) in appropriate host cells depends on environmental factors and the host cell's immunity. The dynamics of each VHSV RNA strand (vRNA, cRNA, and mRNA) in different conditions can provide a clue on the viral replication strategies, which can be a base for the development of efficient control measures. As VHSV is known to be sensitive to temperature and type I interferon (IFN) responses, in this study, we analyzed the effect of temperature difference (15 °C and 20 °C) and IRF-9 gene knockout on the dynamics of the three VHSV RNA strands in Epithelioma papulosum cyprini (EPC) cells using a strand-specific RT-qPCR. The tagged primers designed in this study successfully worked to quantify the three strands of VHSV. In the results of the temperature effect, the higher speed in viral mRNA transcription and the significantly higher (more than 10 times at 12-36 h) copy number of cRNA at 20 °C compared to those at 15 °C suggested the positive effect of high temperature on VHSV replication. In the results of the IRF-9 gene knockout effect, although IRF-9 gene knockout did not bring a dramatic effect on VHSV replication compared to the temperature effect, the increase of mRNA in IRF-9 KO cells was faster than normal EPC cells, which was reflected in the copy numbers of cRNA and vRNA. The IRF-9 gene knockout effect was not dramatic even in the replication of rVHSV-ΔNV-eGFP that harbors eGFP gene ORF instead of NV gene ORF. These results suggest that VHSV may be highly susceptible to pre-activated type I IFN responses but not highly susceptible to post-infection-mediated type I IFN responses or lowered type I IFN before infection. In both experiments of temperature effect and IRF-9 gene knockout effect, the copy number of cRNA never exceeded the copy number of vRNA at all assay times, suggesting that the binding efficiency of the RNP complex to the 3' end of cRNA might be lower than that to the 3' end of vRNA. Further research is needed to elucidate the regulatory mechanism that limits the amount of cRNA at an appropriate level during VHSV replication.
Subject(s)
Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , RNA, Complementary , RNA, Messenger , Temperature , Gene Knockout Techniques , Novirhabdovirus/physiology , Virus ReplicationABSTRACT
Viral hemorrhagic septicemia virus (VHSV) causes a severe and often lethal infection in olive flounder (Paralichthys olivaceus) in Korea, resulting in mass mortality and substantial economic loss. As a potential prevention strategy for infectious viral diseases, this study aimed to evaluate the antiviral activity of three compounds (arctigenin [ARG], ribavirin [RBV], and ivermectin [IVM]) against VHSV infection in vitro and in vivo. In epithelioma papulosum cyprini cells, the expression of both VHSV glycoprotein (G) and nucleoprotein (N) genes were significantly suppressed by the three compounds in a dose-dependent manner (P < 0.05). Also, cell morphology and viability were maintained at the following concentrations: ARG 1.5 mg/L, RBV 2.5 mg/L, and IVM 10 mg/L. The fish that were treated with RBV (8.33 mg/kg) and IVM (0.25 mg/kg) before VHSV infection and those treated with IVM (0.25 mg/kg) after VHSV infection showed significant improvements in the survival rate, a reduction in the viral shedding rate, and downregulation of viral gene expression compared to those seen in fish with naïve VHSV infections. Furthermore, among the innate immune genes studied, persistent expression of Mx and upregulation of tumor necrosis factor-α gene expression in VHSV-infected fish treated with RBV and IVM revealed that these compounds might induce an immunostimulatory effect as one of their antiviral activities. Overall, this study supports the use of RBV and IVM as antiviral agents to control VHSV infections in olive flounder.
Subject(s)
Fish Diseases , Flounder , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , Ribavirin/pharmacology , Antiviral Agents/pharmacology , Ivermectin/pharmacology , Novirhabdovirus/physiologyABSTRACT
Hirame novirhabdovirus (HIRRV) is a fish rhabdovirus belonging to family Rhabdoviridae, genus Novirhabdovirus, which is highly contagious and virulent, and causes hemorrhagic disease in many fish species. In the present work, the whole genome sequence of HIRRV strain CNPo2015 that previously isolated from cultured flounders was obtained using high-throughput sequencing. It consists of 10,998 nucleotides and encodes six viral proteins arranged in order of 3'-N-P-M-G-NV-L-5'. Among Novirhabdovirus, L protein of CNPo2015 possessed the lowest amino acid sequence divergence with HIRRV isolate CA 9703 and HIRRV 080113, and the highest with Snakehead rhabdovirus. Furthermore, the immune response of Hirame natural embryo (HINAE) cell line to HIRRV infection was characterized by RNA-seq, and the results showed that 1976 differentially expressed genes (DEGs) including 1219 up-regulated and 727 down-regulated genes were identified in the HINAE cells infected with HIRRV at 48 h post infection (hpi). Several KEGG pathways were significantly enriched in the viral infected cells, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, cell cycle, apoptosis, RIG-I-like receptors signaling pathway and P13K-AKT signaling pathway. Post viral infection, the flow cytometric Annexin V/PI assay found that apoptotic rate of HINAE cells showed a slight increase within 3 days and then the early and late apoptotic rate were significantly increased to 41 ± 2.65% and 12.37 ± 2.61% at day 4, respectively. Meanwhile, qRT-PCR results also showed that six apoptosis-related genes (BCL2L1, CASPASE 3, CASPASE 10, FAS, AKT and CDK1) were significantly upregulated. This investigation has not only enriched our knowledge of sequence difference characteristics between CNPo2015 and other Novirhabdoviruses, but also provided a data basis for deeper understanding of immune responses in flounder cells post viral infection.
Subject(s)
Fish Diseases , Flounder , Novirhabdovirus , Virus Diseases , Animals , Proto-Oncogene Proteins c-akt/genetics , Novirhabdovirus/physiology , RNA-SeqABSTRACT
Viperin is an important virus-induced protein in animals that negatively participates in RNA viral replication and transcription. The reactive machinery of viperin suggests that it produces a regulatory molecule ddhCTP, which may affect immune regulation. In this study, we investigated the expression pattern of viperin in larval and adult stages of zebrafish by whole-mount in situ hybridization and reverse transcription-quantitative PCR (RT-qPCR). To elucidate the function of viperin, we generated a zebrafish knockout model using the CRISPR/Cas9 method and evaluated the mutation's effects under viral hemorrhagic septicemia virus (VHSV) infections. In zebrafish larvae, viperin was expressed in the brain region, eye, and pharynx, which was confirmed by cryosectioning. In adult zebrafish, blood cells showed the highest levels of viperin expression. In 5 dpf fish challenged with VHSV, the expression of the viral NP protein was significantly enhanced in viperin-/- compared to wild-type fish. In vitro VHSV propagation analysis indicated comparatively higher levels of virus propagation in viperin-/- fish. Mortality analysis confirmed higher mortality rates, and interferon gene expression analysis showed a strong upregulation of interferon (ifn)φ1 and 3 gene in viperin-/- fish infected with VHSV. This study describes the successful generation of a viperin-knockout model and the role of viperin during VHSV infections.
Subject(s)
Fish Diseases , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , Zebrafish/genetics , Zebrafish/metabolism , CRISPR-Cas Systems , Novirhabdovirus/physiology , Viral Proteins/genetics , Mutation , Interferons/geneticsABSTRACT
Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.
Subject(s)
Fish Diseases , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Perciformes , Virus Diseases , Animals , Apoptosis Regulatory Proteins , Mammals , Novirhabdovirus/physiology , Poly I-C/pharmacology , Proto-Oncogene Proteins c-bcl-2ABSTRACT
The present study was conducted to assess the protective efficacy of a trivalent oral vaccine containing chitosan-PLGA encapsulated inactivated viral haemorrhagic septicemia virus (VHSV), Streptococcus parauberis serotype I and Miamiensis avidus antigens, followed by its oral (incorporated in feed) administration to olive flounder (Paralichthys olivaceus) fingerlings for a period of 15-consecutive days. After 35 days of initial vaccination, three separate challenge studies were conducted at the optimal temperature of the targeted pathogens using an intraperitoneal injection route. RPS analysis revealed moderate protection in the immunized group against all the three pathogens viz., VHSV (53.30% RPS), S. parauberis serotype-I (33.30% RPS), and M. avidus (66.75% RPS), as compared to the respective non-vaccinated challenge (NVC) control group. In addition, the immunized fish demonstrated significantly (p < 0.05) higher specific antibody titres in serum and significant (p < 0.05) upregulation in the transcript levels of immune genes of Igs (IgM, IgT, pIgR), TLRs (TLR 2, TLR 7), cytokines (IL-1ß, IL-8) and complement pathway (C3) in the mucosal and systemic tissues than those of NVC control fish, suggesting orchestration of pathogen-specific host immune responses thereby favouring its combativeness against the three pathogens. The expression dynamics of IFN-γ, Mx, caspase 3 genes post VHSV challenge; IFN-γ, TLR 2, caspase 1 genes post S. parauberis serotype I challenge and CD-8α, IL-10, TNF-α genes post M. avidus challenge further substantiates the efficacy of the vaccine in stimulating antiviral, antibacterial and antiparasitic immune responses in the host resulting in their better survival. The findings from the present study reflect that the formulated trivalent oral vaccine incorporating VHSV, S. parauberis serotype I and M. avidus antigens can be a promising prophylactic strategy to prevent the associated disease outbreaks in olive flounder.
Subject(s)
Chitosan , Fish Diseases , Flounder , Hemorrhagic Septicemia, Viral , Hemorrhagic Septicemia , Novirhabdovirus , Oligohymenophorea , Viral Vaccines , Animals , Fish Diseases/prevention & control , Hemorrhagic Septicemia, Viral/prevention & control , Novirhabdovirus/physiology , Streptococcus , Toll-Like Receptor 2ABSTRACT
Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63-97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.
Subject(s)
Fish Diseases , Flounder , Novirhabdovirus , Animals , Female , Novirhabdovirus/physiology , Phylogeny , RNA, Messenger/metabolismABSTRACT
Production losses of olive flounder (Paralichthys olivaceus) have increased owing to viral haemorrhagic septicaemia virus (VHSV) infection. In this study, we determined safe concentrations of orally administered saponin and chitosan by analysing serum enzyme (AST/ALT) levels as biochemical markers of hepatic injury. Furthermore, we demonstrated the efficacy, duration of protection, and safety of saponin and chitosan-based vaccines with inactivated VHSV (IV). Oral administration of saponin, chitosan, and their combination did not induce fish mortality at all tested concentrations (0.29, 1.45, and 2.9 mg/g of fish body weight/day) 10 days after administration. However, AST level was high at a dose >0.29 mg/g of fish body weight/day. Both saponin and chitosan were found to be safe and acceptable for vaccination studies at a dose of 0.29 mg/g of fish body weight/day. Administration of IV alone did not induce protection at 2 and 4 weeks post vaccination (wpv). Olive flounders administered saponin + IV and chitosan + IV vaccines had higher immunity against VHSV with relative percentage survival (RPS) of 12.5-7.5% and 0-20.1%, respectively; however, additional immunisation with combination of saponin + chitosan + IV clearly enhanced the protection with RPS values of 10-15%, 26.7%, 42.9%, and 37.5% at 4, 8, 12, and 20 wpv, respectively. Although the RPS value of oral immunisation was not comparable to that of injectable vaccines, the manufacturing process is simple and oral administration causes less stress to juvenile fish. To investigate the development of a protective immune response, olive flounder were re-challenged with VHSV (107.8 TCID50/fish) at 70 days postinfection; 100% of the previously unexposed fish died, whereas 80-100% of the previously immunised fish survived. Our results showed the possibility of developing preventive measures against VHSV using saponin and chitosan-based oral vaccines with inactivated virus.
Subject(s)
Chitosan , Fish Diseases , Flounder , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Saponins , Viral Vaccines , Animals , Body Weight , Novirhabdovirus/physiologyABSTRACT
Viral hemorrhagic septicemia virus (VHSV) infection is associated with fatal outcomes in the aquaculture production of olive flounder (Paralichthys olivaceus). Olive flounders at low and high temperatures are known to be highly susceptible and resistant to VHSV infection, respectively. To study temperature-dependent innate immune activity, 4-aminobenzoic hydrazide (4-AH), a myeloperoxidase (MPO) inhibitor, was used to treat VHSV-infected olive flounders reared at a high temperature of 20 °C (20VI). Mortality, the MPO transcription, and the proteomic expression pattern of the 20VI group were then compared with those of groups of VHSV-infected flounders reared at 15 °C (15V) and 20 °C (20V). The cumulative mortality rate of the 20VI group was increased by 35% compared with that of the untreated 20V group. The MPO transcription was decreased 5.8-fold in 20VI than in 20V group. Its expression decreased further at a lower temperature and after exposure to VHSV. Histopathological analysis revealed necrosis of splenic tissue in 20VI and 15V, but not in 20V group. Based on clustering analysis, proteins with increased expression in 15V and 20VI groups were associated with viral mRNA translation and reproduction compared with those of 20V group. Increased expression of DHX58, MX1, and UBB was detected in 15V and 20VI groups, suggesting a role in triggering innate immune response. Unfortunately, these genes failed to induce the translocation of GLUT4 to the surface membrane from the intracellular location due to decreased expression of 14-3-3 proteins (YWHAB and YWHAZ) and microtubules (TUBA1A and TUBB4B). Suppression of glucose supply led to inactivation of MPO and suppression of MHC-I and MHC-II-linked immune activity, resulting in high viral infection and spread. In conclusion, this study highlights that defective GLUT4 translocation-dependent glucose uptake increases the mortality of VHSV-infected olive flounders by inhibiting MPO activity.
Subject(s)
Fish Diseases , Flounder , Hemorrhagic Septicemia, Viral , Novirhabdovirus , Animals , Novirhabdovirus/physiology , Proteomics , TemperatureABSTRACT
The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.