• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.3
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• pp.328-331
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• 2018

We developed and subsequently characterized mouse monoclonal antibodies (MAbs) against nervous necrosis virus (NNV, RGNNV genotype). We established six hybridoma clones secreting MAbs against NNV antigen: 2B1, 2B11, 2C12, 13C1-1, 13C1-2 and 14D11. All six MAbs belonged to the IgG2a isotype with a kappa light chain and their reactivity recognized against the 41 kDa coat protein of NNV by Western blot analysis. The affinity constants of the six MAbs were measured by enzyme-linked immunosorbent assay (ELISA). All six MAbs reacted with two NNV isolates (SgNag05 and Gemunodo06), while no reactivity was observed with five know fish viruses, namely marine birnavirus, infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, hirame rhabdovirus, and infectious hematopoietic necrosis virus. Moreover, high ELISA optical density (OD) values (0.87-1.42) were observed in the brain tissues of NNV-infected sevenband grouper, while low OD values (less than 0.12) were recorded in the brain tissues of uninfected fish. These results suggest that these six MAbs are highly competent and useful for the detection of NNV with the RGNNV genotype.

• Journal of fish pathology
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• v.33 no.2
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• pp.103-109
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• 2020

In order to use nervous necrosis virus (NNV) virus-like particles (VLPs) as a delivery tool for heterologous antigens or plasmids, we attempted to produce red-spotted grouper nervous necrosis virus (RGNNV) VLPs displaying a partial region of viral hemorrhagic septicemia virus (VHSV) glycoprotein at the surface and VLPs that are harboring DNA vaccine plasmids within the VLP. A peptide encoding 105 amino acids of VHSV glycoprotein was genetically inserted in the loop region of NNV capsid gene, and VLPs expressing the partial part of VHSV glycoprotein were successfully produced. However, in the transmission electron microscope analysis, the shape and size of the partial VHSV glycoprotein-expressing NNV VLPs were irregular and variable, respectively, indicating that the normal assembly of capsid proteins was inhibited by the relatively long foreign peptide (105 aa) on the loop region. To encapsulate by simultaneous transformation with both NNV capsid gene expressing plasmids and DNA vaccine plasmids (having an eGFP expressing cassette under the CMV promoter), NNV VLPs containing plasmids were produced. The encapsulation of plasmids in the NNV VLPs was demonstrated by PCR and cells exposed to the VLPs encapsulating DNA vaccine plasmids showed fluorescence. These results suggest that the encapsulation of plasmids in NNV VLPs can be done with a simple one-step process, excluding the process of disassembly-reassembly of VLPs, and NNV VLPs can be used as a delivery tool for DNA vaccine vectors.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.4
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• pp.403-410
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• 2015

Viral nervous necrosis (VNN) is a serious disease of sevenband grouper Epinephelus septemfasciatus in Korean aquaculture farms. However, we suggest the following preventative methods for hatcheries: 1) disinfecting rearing water, 2) selecting spawners via ELISA and PCR, 3) selecting eggs via PCR, 4) disinfecting fertilized eggs, and 5) proper facilities management. When these methods are implemented, nervous necrosis virus (NNV)-free fish are produced because vertical and horizontal transmission is prevented. However, horizontal transmission of NNV through rearing seawater sourced from the environment during grow-out stages in sea cages can still occur. Live NNV vaccines with a low rearing temperature or Poly(I:C) immunization are very effective at preventing horizontal transmission of NNV in rearing farms. Furthermore, even after VNN is contracted, fish mortality can be reduced by administering Poly(I:C).

• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.5
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• pp.527-533
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• 2017

We investigated the infection of nervous necrosis virus (NNV) in seven band grouper Hyporthodus septemfasciatus broodstocks, which have been reared in aquaculture farms in South Korea during 2012-2014. To investigate the prevalence of NNV within the broodstock, egg, sperm, and blood were sampled in the spawning season. The egg and sperm samples were subjected to a nested reverse transcription (RT) polymerase chain reaction (PCR) assay to detect NNV and were inoculated on SSN-1 cells to culture the virus. Blood samples were used to detect antibodies against NNV using enzyme linked immunosorbent assay (ELSIA). Positive values from ELISA were found in 39 of 162 samples (24%) in 2012, and 13 of 28 samples (46%) in 2014. Additionally, 4 of 34 broodstocks (11%) investigated in 2013-2014 were determined to be carriers from the nested RT-PCR and in vitro cultivation. The broodstocks in which antibodies against NNV were detected by ELISA, or in which NNV was detected by the nested RT-PCR assay, posed a risk of vertical transmission of NNV. Therefore, it is necessary to select virus-free broodstocks in seed production to reduce the possibility of the vertical transmission of NNV.

• Journal of fish pathology
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• v.19 no.1
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• pp.65-72
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• 2006

Nervous necrosis virus (NNV) that causes severe mortality during seed production of red drum (Sciaenops ocellatus) is known to be vertically transmitted from infected spawners. This study was conducted to produce NNV free seeds by testing spawners for NNV infection and using virus free eggs for seed production. RT-PCR analysis of 40 spawners showed 18 positives and 22 negatives. NNV was not detected from eggs obtained from the negative spawners but was detected from those obtained from the positives. NNV was not detected from culturing seawater in tanks and Chlorella spp., Brachionus plicatilis., and Brine shrimp those were provided as live feed. The survival rates of fry from NNV positive and negative spawners were 80% and 85%, respectively by two weeks after hatching. The mortality increased from 25days after hatching and the final survival rate of seeds from NNV positive and negative spawners were 0% and 18.3%, respectively on 41 days after hatching. These results suggested that virus free red drum seeds can be obtained by using virus-free spawners.

• Journal of fish pathology
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• v.31 no.1
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• pp.9-13
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• 2018

We previously monitored nervous necrosis virus (NNV) in brain samples of artificially produced walleye Pollock (Gadus chalcogrammus) seedlings, with a low prevalence (1.8%, 1/55) but no clinical symptoms. Given that this virus is considered one of the most serious viral threats for almost all marine aquaculture fish species and characterized by both vertical and horizontal transmission, it would be interesting to monitor NNV in the fertilized eggs as well. We collected fertilized walleye pollock eggs from the farms located in Goseong during January to March, 2017. Approximately 50 mg of eggs were periodically taken from 4 each different batches, and 37 different pooled sample sets in total were made during sampling period. RNA was extracted from the eggs by using Trizol and cDNA was synthesized for RT-PCR for detecting NNV. Primers and PCR conditions are the same as previously described. As a result, NNV was not detected from any of the sample sets by one step PCR (0%, 0/37), suggesting NNV may not be a threat in walleye pollock aquaculture in Korea at present time. However, continuous monitoring for NNV should be conducted because introducing a new species into aquaculture industry involves potentials of disease outbreak and NNV is already known to cause outbreaks in gadoid fishes.

• Journal of Life Science
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• v.30 no.4
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• pp.402-409
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• 2020

Nervous necrosis virus (NNV) contains a bi-segmented viral genome, RNA1 (3.4 kb, RdRp), and RNA2 (1.4 kb, capsid protein) in a small particle (25 nm). Despite its extremely compact size, NNV has caused serious damage by infecting approximately 120 fish species worldwide since it was first reported in the late 1980s. In order to minimize the damage caused by NNV infection and develop effective vaccines, it is necessary to understand the intra cellular signaling system according to NNV infection. NNV infection induces cell cycle arrest at the G1 phase via the p53-dependent pathway to use the cellular system for its replication. Otherwise, host cells recognize NNV infection through the RIG-1-like receptor (RLR) signaling pathway to control the virus and infected cells, and then ISGs required for antiviral action are activated via the IFN signaling pathway. Moreover, apoptosis of infected cells is triggered by the unfolded protein response (UPR) through ER stress and mitochondria-mediated cell death. Cell signaling studies on the NNV infection mechanisms are still at an early stage and many pathways have yet to be identified. Understanding the various disease-specific cellular signaling systems associated with NNV infection is essential for rapid and accurate diagnosis and vaccine development.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1761-1767
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• 2015

Sevenband grouper, Epinephelus septemfasciatus, is becoming an important aquaculture species in Korea. However, viral nervous necrosis disease is a large problem causing mass mortality in sevenband grouper aquaculture. Recombinant protein vaccines are one of the best methods to reduce these economic losses. However, the cell-based expression method mainly produces inclusion bodies and requires additional procedures. In this study, we expressed a recombinant viral coat protein of sevenband grouper nervous necrosis virus (NNV) using a cell-free protein synthesis system. The purified recombinant NNV coat protein (rNNV-CP) was injected into sevenband grouper at different doses followed by a NNV challenge. Nonimmunized fish in the first trial (20 μg/fish) began to die 5 days post-challenge and reached 70% cumulative mortality. In contrast, immunized fish also starting dying 5 days postchallenge but lower cumulative mortality (10%) was observed. Cumulative morality in the second trial with different doses (20, 4, and 0.8 μg/fish) was 10%, 40%, and 50%, respectively. These results suggest that rNNV-CP can effectively immunize sevenband grouper depending on the dose administered. This study provides a new approach to develop a recombinant vaccine against NNV infection for sevenband grouper.

• Journal of fish pathology
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• v.34 no.2
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• pp.177-184
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• 2021

Vaccines based on single-cycle viruses that are replication-incompetent due to knockout of replication-related structural gene(s) are more immunogenic than inactivated or subunit vaccines and can be used as delivery vehicles for foreign antigens without concerns on the reverting to virulent forms. The aim of this study was to develop a delivery vehicle for nervous necrosis virus (NNV)-like particles (VLPs) using G gene deleted single-cycle VHSV (rVHSV-𝚫G). Recombinant single-cycle VHSVs carrying NNV capsid protein gene between N and P gene of rVHSV-𝚫G genome (rVHSV-𝚫G-NNV_Cap) were rescued by reverse genetic technology. The successful expression of NNV capsid protein in cells infected with rVHSV-𝚫G-NNV_Cap was demonstrated by Western blot analysis, and the production of NNV VLPs in infected cells was confirmed using an electron microscopy. The results suggest that single-cycle VHSVs can be used as a safe delivery vehicle for NNV VLPs, and can be extended to other pathogens for the development of prophylactic vaccines.

• Journal of fish pathology
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• v.30 no.1
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• pp.1-9
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• 2017

This study was conducted to monitor the prevalence of viral hemorrhagic septicemia virus (VHSV), nervous necrosis virus (NNV) and marine birnavirus (MABV) in cultured walleye pollock Gadus chalcogrammus by RT-PCR. All of the viruses tested were not detected by one-step PCR in 62 spleen sample sets, except for NNV in one brain sample set (1/55). By two-step PCR, VHSV was detected in 51.6%(32/62) and NNV was detected in 1.6%(1/62) spleen sample set, but MABV was not detected. In the brain sample sets, the detection rate of NNV was 3.6%(2/55). VHSV and NNV were detected for the first time in cultured walleye pollock in this study. However, the titers of viruses in these sample sets are thought to be very low, because most of the positive sample sets were detected by two-step PCR and none of the fish showed any clinical symptoms of each virus. Continuous monitoring, subsequent virus isolation and validation of carrier fish will be necessary.