Agent Of Viral Nervous Necrosis Research Articles

Identification of host-specificity determinants in betanodaviruses by using reassortants between striped jack nervous necrosis virus and sevenband grouper nervous necrosis virus.

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.

Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer

Betanodaviruses, members of the Nodaviridae family, are the causative agents of viral nervous necrosis in fish and infection by which cause high mortality in larvae and juveniles in a wide range of marine fish species in Asia, Europe, Australia, Martinique, and Tahit. Greasy grouper ( Epinephelus tauvina) nervous necrosis viruses (GGNNV) were investigated for their apoptotic activity in culture cells. GGNNV infection of sea bass (SB) cells appeared to induce a typical cytopathic effect (CPE), i.e., cytoplasmic vacuolation, thinning, rounding up, detachment of infected cells from the cultured dish, and eventually cell lysis and death. The infected SB cells underwent DNA fragmentation and stained positive in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay, suggesting that GGNNV infection induced apoptosis in SB cells. In addition, GGNNV-infected SB cells showed an increased activity of caspase-8-like proteases (IETDase) and caspase-3-like proteases (IETDase), whereas inhibitor of caspase-8 and caspase-3 reduced GGNNV-induced apoptosis. This suggests that GGNNV may promote apoptosis via the extrinsic pathway in SB cells. Protein α, the precursor of GGNNV capsid proteins, was transiently expressed in SB and Cos-7 cells. The DNA fragmentation and TUNEL positive signal were apparent in SB and Cos-7 cells expressing protein α, suggesting that protein α may serve as an apoptotic inducer in these cells. Moreover, expression of protein α resulted in the activation of caspase-3-like proteases in both cells, which could be inhibited by a caspase-3-like protease specific inhibitor DEVD-CHO peptide. These results suggest that fish caspases are important elements in GGNNV-meditated apoptosis.

Nodavirus Infection in Hatchery-Reared Orange-Spotted Grouper Epinephelus coioides: First Record of Viral Nervous Necrosis in the Philippines.

Mass mortality occurred in 34-day old larval orange-spotted grouper Epinephelus coioides reared at a hatchery in the Philippines with clinical signs such as anorexia and abnormal swimming behavior. Histopathology of moribund fish demonstrated marked vacuolation of the brain, spinal cord and retina. Cytopathic effects were observed in SSN-1 cells inoculated with the tissue filtrate of affected grouper. Electron microscopy revealed non-enveloped virus particles measuring 20 to 25 nm in diameter in the cytoplasm of degenerated SSN-1 cells. Piscine nodavirus (betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues and SSN-1 cells inoculated with the tissue filtrate of affected fish by RT-PCR. This is the first record of VNN in the Philippines.

Nodavirus infection of juvenile white seabass, Atractoscion nobilis, cultured in southern California: first record of viral nervous necrosis (VNN) in North America

The viral aetiology of mass mortalities of white seabass, Atractoscion nobilis, cultured in southern California, USA was examined. Disease outbreaks occurred in juvenile fish reared at two culture facilities from June to December 1999, with clinical signs such as anorexia and erratic swimming motion. Microscopic lesions observed in moribund fish included marked vacuolation of brain, spinal cord and retina. The piscine nodavirus (Betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues by electron microscopy, indirect fluorescent antibody test (IFAT), reverse transcription–polymerase chain reaction (RT–PCR), and isolation in cell culture. The agent was identified as one of the four known genotypes of piscine nodavirus. In addition, a similar nodavirus was also detected in fish samples from disease outbreaks at the same facility in 1992. In the last decade, VNN has been reported among cultured populations of marine fish worldwide and this paper is the first record of the agent in North America.

A combined cell‐culture and RT–PCR method for rapid detection of piscine nodaviruses

A rapid method to detect piscine nodaviruses, the causal agents of viral nervous necrosis (VNN), at low virus levels is described. The reverse transcription–polymerase chain reaction (RT–PCR) technique required 104–105 TCID50 for detection of four strains representing different genotypes of piscine nodavirus: striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV) and barfin flounder nervous necrosis virus (BFNNV). All the genotypic variants were isolated at the lowest titre (100 TCID50) by the E‐11 cell line, a clone derived from the SSN‐1 cell line, but it took approximately 10 days until the cytopathic effect (CPE) appeared in cells inoculated at low virus levels. When the virus was inoculated at various concentrations in E‐11 cells and incubated at 20 or 25 °C, cells inoculated previously with virus at a dose of 100 TCID50 became positive within 48 h incubation in the RT–PCR test. The present combined procedure of cell‐culture and RT–PCR techniques will be useful as a rapid and convenient method to detect infective viral particles from asymptomatic carriers or samples with low virus levels.

Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia

Nodaviruses are widespread causative agents of viral nervous necrosis in fish. Based on the coat protein sequence, fish nodaviruses are categorized into four different genotypes. In this study, we present data on the phylogenetic and antigenic characterization of 12 new isolates, eight European and four of Asian origin, from farmed and wild species of fish. Phylogenetic analysis based on the nucleotide sequence (688 bases) of the coat protein classified the majority of these new isolates to the RGNNV genotype. Geographic or host-species specificities were not revealed by this study. Neutralizing assay experiments, further confirmed the genotypic classification, supporting the possibility that the different nodavirus genotypes can also be serologically distinguishable.

Protective immunity of sevenband grouper, Epinephelus septemfasciatus Thunberg, against experimental viral nervous necrosis

This paper describes the protective immune responses of sevenband grouper, Epinephelus septemfasciatus Thunberg, immunized with live piscine nodavirus, the causative agent of viral nervous necrosis (VNN), or the Escherichia coli– expressed recombinant coat protein. Nodavirus‐neutralizing antibodies were detected at titres ranging from 1:158 to 1:1257 in serum of sevenband grouper which survived intramuscular injection with the virus, by a cell culture assay system. The virus‐neutralizing ability of immune serum was also confirmed by injecting virus previously treated with serum into fish. This indicates establishment of acquired immunity in survivors and thus explains why survivors from natural infection are resistant to recurrence of the disease. Young sevenband grouper were immunized twice by intramuscular injections with the recombinant coat protein. Immunized fish produced neutralizing antibodies at high titres for at least 110 days and showed significantly lower mortalities in virus challenge tests. These results suggest the potential for vaccination against VNN in sevenband grouper, which is susceptible to piscine nodavirus at all life‐stages.

Selection of brood stock candidates of barfin flounder using an ELISA system with recombinant protein of barfin flounder nervous necrosis virus.

Barfin flounder nervous necrosis virus (BFNNV), the causative agent of viral nervous necrosis (VNN) of barfin flounder, is vertically transmitted from spawners to larvae. In the present study, an ELISA with a recombinant protein of BFNNV was performed for the detection of antibodies against BFNNV and applied for the selection of brood fish in order to prevent viral vertical transmissions. Brood stocks were divided into 4 groups based on ELISA antibody titers (< or = 10, 20, 40 and >40), and the BFNNV status of the brood stocks was determined by PCR. BFNNV was detected from the brood fish in the group with an antibody titer of >40 but not from those with titers < or = 10, 20 and 40. The offspring obtained from PCR-negative brood fish pairs in each group of ELISA antibody titers were subsequently reared for observation of VNN occurrence. VNN occurred in juveniles from 2 of 9 pairs of spawners with an antibody titer > or = 40, but did not occur in spawners with an antibody titer of < or = 10. Therefore, it was concluded that selection of brood fish using both the PCR test and ELISA antibody titers could help prevent vertical transmission of BFNNV in larval production of barfin flounder.

Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus.

Striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis in marine fish, is a member of the family Nodaviridae whose genome consists of two positive-sense RNA molecules encapsidated in a single virion. In this study, the nucleotide sequence of SJNNV RNA1 was determined. The SJNNV RNA1 was 3081 bases long and contained a single ORF encoding 983 aa of approximately 110 kDa. The sequence identities between RNA1 of SJNNV and RNA1 of insect nodaviruses were 28% at the nucleotide and amino acid levels, although the conserved motifs for the RNA-dependent RNA polymerase were located at almost the same positions in the amino acid sequences. The present study, together with our previous work on SJNNV RNA2, suggests that a new genus, Piscinodavirus, should be created in the family Nodaviridae.

Mapping a neutralizing epitope on the coat protein of striped jack nervous necrosis virus.

Striped jack nervous necrosis virus (SJNNV), a fish nodavirus, is the causative agent of viral nervous necrosis in marine fishes. The fish nodaviruses are divided into four different genotypes based on the nucleotide sequence of the coat protein gene. In the present study, partial coat protein genes of fish nodaviruses were expressed. This allowed the serological relationship among the different virus genotypes to be analysed and neutralizing epitopes on the coat protein to be mapped. Western blot analysis revealed that SJNNV and other fish nodavirus genotypes shared a significant number of common antigenic determinants, although SJNNV was serologically distinguishable. The results suggested that the SJNNV determinant for neutralizing MAbs was a linear epitope, which consisted of a repeated amino acid sequence within the coat protein. One of the neutralizing epitopes of SJNNV was deduced to be PAN at aa 254-256 in the coat protein.

Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene.

A molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. These fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.

Failure of the Polymerase Chain Reaction (PCR) Method to Detect Striped Jack Nervous Necrosis Virus (SJNNV) in Striped JackPseudocaranx dentexSelected as Spawners

Abstract The causative agent of viral nervous necrosis (VNN) in striped jack Pseudocaranx dentex is transmitted from female and male spawners to larvae, and elimination of carrier spawners, determined by the detection of striped jack nervous necrosis virus (SJNNV) via polymerase chain reaction (PCR), has been used to prevent transmission of the disease in larval production facilities. However, some outbreaks of VNN occurred in larvae obtained from SJNNV-negative spawners. We compared the occurrence of infection between groups of larvae obtained from SJNNV-negative spawners and those from SJNNV-positive spawners. Viral nervous necrosis occurred in all seven groups of larvae obtained from the virus-positive spawners between the 3rd and 7th day of rearing. The virus was detected only occasionally after 2 weeks in four of six groups obtained from the virus-negative spawners. These results confirmed vertical transmission of the virus and revealed that a very small amount of SJNNV in spawners escaped PCR detect...

Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 24:99-105 (1996) - doi:10.3354/dao024099 Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae Nguyen HD, Nakai T, Muroga K The progress of infection with SJNNV (nodavirus), the causative agent of viral nervous necrosis (VNN), was investigated by using naturally infected (acute and subacute groups) and experimentally infected striped jack larvae at different stages of infection. Although there were slight differences in the progress of infection among the 3 groups of fish examined, the general features of infection were quite similar. Necrosis and vacuolation of the nerve cells were first observed in the spinal cord, particularly in the area just above the swimbladder, later in the brain, and then in the retina. Mortalities occurred 1 to 2 d after the commencement of lytic degeneration of the cells, which resulted in heavy vacuolation in these nervous tissues. Virus antigens were detectable in the nervous tissues by the fluorescent antibody technique (FAT) when conspicuous vacuolation appeared in the cytoplasm. Virus particles were detectable by electron microscopy in concurrence with the appearance of heavy tissue vacuolation. These results indicate that SJNNV exhibits a tropism for nerve cells and its initial multiplication site is the spinal cord, from which the virus spreads to the brain and finally to the retina. Hyperplasia was observed in the skin of 1 naturally infected larval group (acute infection) and virus multiplication was observed in these affected epithelial cells. However, the role of skin as a portal of entry for SJNNV remains unclear. Viral nervous necrosis . Nodavirus . Striped jack . In vivo multiplication Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 24, No. 2. Publication date: February 01, 1996 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1996 Inter-Research.

Detection of Striped Jack Nervous Necrosis Virus(SJNNV) by Enzyme-Linked Immunosorbent Assay(ELISA).

The present paper describes the detection of striped jack nervous necrosis virus (SJNNV), the causative agent of viral nervous necrosis, in eggs, larvae, and brood stocks of hatchery-reared and captured striped jack Pseudocaranx dentex by indirect ELISA. An appropriate condition for the ELISA was determined using purified SJNNV and the detection limit of virus antigens was approximately 5 ng/well. All samples of larvae supposed to be affected with the disease were proved positive for the presence of SJNNV antigens in this ELISA test. The viral antigens were also detected in some samples of fertilized eggs and ovaries of spawners but not in the testis or brain. Thus, it was indicated that spawners were an inoculum source of this viral disease in striped jack.

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

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 μl 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

Portal Entry and Progression of Betanoda Virus Causing Viral Nervous Necrosis in Sevenband Grouper &lt;i&gt;Epinephelus septemfaciatus&lt;/i&gt;

A study was conducted to identify the entry site for the betanodavirus, the causative agent of viral nervous necrosis (VNN) in 375 fish (150 spawn, 150 fry and 75 fingerling) during the period from March to August 2003. Highly susceptible fish, sevenband grouper Epinephelus septemfasciatus to sevenband grouper nervous necrosis virus (SGNNV) was used as experimental fish. Fish were placed into the seawater containing 104 TCID50 mL-1 of the viral isolate for 1 h. Samples for virus isolation were randomly collected at an interval of 3 h, 12 h, 24 h and daily, and continued until 5 to14 days depending on the mortality. The inoculated virus could first recover from the mouth including nostril at 3 h of exposure and then from fin at 12 h, and within 1 d it was detected from all the organs examined at titers ranging from 104 to 107 TCID50 g-1. The highest titer was found at day 5 in the target organs of central nervous system (CNS), i.e., the brain, bone including spinal cord, and eye. The results suggest that nasal route the initial route for the portal entry of betanodavirus into fish. Key words: Betanodavirus; viral nervous necrosis; virus titration; portal entry, nasal route; sevenband grouper doi: 10.3329/bjvm.v2i1.1942 Bangl. J. Vet. Med. (2004). 2 (1) : 83-87