Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Tanaka, Shinji; Kuriyama, Isao; Nakai, Toshiko; Miyazaki, Teruo

doi:10.1046/j.1365-2761.2003.00440.x

Cited by 26 publications

(23 citation statements)

References 17 publications

(27 reference statements)

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“…Histopathological changes include extensive cellular vacuolation and necrotic neuronal degeneration in the central nervous system and retina (44). The molecular mechanisms involved in the pathogenesis of this disease are still unknown.…”

Section: Discussionmentioning

confidence: 99%

“…Briefly, recombinant plasmid was amplified by Taq/Pfu DNA polymerase with the aid of the designed primers. After digestion with EcoRI and BglII or BamHI restriction enzyme at 37°C for 3 h, the PCR products were ligated with each vector to create recombinant plasmids including pEYFP-B2, pEYFP-B2(1-27), pEYFP-B2(1-40), pEYGP-B2(1-50), pEYFP-B2(28 -75), pEYFP-B2(51-75), pEYFP-B2(10-50), pEYFP-B2-del [41][42][43][44][45][46][47][48][49][50] , and pEYFP-B2-PM (containing four specific point mutation sites on V44A, I45A, R52A, and R53A).…”

Section: Methodsmentioning

confidence: 99%

“…4A, d-f, indicated by arrows) but not FLAG (Fig. 4A, a-c; negative control) or FLAG-B2-del [41][42][43][44][45][46][47][48][49][50] (Fig. 4A, g-i, function-less control) was able to induce MMP loss in trans- fected GF-1 cells.…”

Section: Protein B2 Mitochondrial Targeting Can Induce Cell Death Medmentioning

confidence: 98%

“…Consistent with the results of the MMP loss assay (Fig. 4, A and B), the annexin-V assay showed induction of cell death by EYFP-B2 (33%) and B2 protein fragments containing the mitochondrial targeting sequence (1-50 (39%), 28 -75 (46%), and 10 -50 (48%)) but not by fragments not containing the sequence (1-27 (2%), 1-40 (2%), 51-75 (1%), EYFP-B2-del [41][42][43][44][45][46][47][48][49][50] levels slightly in GF-1 (91%) and 293T cells (93%). On the other hand, complex I inhibitor DPI (100 mM) and complex II inhibitor 3-NP (10 mM), used as positive controls, limited the amount of ATP depletion to 22 (GF-1 cells) and 31% (293T cells) for DPI and to 53 (GF-1 cells) and 56% (293T cells) for 3-NP.…”

Section: Protein B2 Mitochondrial Targeting Can Induce Cell Death Medmentioning

confidence: 99%

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

Hong

2010

Journal of Biological Chemistry

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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 postfertilization. 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.Betanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina and by clinical signs (e.g. abnormal swimming behavior and development of a darker body color) (1). This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance (2).The family Nodaviridae is comprised of the genera Alphanodavirus and Betanodavirus. Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3-5). Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated (3). RNA1 encodes an ϳ110-kDa non-structural protein that has been designated RNA-dependent RNA polymerase or protein A. This protein is vital for replication of the viral genome. RNA2 encodes a 42-kDa capsid protein (6, 7), which may also function in the induction of cell death (8, 9). Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3Ј terminus of RNA1. RNA3 contains two putative open reading frames that potentially encode an 111-amino acid protein B1 and a 75-amino acid protein B2 (3, 10, 11). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10). In contrast, betanodaviru...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Protein B2 Mitochondrial Targeting Can Induce Cell Death Medmentioning

confidence: 98%

Section: Protein B2 Mitochondrial Targeting Can Induce Cell Death Medmentioning

confidence: 99%

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

Hong

2010

Journal of Biological Chemistry

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“…As juvenile Japanese flounder less than approximately 20 mm in total length or 0.1 g in body weight are so susceptible to betanodavirus isolates (RGNNV) in experimental and natural infections (Nguyen et al, 1994;Tanaka et al, 2003), this fish species seemingly has a strongly age-dependent protection against betanodavirus infection. Following our previous report (Pakingking et al, 2004), we tested the antiviral activity in the sera of ABV-inoculated flounder against RGNNV by an in vitro cell protection assay employing E-11 cells.…”

Section: Discussionmentioning

confidence: 99%

Aquabirnavirus-induced Protection of Marine Fish against Piscine Nodavirus Infection

et al. 2005

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ABSTRACT--Experimental dual-infections with a non-lethal aquabirnavirus (ABV) and a lethal betanodavirus (redspotted grouper nervous necrosis virus: RGNNV) were carried out in Japanese flounder Paralichthys olivaceus and sevenband grouper Epinephelus septemfasciatus.In the dual-infection group, ABV was intramuscularly (IM) injected into fish seven days before the IM-injec tion with RGNNV. In the experiments with flounder, a high expression of an Mx gene, a molecular marker for type I interferon(s) (IFN) production, occurred in the head kidneys and brains at Day 7 post-ABV injection. Although no mortality was found not only in the dual-infected group but also in the single infection group with RGNNV (control group), the infective titers of RGNNV in the tissues of the dual-infected group were significantly lower at any sampling times than those in the control group. In the experiments with grouper, the preceding ABV infection resulted in complete protec tion against RGNNV infection. The infective titers of RGNNV in the tissues were also lower in the dual-infected group than in the control group throughout the experiments, and finally the virus dis appeared from the head kidneys and brains of the dual-infected group at Day 14 and Day 56 post injections, respectively. These results suggest that an ABV-induced IFN(s) effectively suppresses the progression of secondary betanodavirus infection.

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Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study

Wang

Zhang

Wang

et al. 2017

Fish Physiol Biochem

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In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

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Susceptibility of cultured juveniles of several marine fish to the sevenband grouper nervous necrosis virus

Cited by 26 publications

References 17 publications

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

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

Aquabirnavirus-induced Protection of Marine Fish against Piscine Nodavirus Infection

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study

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