Nervous Necrosis Virus Infection Research Articles

Real-Time Quantitative PCR Assay for Monitoring of Nervous Necrosis Virus Infection in Grouper Aquaculture

Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

Antiviral activity by fish antimicrobial peptides of epinecidin-1 and hepcidin 1–5 against nervous necrosis virus in medaka

The nervous necrosis virus (NNV)-medaka infection model was used in this study for analysis of NNV infection and treatment of NNV with the antimicrobial peptides (AMPs) of epinecidin-1 and hepcidin 1–5 at the organismal level. Our results showed that co-treatment of AMPs with the virus was effective in promoting a significant increase in medaka survival. Re-challenge with the virus also showed high survival suggesting that these two AMPs enhanced fish survival. However, pretreatment or post-treatment with AMPs showed that both of these AMPs increased medaka survival and suggested that AMPs can be used as drugs to rescue infected medaka. The data presented here indicate that epinecidin-1 and hepcidin 1–5 have in vivo antivirus activity against the NNV, and hepcidin 1–5 functions like a lytic peptide after an in vitro assay. Infection after pretreatment, co-treatment, and post-treatment with epinecidin-1 or hepcidin 1–5 was verified by RT-PCR which showed both peptides can downregulate NNV and interferon gene expressions. In addition, our results suggest that epinecidin-1 or hepcidin 1–5 may prove to be an effective chemotherapeutic agent for aquaculture in the future.

Anti-viral mechanism of barramundi Mx against betanodavirus involves the inhibition of viral RNA synthesis through the interference of RdRp

Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation.

Enhanced viability of a nervous necrosis virus‐infected stable cell line over‐expressing a fusion product of the zfBcl‐xL and green fluorescent protein genes

Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.

Persistence of betanodavirus in Barramundi brain (BB) cell line involves the induction of Interferon response

The BB cell line derived from the brain tissue of a barramundi ( Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (≦1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line.

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.

Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae

This preliminary study elucidates the in vitro and in vivo effects of temperature on grouper nervous necrosis virus (GNNV) infection. A novel continuous cell line derived from the fin tissue of a grouper ( Epinephelus coioides, Hamilton), named as GF-1 cell line, was used. Cytopathic effect was observed in GNNV-infected GF-1 cells incubated at 24–32°C after viral adsorption, but not at 20°C or 37°C even though the viral adsorption temperature was 28°C. Viral protein could be detected in the pellets of GNNV-infected GF-1 cells cultured at 20–32°C, but not at 37°C. In a challenge test, GNNV-challenged larvae which were maintained at a constant 28°C began to die 1 day post challenge (p.c.) with a death rate of 80%. Mortality reached 100% by 50 h p.c., while the mortality of negative control fish was only 5%. The cumulative mortality of GNNV-challenged larvae at ambient temperature, i.e. 28°C at noon and 24°C at midnight, was 10% 1 day p.c., and increased to 100% by 80 h p.c. Based on the results, we concluded that temperature plays an important role in GNNV infection and pathogenicity.

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.