GF-1 Cells Research Articles

Molecular cloning of orange-spotted grouper (Epinephelus coioides) heat shock transcription factor 1 isoforms and characterization of their expressions in response to nodavirus

Heat shock transcription factor 1 (HSF1) regulates heat shock proteins (HSPs), which assist in protein folding and inhibit protein denaturation following stress. HSF1 was firstly cloned from orange-spotted grouper and exists as two isoforms, one with (osgHSF1a) and one without (osgHSF1b) exon 11. Heat exposure increased the expression of osgHSF1b while cold exposure increased that of osgHSF1a. Both isoforms were mainly expressed in the brains, eyes, and fins. Expression of osgHSF1b was higher than osgHSF1a during development. Poly I:C and LPS could also induce osgHSF1 isoforms expression differentially. Exposure to nervous necrosis virus (NNV) increased the level of both osgHSF1 isoforms at 12 h. GF-1 cells with overexpression of osgHSF1 isoforms enhanced viral loads within 24 h, whereas both pharmacological inhibition and RNA interference of HSF1 reduced virus infection. This study shows that osgHSF1 can support the early stage of virus infection and provides a new insight into the molecular regulation of osgHSF1 between the influence of temperatures and immunity.

Abstract 4099: Oligodendrocyte stimulates the invasion ability of glioblastoma cells

Abstract Background & Aims: Glioblastoma (GBM, WHO Grade IV) is characterized by rapid cancer cell infiltration, and is one of the most lethal neoplasms among solid cancers. GBM might arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interaction between GBM cells and glial cells. Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived from surgical tissues of each patient with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived from surgical tissues of each patient with GBM. The culture medium was composed of DMEM with 10% fetal calf serum. We examined the effects of glial cells on the invasion ability of GBM cells by wound-healing assay and invasion assay. Using IncuCyte invasion assay and matrigel invasion assay, the invasiveness of T98G and U251 cells was analyzed in the presence or absence of conditioned medium from oligodendrocyte cells or fibroblasts. The effect of the proliferation of T98G and U251 cells was examined by MTT assay or cell count. Results: ODC1 and ODC2 cells were positive for Olig2 staining but not for GFAP staining, suggesting that ODC1 and ODC2 were oligodendrocyte cells. GF1 and GF2 cells were spindle-shape and were negative for both Olig2 staining and GFAP staining, suggesting that GF1 and GF2 cells were fibroblasts. Oligodendrocyte cells, ODC1 and ODC2 cells, from grade II glioma significantly (p<0.01) increased the migration and invasion ability of GBM cells, T98G and U251 cells. In contrast, GF1 and GF2 fibroblasts from GBM did not affect on the migration and invasion ability of GBM cells. Both oligodendrocyte cells and fibroblasts did not stimulate the proliferation of T98G and U251 cells. Conclusion: Oligodendrocytes might up-regulate the invasiveness of malignant glioma cells. An invasion-stimulating factor(s) from oligodendrocyte cells might be partially associated with the progression from lowe-grade gliomas to GBM. Citation Format: Toshiyuki Kawashima. Oligodendrocyte stimulates the invasion ability of glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4099.

Molecular and functional characterization of autophagy marker LC3 in orange-spotted grouper (Epinephelus coioides)

Autophagy is a conserved cellular process in response to stress, sustain normal cell growth by degrade and recycle unnecessary cytosolic components in autophagosome and autolysosome. Microtubuleassociated protein light chain 3 known as LC3 is a major constituent of the autophagosome and used as a reliable marker for autophagy. In this study, a full length of orange-spotted grouper LC3 (osgLC3) mRNA had been cloned with a size of 1,440 bp including the sequence encoded for a 126 a.a. protein. The phylogenetic relationship and relative tissue mRNA expression levels for osgLC3 has been evaluated. The punctate structures increased in the GF-1 cells with over-expressed osgLC3 under nutrient deprivation revealed that starvation induced autophagy formation and the colocalization of punctate structures and lysosome indicated autolysosome formation during autophagy induction. Additionally, it has found that autophagy formation in GF-1 cells induced by nervous necrosis virus (NNV) infection through quantitation of endogenous osgLC3-positive punctate structures. In grouper larvae, the mRNA expression of osgLC3 were increased under NNV infection and the osgLC3 was able to convert to lipidated form of LC3 (osgLC3-II) for synthesis of autophagosome. Moreover, it has been verified that Hsp90 plays a role in autophagy and here the osgLC3 and Hsp90AB had higher FRET efficiency that implied the interaction between osgLC3 and Hsp90AB. Based on these results, osgLC3 contributed to the autophagy formation in response to NNV infection in grouper and the working mechanisms of osgLC3 and Hsp90AB during virus infection need further studies.

Infectious spleen and kidney necrosis virus (ISKNV) used a novel strategy to modulate the mitochondria/ caspases -mediated cell death pathway via ROS signaling in a fish cell line

Iridoviruses are large DNA viruses that infect invertebrates and poikilothermic vertebrates, such as insects, fish, amphibians, reptiles, crustaceans, and mollusks. The emergence of deadly iridoviral disease in aquatic fish has led to a growing interest in the mechanism of iridovirus-induced host cell death. In this present, we found that infectious spleen and kidney necrosis virus (ISKNV) can trigger a host cellular death via ROS signaling in grouper fin (GF-1) cells. ISKNV infection can induce reactive oxygen species (ROS) production by using H2DCFDA assay at early replication stage. Then, this ROS signaling can upregulate the pro-apoptotic genes Bax and Bak expressions at middle replication stage. Moreover, ISKNV-induced ROS-mediated signals triggered the mitochondrial membrane potential (MMP) loss, which correlated to activate downstream caspases such as caspase-8, caspase-9 and caspase-3 at late replication stage. Final, we found that antioxidants treatment dramatically reduced ISKNV-induced host cellular death in GF-1 cells. Taken together our data show that ISKNV used a novel strategy to regulate the mitochondria/caspase-mediated cell death pathway via ROS signaling, which may provide new insight into DNA virus control and treatment.

GSIV serine/threonine kinase can induce apoptotic cell death via p53 and pro-apoptotic gene Bax upregulation in fish cells.

Previous studies have shown that GSIV induces apoptotic cell death through upregulation of the pro-apoptotic genes Bax and Bak in Grouper fin cells (GF-1 cells). However, the role of viral genome-encoded protein(s) in this death process remains unknown. In this study, we demonstrated that the Giant seaperch iridovirus (GSIV) genome encoded a serine/threonine kinase (ST kinase) protein, and induced apoptotic cell death via a p53-mediated Bax upregulation approach and a downregulation of Bcl-2 in fish cells. The ST kinase expression profile was identified through Western blot analyses, which indicated that expression started at day 1h post-infection (PI), increased up to day 3, and then decreased by day 5 PI. This profile indicated the role of ST kinase expression during the early and middle phases of viral replication. We then cloned the ST kinase gene and tested its function in fish cells. The ST kinase was transiently expressed and used to investigate possible novel protein functions. The transient expression of ST kinase in GF-1 cells resulted in apoptotic cell features, as revealed with Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) assays and Hoechst 33258 staining at 24h (37%) and 48h post-transfection (PT) (49%). Then, through studies on the mechanism of cell death, we found that ST kinase overexpression could upregulate the anti-stress gene p53 and the pro-apoptotic gene Bax at 48h PT. Interestingly, this upregulation of p53 and Bax also correlated to alterations in the mitochondria function that induced loss of mitochondrial membrane potential (MMP) and activated the initiator caspase-9 and the effector caspase-3 in the downstream. Moreover, when the p53-dependent transcriptional downstream gene was blocked by a specific transcriptional inhibitor, it was found that pifithrin-α not only reduced Bax expression, but also averted cell death in GF-1 cells during the ST kinase overexpression. Taken altogether, these results suggested that aquatic GSIV ST kinase could induce apoptosis via upregulation of p53 and Bax expression, resulting in mitochondrial disruption, which activated a downstream caspases-mediated cell death pathway.

Giant seaperch iridovirus infection upregulates Bas and Bak expression, leading to apoptotic death of fish cells

The giant seaperch iridovirus (GSIV) induces host cell apoptosis by a poorly-understood process. In this study, GSIV is shown to upregulate the pro-apoptotic death genes Bax and Bak at the middle replication stage, and factors in the grouper fin cell line (GF-1) are shown to modulate this process. Studying the mechanism of cell death, we found that upregulated, de novo-synthesized Bax and Bak proteins formed heterodimers. This up-regulation process correlated with mitochondrial membrane potential (MMP) loss, increased caspase-3 activity, and increased apoptotic cell death. All effects were diminished by treatment of infected GF-1 cells with the protein synthesis inhibitor cycloheximide. Interestingly, overexpression of the anti-apoptotic gene Bcl-xL also diminished GSIV-induced mitochondria-mediated cell death, increasing host cell viability and decreasing MMP loss at the early replication stage. Our data suggest that GSIV induces GF-1 apoptotic cell death through up-regulation of the pro-apoptotic genes Bax and Bak, which are regulated by Bcl-xL overexpression on mitochondria in GF-1 cells.

Grouper voltage-dependent anion selective channel protein 2 is required for nervous necrosis virus infection.

Nervous necrosis virus (NNV) is a non-enveloped virus with 2 segmented positive-sense single-stranded RNAs. NNV-induced mass mortality has occurred worldwide in many species of cultured marine fish and resulted in substantial economic losses. In our previous study, we cloned the gene of voltage-dependent anion selective channel protein 2 (GVDAC2), and the NNV RNA2 expression level decreased in GVDAC2-knockdown GF-1 cells 24 h after infection. Here, we investigated the role of GVDAC2 in the NNV infection in GF-1 cells. NNV infection did not considerably affect GVDAC2 gene expression. After performing immunostaining, we detected GVDAC2 at the mitochondria and GVDAC2 was colocalized with NNV-RNA-dependent RNA polymerase. However, these 2 proteins did not interact with each other in immunoprecipitation assay. The cellular ATP level in GVDAC2-downregulated cells was lower than that in control cells, and NNV-induced apoptosis was delayed in GVDAC2-siRNA-transfected cells. Therefore, we suggest that GVDAC2 is required for NNV infection for maintaining the cellular ATP level and has positive impact on virus-induced apoptosis.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC–MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay.

GHSC70 is involved in the cellular entry of nervous necrosis virus.

Nervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses of Nodaviridae and is a nonenveloped icosahedral particle with 2 single-stranded positive-sense RNAs. To date, knowledge regarding NNV entry into the host cell remains limited, and no NNV-specific receptor protein has been published. Using grouper fin cell line GF-1 and purified NNV capsid protein in a virus overlay protein binding assay (VOPBA), grouper heat shock cognate protein 70 (GHSC70) and grouper voltage-dependent anion selective channel protein 2 (GVDAC2) were investigated as NNV receptor protein candidates. We cloned and sequenced the genes for GHSC70 and GVDAC2 and expressed them in Escherichia coli for antiserum preparation. Knockdown of the expression of GHSC70 and GVDAC2 genes with specific short interfering RNAs (siRNAs) significantly downregulated viral RNA expression in NNV-infected GF-1 cells. By performing an immunoprecipitation assay, we confirmed that GHSC70 interacted with NNV capsid protein, while VDAC2 did not. Immunofluorescence staining and flow cytometry analysis revealed the presence of the GHSC70 protein on the cell surface. After a blocking assay, we detected the NNV RNA2 levels after 1 h of adsorption to GF-1 cells; the level was significantly lower in the cells pretreated with the GHSC70 antiserum than in nontreated cells. Therefore, we suggest that GHSC70 participates in the NNV entry of GF-1 cells, likely functioning as an NNV receptor or coreceptor protein. Fish nodavirus has caused mass mortality of more than 40 fish species worldwide and resulted in huge economic losses in the past 20 years. Among the four genotypes of fish nodaviruses, the red-spotted grouper nervous necrosis virus (RGNNV) genotype exhibits the widest host range. In our previous study, we developed monoclonal antibodies with high neutralizing efficiency against grouper NNV in GF-1 cells, indicating that NNV-specific receptor(s) may exist on the GF-1 cell membrane. However, no NNV receptor protein has been published. In this study, we found GHSC70 to be an NNV receptor (or coreceptor) candidate through VOBPA and provided several lines of evidence demonstrating that GHSC70 protein has a role in the NNV entry step of GF-1 cells. To the best of our knowledge, this is the first report identifying grouper HSC70 and its role in NNV entry into GF-1 cells.

Beta-nodavirus B2 protein induces hydrogen peroxide production, leading to Drp1-recruited mitochondrial fragmentation and cell death via mitochondrial targeting.

Because the role of the viral B2 protein in the pathogenesis of nervous necrosis virus infection remains unknown, the aim of the present study was to determine the effects of B2 protein on hydrogen peroxide (H2O2)-mediated cell death via mitochondrial targeting. Using a B2 deletion mutant, the B2 mitochondrial targeting signal sequence (41RTFVISAHAA50) correlated with mitochondrial free radical production and cell death in fish cells, embryonic zebrafish, and human cancer cells. After treatment of grouper fin cells (GF-1) overexpressing B2 protein with the anti-oxidant drug, N-acetylcysteine (NAC), and overexpression of the antioxidant enzymes, zfCu/Zn superoxide dismutase (SOD) and zfCatalase, decreased H2O2 production and cell death were observed. To investigate the correlation between B2 cytotoxicity and H2O2 production in vivo, B2 was injected into zebrafish embryos. Cell damage, as assessed by the acridine orange assay, gradually increased over 24 h post-fertilization, and was accompanied by marked increases in H2O2 production and embryonic death. Increased oxidative stress, as evidenced by the up-regulation of Mn SOD, catalase, and Nrf2, was also observed during this period. Finally, B2-induced dynamin-related protein 1 (Drp1)-mediated mitochondrial fragmentation and cell death could be reversed by NAC and inhibitors of Drp1 and Mdivi in GF-1 cells. Taken together, betanodavirus B2 induces H2O2 production via targeting the mitochondria, where it inhibits complex II function. H2O2 activates Drp1, resulting in its association with the mitochondria, mitochondrial fission and cell death in vitro and in vivo.

EcVig, a novel grouper immune-gene associated with antiviral activity against NNV infection

VHSV-induced genes (VIGs) were first identified in rainbow trout (Oncorhynchus mykiss) and subsequently isolated in a variety of fish. Recent studies have shown that most VIGs have immunological functions against pathogenic infections. However, most research has focused on Vig1, such that our present understanding of these genes in other fish species remains limited. This study isolated a homologue of the uncharacterized O. mykiss Vig-B319 (EcVig) from orange-spotted grouper (Epinephelus coioides). Genomic organization suggests that four EcVig isoforms (EcVig A–D), are generated through alternative splicing. Due to the encoding of 2 immunoglobulin (Ig) domains, the EcVig protein can be considered a member of the immunoglobulin superfamily. The expression of EcVig increased 3days after hatching (dph) and peaked at 9dph. This pattern is similar to that displayed by EcMx, an important grouper antiviral gene. Additionally, a tissue tropism assay revealed that EcVig A is the major EcVig isoform present in the tissues considered by this study, with the expression of EcVig A exceeding that of EcVig B. We subsequently investigated whether EcVig expression was induced by the viral pathogen nervous necrosis virus (NNV) or the bacterial pathogen Vibrio anguillarum. Following injection with NNV, the expression levels of EcVig showed significant up-regulation. Conversely, a significant reduction was observed in EcVig expression in brain samples collected from V. anguillarum injected grouper. The overexpression of EcVig A suppressed the replication of NNV in grouper GF-1 cell lines, suggesting that EcVig is an important antiviral factor in the grouper immune responses.

Atlantic Salmon Reovirus Infection Causes a CD8 T Cell Myocarditis in Atlantic Salmon (Salmo salar L.)

Heart and skeletal inflammation (HSMI) of farmed Atlantic salmon (Salmo salar L.) is a disease characterized by a chronic myocarditis involving the epicardium and the compact and spongious part of the heart ventricle. Chronic myositis of the red skeletal muscle is also a typical finding of HSMI. Piscine reovirus (PRV) has been detected by real-time PCR from farmed and wild salmon with and without typical changes of HSMI and thus the causal relationship between presence of virus and the disease has not been fully determined [1]. In this study we show that the Atlantic salmon reovirus (ASRV), identical to PRV, can be passaged in GF-1 cells and experimental challenge of naïve Atlantic salmon with cell culture passaged reovirus results in cardiac and skeletal muscle pathology typical of HSMI with onset of pathology from 6 weeks, peaking by 9 weeks post challenge. ASRV replicates in heart tissue and the peak level of virus replication coincides with peak of heart lesions. We further demonstrate mRNA transcript assessment and in situ characterization that challenged fish develop a CD8+ T cell myocarditis.

Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells.

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12–24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24–48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection.Furthermore, localization of ROS production using esterase activity and Mitotracker staining assays found that the ROS generated can affect mitochondrial morphology changes and causes ΔΨ loss, both of which can be reversed by antioxidant treatment. Taken together, our data suggest that RGNNV induced oxidative stress response for playing dual role that can initiate the host oxidative stress defense system to upregulate expression of antioxidant enzymes and induces cell death via disrupting the mitochondrial morphology and inducing ΔΨ loss, which can be reversed by anti-oxidants and zfcatalase, which provide new insight into betanodavirus-induced ROS-mediated pathogenesis.

Grouper (Epinephelus coioides) CXCR4 is expressed in response to pathogens infection and early stage of development

Chemokine (C-X-C motif) receptor 4 (CXCR4) from orange-spotted grouper (Epinephelus coioides) was identified and characterized in this study. gCXCR4 shared common features in protein sequence and predicted structure of CXCR4 family. This suggested that gCXCR4 is a member of G protein-coupled receptors with seven transmembrane domains. The expression patterns revealed that gCXCR4 may play a key role in early development of grouper. Furthermore, overexpression of gCXCR4-GFP for 48 h had significant effects on the GF-1 cell viability. gCXCR4 protein was mainly expressed in the marginal zone of head kidney and on the surface of intestinal villi. gCXCR4 expression can be detected in all the examined tissues and significantly up-regulated in eye and brain, which are the main targets for nervous necrosis virus (NNV) infection and replication. gCXCR4 gene expression can be induced in the spleen and eye by lipopolysaccharide and NNV, respectively. Our data suggested that gCXCR4 may not only play a role in the early immune response to microbial infection but also restrain to the immune system and central nervous system.

Betanodavirus B2 Causes ATP Depletion-induced Cell Death via Mitochondrial Targeting and Complex II Inhibition in Vitro and in Vivo

The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide ((41)RTFVISAHAA(50)) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h post-fertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis.

The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro-apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)-beta1 in vitro. bFGF induced caspase-3 activation and apoptosis in TGF-beta1-pretreated granulation tissue-derived fibroblasts (GF-1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF-1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF-1 cells pretreated with TGF-beta1. Pretreatment of GF-1 with TGF-beta1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF-beta1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF-1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF-injected skin wounds. These results led to the hypothesis that temporal activation of TGF-beta1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation.

Antimicrobial peptides (AMP) with antiviral activity against fish nodavirus

Nervous necrosis virus (NNV) is classified as betanodavirus of Nodaviridae, and has caused mass mortality of numerous marine fish species at larval stage. Antimicrobial peptides (AMPs) play an important role of innate immunity either against bacterial pathogens or viruses. Up to date, little is known if any AMP could effectively inhibit fish nodaviruses and its mechanism. In this study, the antiviral activities of three antimicrobial peptides (AMPs) against grouper NNV (GNNV) were screened in the fish cell line. Two of the three AMPs, tilapia hepcidin 1-5 (TH 1-5) and cyclic shrimp anti-lipopolysaccharide factor (cSALF), were able to agglutinate purified NNV particles into clump, and the clumps were further confirmed to be viral proteins by TEM and Western blot. The NNV solution, separately pre-mixed with AMP (TH 1-5 or cSALF) or deionized-distilled water for 1 h, was used to infect GF-1 cells, and the levels of capsid protein in the GNNV-AMP-infected cells at 1 h post infection were much lower than that in the GNNV-H 2O-infected cells, indicating that only a small portion of viral particles in the GNNV-AMP mixture could successfully infected the cells. Treatment of cBB cells with TH 1-5 and cSALF did not induce Mx gene expression; however, grouper epinecidin-1 (CP643-1) could induce the expression of Mx in the pre-treated cBB cells. This study revealed three AMPs with anti-NNV activity through two different mechanisms, and shed light on the future application in aquaculture.

Zebrafish anti-apoptotic protein zfBcl-x L can block betanodavirus protein α-induced mitochondria-mediated secondary necrosis cell death

Betanodavirus protein α induces cell apoptosis or secondary necrosis by a poorly understood process. In the present work, red spotted grouper nervous necrosis virus (RGNNV) RNA 2 was cloned and transfected into tissue culture cells (GF-1) which then underwent apoptosis or post-apoptotic necrosis. In the early apoptotic stage, progressive phosphatidylserine externalization was evident at 24 h post-transfection (p.t.) by Annexin V-FLUOS staining. TUNEL assay revealed apoptotic cells at 24–72 h p.t, after which post-apoptotic necrotic cells were identified by acridine orange/ethidium bromide dual dye staining from 48 to 72 h p.t. Protein α induced progressive loss of mitochondrial membrane potential (MMP) which was detected in RNA2-transfected GF-1 cells at 24, 48, and 72 h p.t., which correlated with cytochrome c release, especially at 72 h p.t. To assess the effect of zfBcl-x L on cell death, RNA2-transfected cells were co-transfected with zfBcl-x L . Co-transfection of GF-1 cells prevented loss of MMP at 24 h and 48 h p.t. and blocked initiator caspase-8 and effector caspase-3 activation at 48 h p.t. We conclude that RGNNV protein α induces apoptosis followed by secondary necrotic cell death through a mitochondria-mediated death pathway and activation of caspases-8 and -3.

Production of monoclonal antibodies against grouper nervous necrosis virus (GNNV) and development of an antigen capture ELISA

Five (2 IgG, 3 IgM) monoclonal antibodies (MAbs) against the G9508KS strain of grouper nervous necrosis virus (GNNV) were produced and characterized. All 5 MAbs showed positive signals in the retina of GNNV-infected grouper larvae and in the cytoplasm of GNNV-infected GF-1 cells using immunohistochemistry staining. Two MAbs reacted with the denatured capsid protein derived from GNNV-infected GF-1 cells in Western blot analysis, but did not react with the GNNV recombinant capsid protein expressed by E. coli in an indirect immnunosorbent assay (ELISA). All 5 MAbs were able to neutralize GNNV, tiger puffer NNV (TPNNV) and barfin flounder NNV (BFNNV), while only 2 of the MAbs neutralized striped jack NNV (SJNNV). A capture ELISA system based on the use of MAbs for capture and a rabbit polyclonal antibody for detection was developed. When absorbance values higher than 0.5 were judged to be positive, the sensitivity of the capture ELISA system was 2.5 ng per well of purified GNNV protein or 6.5 x 10(4) TCID50 per well of GNNV supernatant from culture cells. This capture ELISA system could become a more specific and sensitive tool for NNV diagnosis in the field and in routine laboratories.

Genetic and antigenic analysis of betanodaviruses isolated from aquatic organisms in Taiwan.

Viral nervous necrosis (VNN) is a worldwide disease among marine fishes. In Taiwan, NNN disease was first identified in 2 species of hatchery-reared grouper, Epinephelus fuscogutatus and E. akaaya in 1994. Since then, increasing mortalities have occurred among groupers Epinephelus spp., and also among European eels Anguilla anguilla L., yellow-wax pompano Trachinotus falcatus, firespot snapper Lutaanus erythropterus B., barramundi Lates calcarifer, cobias Rachycentron canadum, humpback groupers Cromileptes altivelis and Chinese catfish Parasilurus asotus. In the present study, samples were collected from affected fishes and processed for reverse transcriptase (RT) PCR amplification and virus isolation in cell culture. Infected cells (GF-1 cell line) exhibited cytopathic-effect characteristics of grouper nervous necrosis virus (GNNV). A RT-PCR product of approximately 830 bp was amplified from the brain homogenate of tested samples and sequenced. The nucleotide and deduced amino acid sequences of the amplified RT-PCR products from all isolates were strongly homologous (> 97 %) with the corresponding region of the published sequence of red-spotted grouper nervous necrosis virus (RGNVV). Therefore, all Taiwan NNV (nervous necrosis virus) isolates studied in this report belong to the RGNNV genotype. We used 5 neutralizing monoclonal antibodies (MAbs) against GNNV to analyze the antigenic relationship of Taiwan NNV isolates and striped jack nervous necrosis virus (SJNNV). The results of neutralization tests revealed that all Taiwan NNV isolates were closely related, but antigenically different from SJNNV in 3 neutralizing epitopes. To our knowledge, this is the first description of NNV infection in European eels, yellow-wax pompano, firespot snapper, cobia and Chinese catfish, and the first reported instance of natural NNV infection in freshwater fishes causing high mortality.