Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

ABSTRACTNervous necrosis virus (NNV) is a member of theBetanodavirusgenus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively.

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Nervous Necrosis Virus Coat Protein Mediates Host Translation Shutoff through Nuclear Translocalization and Degradation of Polyadenylate Binding Protein

Journal of Virology ◽

10.1128/jvi.02364-20 ◽

2021 ◽

Author(s):

Chao-An Cheng ◽

Jia-Ming Luo ◽

Ming-Hsien Chiang ◽

Kuei-Yuan Fang ◽

Chen-Hung Li ◽

...

Keyword(s):

Coat Protein ◽

Marine Fish ◽

Molecular Mechanisms ◽

Fish Larvae ◽

Binding Protein ◽

Economic Damage ◽

Brain Cells ◽

Nervous Necrosis Virus ◽

Epinephelus Coioides ◽

Necrosis Virus

Nervous necrosis virus (NNV) belongs to the Betanodavirus genus of the Nodaviridae family and is the main cause of viral nervous necrosis disease in marine fish larvae and juveniles worldwide. The NNV virion contains two positive-sense, single-stranded RNA genomes, which encode RNA-dependent RNA polymerase, coat protein and B2 protein. Interestingly, NNV infection can shut off host translation in orange-spotted grouper ( Epinephelus coioides ) brain cells, however, the detailed mechanisms of this action remain unknown. In this study, we discovered that the host translation factor, polyadenylate binding protein (PABP), is a key target during NNV takeover of host translation machinery. Additionally, ectopic expression of NNV coat protein is sufficient to trigger nuclear translocalization and degradation of PABP, followed by translation shutoff. A direct interaction between NNV coat protein and PABP was demonstrated, and this binding requires the NNV coat protein N-terminal shell domain and PABP proline-rich linker region. Notably, we also showed that degradation of PABP during later stages of infection is mediated by the ubiquitin-proteasome pathway. Thus, our study reveals that the NNV coat protein hijacks host PABP, causing its relocalization to the nucleus and promoting its degradation to stimulate host translation shutoff. IMPORTANCE Globally, more than 200 species of aquacultured and wild marine fish are susceptible to NNV infection. Devastating outbreaks of this virus have been responsible for massive economic damage in the aquaculture industry, but the molecular mechanisms by which NNV affects its host remain largely unclear. In this study, we show that NNV hijacks translation in host brain cells, with the viral coat protein binding to host PABP to promote its nuclear translocalization and degradation. This previously unknown mechanism of NNV-induced host translation shutoff greatly enhances the understanding of NNV pathogenesis and provides useful insights and novel tools for development of NNV treatments, such as the use of orange-spotted grouper brain cells as an in vitro model system.

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Identification of Host-Specificity Determinants in Betanodaviruses by Using Reassortants between Striped Jack Nervous Necrosis Virus and Sevenband Grouper Nervous Necrosis Virus

Journal of Virology ◽

10.1128/jvi.78.3.1256-1262.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1256-1262 ◽

Cited By ~ 74

Author(s):

Tokinori Iwamoto ◽

Yasushi Okinaka ◽

Kazuyuki Mise ◽

Koh-Ichiro Mori ◽

Misao Arimoto ◽

...

Keyword(s):

Coat Protein ◽

Host Specificity ◽

Polyclonal Antibodies ◽

Infected Fish ◽

Nervous Necrosis Virus ◽

Genomic Segment ◽

Necrosis Virus ◽

Reassortant Virus ◽

Transcription System ◽

Viral Nervous Necrosis

ABSTRACT Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.

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Surveillance of Viruses in Wild Fish Populations in Areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula)

Applied and Environmental Microbiology ◽

10.1128/aem.02090-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6560-6571 ◽

Cited By ~ 10

Author(s):

Patricia Moreno ◽

José G. Olveira ◽

Alejandro Labella ◽

Juan Manuel Cutrín ◽

Jorge C. Baro ◽

...

Keyword(s):

Iberian Peninsula ◽

River Mouth ◽

Wild Fish ◽

Viral Hemorrhagic Septicemia Virus ◽

Nervous Necrosis Virus ◽

Gulf Of Cadiz ◽

Necrosis Virus ◽

Content Type ◽

Gulf Of Cádiz ◽

Common Pandora

ABSTRACTThis report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.

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Fate of Betanodaviruses in the Experimentally Infected Natural and Non-Natural Host

Bangladesh Journal of Microbiology ◽

10.3329/bjm.v24i2.1252 ◽

1970 ◽

Vol 24 (2) ◽

pp. 100-104 ◽

Cited By ~ 2

Author(s):

Ghausiatur Reza Banu ◽

Koh-Ichiro Mori ◽

Misao Arimoto ◽

Toshihiro Nakai

Keyword(s):

Spinal Cord ◽

Tissue Distribution ◽

Experimental Period ◽

Infected Fish ◽

Natural Host ◽

Nervous Necrosis Virus ◽

Necrosis Virus ◽

Infectious Dose ◽

Viral Nervous Necrosis ◽

Natural Host Species

To investigate the fate of betanodavirus, the causative agent of viral nervous necrosis (VNN) in cultured fish, the tissue distribution of virus in experimentally infected fish was investigated. Two genetically different betanodaviruses, striped jack nervous necrosis virus (SJNNV; SJNNV-genotype) and kelp grouper nervous necrosis virus (KGNNV) belonging to red spotted grouper nervous necrosis virus (RGNNV)-genotype, were intramuscularly cross-injected with a dose of 106 TCID50/fish/200 Î¼l in striped jack Pseudocaranx dentex or kelp grouper Epinephelus moara and sacrificed at scheduled to titre the virus in the spinal cord, brain, eye, kidney and blood at day 0, 1, 3, 5, 14 and 21. As a result, intramuscularly inoculated virus was recovered at high titres from the brain, spinal cord, and eye of their natural host species, i.e., striped jack for SJNNV and kelp grouper for KGNNV during 3-week experimental period, while the virus titres were relatively low in the organs of non-natural hosts, particularly in kelp grouper injected with SJNNV. In every case, no virus was detected in blood samples, suggesting that infection did not develop to be systemic. Keywords: Betanodavirus, Viral nervous necrosis, Fate, Tissue distribution, Pseudocaranx dentex, Epinephelus moara, 50% tissue culture infectious dose (TCID50)DOI: http://dx.doi.org/10.3329/bjm.v24i2.1252 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 100-104

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Near-Complete Genome Sequence of a Fish Nervous Necrosis Virus Isolated from Hybrid Grouper in China

Microbiology Resource Announcements ◽

10.1128/mra.01453-19 ◽

2020 ◽

Vol 9 (15) ◽

Author(s):

Peng Jia ◽

Xiaoqi Chen ◽

Jiajie Fu ◽

Meisheng Yi ◽

Wenbo Chen ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Nervous Necrosis Virus ◽

Necrosis Virus ◽

Virus Genotype ◽

Content Type ◽

Hybrid Grouper ◽

Epinephelus Fuscoguttatus

The genome sequence of nervous necrosis virus strain HGN1910, isolated from hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), was cloned, sequenced, and characterized. Two near-complete gene segments were obtained, RNA1 and RNA2. Phylogenetic analysis shows that the virus belongs to the red-spotted grouper nervous necrosis virus genotype of betanodavirus.

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GHSC70 Is Involved in the Cellular Entry of Nervous Necrosis Virus

Journal of Virology ◽

10.1128/jvi.02523-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 61-70 ◽

Cited By ~ 31

Author(s):

Jui-Shin Chang ◽

Shau-Chi Chi

Keyword(s):

Capsid Protein ◽

Fish Species ◽

Receptor Protein ◽

Economic Losses ◽

Nervous Necrosis Virus ◽

Specific Receptor ◽

Necrosis Virus ◽

Content Type ◽

Voltage Dependent ◽

Selective Channel

ABSTRACTNervous necrosis virus (NNV) is a devastating pathogen of cultured marine fish and has affected more than 40 fish species. NNV belongs to the betanodaviruses ofNodaviridaeand 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 inEscherichia colifor 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.IMPORTANCEFish 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.

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Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection

PLoS Pathogens ◽

10.1371/journal.ppat.1009665 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009665

Author(s):

Qing Wang ◽

Cheng Peng ◽

Min Yang ◽

Fengqi Huang ◽

Xuzhuo Duan ◽

...

Keyword(s):

Single Cell ◽

Immune Cells ◽

Neuronal Cell ◽

Cell Types ◽

Infected Fish ◽

Nerve Tissue ◽

Nervous Necrosis Virus ◽

Epinephelus Coioides ◽

Rna Seq ◽

Necrosis Virus

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

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Interaction of Infectious Spleen and Kidney Necrosis Virus ORF119L with PINCH Leads to Dominant-Negative Inhibition of Integrin-Linked Kinase and Cardiovascular Defects in Zebrafish

Journal of Virology ◽

10.1128/jvi.01955-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 763-775 ◽

Cited By ~ 4

Author(s):

Ji-Min Yuan ◽

Bai-Liang He ◽

Lu-Yun Yang ◽

Chang-Jun Guo ◽

Shao-Ping Weng ◽

...

Keyword(s):

Dominant Negative ◽

Zebrafish Embryos ◽

Mandarin Fish ◽

Necrosis Virus ◽

Siniperca Chuatsi ◽

Content Type ◽

Dominant Negative Form ◽

A Genome ◽

Integrin Linked Kinase ◽

Negative Form

ABSTRACTInfectious spleen and kidney necrosis virus (ISKNV) is the type species of theMegalocytivirusgenus,Iridoviridaefamily, causing a severe systemic disease with high mortality in mandarin fish (Siniperca chuatsi) in China and Southeast Asia. At present, the pathogenesis of ISKNV infection is still not fully understood. Based on a genome-wide bioinformatics analysis of ISKNV-encoded proteins, we found that ISKNV open reading frame 119L (ORF119L) is predicted to encode a three-ankyrin-repeat (3ANK)-domain-containing protein, which shows high similarity to the dominant negative form of integrin-linked kinase (ILK); i.e., viral ORF119L lacks the ILK kinase domain. Thus, we speculated that viral ORF119L might affect the host ILK complex. Here, we demonstrated that viral ORF119L directly interacts with particularly interesting Cys-His-rich protein (PINCH) and affects the host ILK-PINCH interactionin vitroin fathead minnow (FHM) cells.In vivoORF119L overexpression in zebrafish (Danio rerio) embryos resulted in myocardial dysfunctions with disintegration of the sarcomeric Z disk. Importantly, ORF119L overexpression in zebrafish highly resembles the phenotype of endogenous ILK inhibition, either by overexpressing a dominant negative form of ILK or by injecting an ILK antisense morpholino oligonucleotide. Intriguingly, ISKNV-infected mandarin fish develop disorganized sarcomeric Z disks in cardiomyocytes. Furthermore, phosphorylation of AKT, a downstream effector of ILK, was remarkably decreased in ORF119L-overexpressing zebrafish embryos. With these results, we show that ISKNV ORF119L acts as a domain-negative inhibitor of the host ILK, providing a novel mechanism for the megalocytivirus pathogenesis.IMPORTANCEOur work is the first to show the role of a dominant negative inhibitor of the host ILK from ISKNV (an iridovirus). Mechanistically, the viral ORF119L directly binds to the host PINCH, attenuates the host PINCH-ILK interaction, and thus impairs ILK signaling. Intriguingly, ORF119L-overexpressing zebrafish embryos and ISKNV-infected mandarin fish develop similar disordered sarcomeric Z disks in cardiomyocytes. These findings provide a novel mechanism for megalocytivirus pathogenesis.

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