• 한국어류학회지
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• 제27권3호
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• pp.183-188
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• 2015

본 연구에서는 무지개송어의 해수면 양식장으로의 이동에 따라 나타날 수 있는 어류 병원성 바이러스의 영향을 알아보기 위해, 담수 무지개송어 유래 병원체인 infectious hematopoietic necrosis virus (IHNV)와 infectious pancreatic necrosis virus (IPNV)를 사용하여 주요 해산 양식 어종인 넙치, 조피볼락, 돌돔, 참돔 및 능성어에 주사한 후 병원성 및 감염 여부를 조사하였으며, 또한 해산 양식 어종 유래 병원체인 marine birnavirus (MABV), hirame rhabdovirus (HIRRV) 및 nervous necrosis virus (NNV)를 사용하여 무지개송어에 대한 병원성 및 감염 여부를 조사하였다. IHNV와 IPNV는 주요 해산 양식 어종에 감염되는 것으로 확인되었으며, 또한 무지개송어는 해산 유래 위해 병원체인 MABV와 NNV에 감염되거나 HIRRV에 의해 폐사되는 것으로 확인되었다. 이상의 연구 결과, 해수사육 무지개송어는 어류 병원성 바이러스들에 의해 영향 받을 수 있을 것으로 사료되었다.

• Journal of Microbiology and Biotechnology
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• 제22권7호
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• 한국수산과학회지
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• 제35권3호
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• pp.237-241
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• 2002

This study was performed both to explore the host of nervous necrosis virus (NNV) between mariculturing fish species and to examine the phylogenic position of the NNV in Korea. NNV was confirmed on the basis of histopathological and molecular biological examination, then VNN infection was Preyed from either moribund or dead fishes including red drum, Sciaenops ocellatus; oblong rock fish, Sebastes oblongus and flounder, Paralichthys olivaceus. As a result of sequencing for a part of ms, virus from red drum was showed $98\%, $97\%, $86\% and $74\% homology with oblong rock fish, grouper, Japanese flounder and striped jack, respectively. On the other hand, NNV from oblong rock fish was demonstrated $96\%, $85\% and $72\% homology with grouper, Japanese flounder and striped jack, respectively. NNV from red drum and oblong rock fish was exhibited phylogenically distant from the representative NNV, SJNNV originated from striped jack. On the contrary, the viruses appeared to be similar species with Taiwan NNV isolated from culturing grouper.

• Fisheries and Aquatic Sciences
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• 제22권12호
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• pp.28.1-28.8
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• 2019

Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.

• 한국어병학회지
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• 제37권1호
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• pp.1-8
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• 2024

The advantages of replicon vectors of RNA viruses include a high ability to stimulate innate immunity and exponential amplification of target mRNA leading to high expression of foreign antigens. The present study aimed to construct a DNA-layered nervous necrosis virus (NNV) replicon vector system in which the capsid protein gene was replaced with a foreign antigen gene and to compare the efficiency of foreign antigen expression between the conventional DNA vaccine vector and the present replicon vector. We presented the first report of a nodavirus DNA replicon-based foreign antigen expression system. Instead of a two-vector system, we devised a one-vector system containing both an NNV RNA-dependent RNA polymerase cassette and a foreign antigen-expressing cassette. This single-vector approach circumvents the issue of low foreign protein expression associated with the low co-transfection efficiency of a two-vector system. Cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 (with the capsid gene ORF replaced with VHSV glycoprotein ORF) exhibited significantly higher transcription of the VHSV glycoprotein gene compared to cells transfected with either a vector without hammerhead ribozyme or a conventional DNA vaccine vector expressing the VHSV glycoprotein. Furthermore, the transcription level of the VHSV glycoprotein in cells transfected with a vector harboring hammerhead ribozyme-fused RNA1 and RNA2 showed a significant increase over time. These results suggest that NNV genome-based DNA replicon vectors have the potential to induce stronger and longer expression of target antigens compared to conventional DNA vaccine vectors.

• Genomics & Informatics
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• 제20권3호
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• pp.33.1-33.17
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• 2022

Nervous necrosis virus (NNV) is a deadly infectious disease that affects several fish species. It has been found that the NNV utilizes grouper heat shock cognate protein 70 (GHSC70) to enter the host cell. Thus, blocking the virus entry by targeting the responsible protein can protect the fishes from disease. The main objective of the study was to evaluate the inhibitory potentiality of 70 compounds of Azadirachta indica (Neem plant) which has been reported to show potential antiviral activity against various pathogens, but activity against the NNV has not yet been reported. The binding affinity of 70 compounds was calculated against the GHSC70 with the docking and molecular dynamics (MD) simulation approaches. Both the docking and MD methods predict 4 (PubChem CID: 14492795, 10134, 5280863, and 11119228) inhibitory compounds that bind strongly with the GHSC70 protein with a binding affinity of -9.7, -9.5, -9.1, and -9.0 kcal/mol, respectively. Also, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of the compounds confirmed the drug-likeness properties. As a result of the investigation, it may be inferred that Neem plant compounds may act as significant inhibitors of viral entry into the host cell. More in-vitro testing is needed to establish their effectiveness.

• 한국어병학회지
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• 제31권2호
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• pp.71-79
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• 2018

2015년 2월부터 2018년 8월까지 총 1,253 마리의 자연산 명태 (Gadus chalcogrammus)를 강원도 고성 아야진항 근해에서 정치망을 사용하여 포획한 후, 바이러스 (viral hemorrhagic septicemia virus, VHSV; nervous necrosis virus, NNV; marine birnavirus, MABV) 모니터링을 RT-PCR법으로 수행하였다. One-step PCR법으로 비장시료 및 뇌시료에서는 대상 바이러스가 모두 검출되지 않았으며, 일부 시료를 two-step PCR법으로 검사한 결과 VHSV는 19.7% (36/183)의 비장시료에서 검출되었다. 또한, NNV는 4.4% (8/183)의 비장시료, 1.2% (3/259)의 뇌시료에서 검출되었다. 검출된 바이러스의 계통분석 결과, 기존의 국내에서 분리되는 바이러스의 유전형에 각각 속하는 것으로 나타났다 (Genotype IVa, RGNNV genotype). 바이러스의 분리를 시도하지 않아 검출된 바이러스의 활성은 알 수 없지만, 모든 양성 시료가 two-step PCR법으로 검출되었으므로 매우 낮을 것으로 추측된다.

• 한국어병학회지
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• 제34권1호
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• pp.99-104
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• 2021

In this study, we investigated safety and efficacy of a low temperature immunization protocol with NNV in red spotted grouper, Epinephelus akaara and long tooth grouper, Epinephelus bruneus and seven band grouper, Hyporthodus septemfasciatus. Further, growth rate between immunized and naïve fish was evaluated during the experiment to check side effect of immunization. Three grouper species were immunized by immersion method with live NNV at 10^5.0 TCID₅₀/mL at 16.5℃ for 30 min and reared for 120 days at natural sea water temperature. To evaluate growth rate, total length and wet weight was measured 7 times after immunization. Immunized three grouper species were challenged by intramuscular inoculation with NNV at 10^4.2 TCID₅₀/100 µL/fish. Immunization at low temperature with live NNV did not show any clinical symptoms of infection, mortality and inhibition of growth. After challenge, cumulative mortality of naïve seven band grouper, red spotted grouper, long tooth grouper were 45, 10, 20 %, respectively. However no mortality was observed at immunized groupers. Thus, it was demonstrated that immunization at low temperature with live NNV are able to protect three different species of groupers without inhibition of growth.

• 한국어병학회지
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• 제25권2호
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• pp.111-116
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• 2012

본 연구에서는 2006-2008년 남해안 일대의 해상가두리에서 사육중인 능성어에서 발생하는 viral nervous necrosis (VNN)의 발생양상을 조사하였다. VNN은 사육 수온이 $24-26^{\circ}C$ 범위인 8월부터 발생하기 시작하여 수온이 $20-25^{\circ}C$ 범위인 9-10월까지 지속되었고, 성어보다는 치어에서 폐사율이 높게 나타나는 것으로 확인되었다. 폐사되는 패턴으로는 급성으로 인한 대량폐사와 소량으로 지속적으로 폐사되는 경우가 확인되었다. 능성어로부터 분리된 NNV 분리주들의 coat protein gene을 계통분석한 결과, 분리주들은 모두 RGNNV 유전자형에 속하였다. 이상의 결과로 능성어 양식장에서의 VNN은 RGNNV type의 NNV에 의해 여름철 7-9월(사육수온: 약 $24^{\circ}C$)에 치어뿐만 아니라 성어에서 발생하는 것으로 확인되었다.

• 한국어병학회지
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• 제35권1호
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• pp.121-128
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• 2022

Recent studies revealed that histone proteins are involved in innate immune responses during pathogen invasion as well as DNA packing. This study characterized the histone genes (H2A.V) of sevenband groupers and analyzed gene expression in NNV-infected sevenband groupers. The open reading frame (ORF) of H2A.V is 387 bp which encoded 128 amino acid residues. The deduced amino acid sequence of H2A.V harbor a highly conserved domain for H2A/H2B/H3 and H2A_C binding domain. Quantitative real-time PCR analysis showed that H2A.V had a high gene expression level in the brain and blood after being NNV-infected. An increase in extracellular histone protein in the blood has been identified as a biomarker for vascular function in humans. More research is required to understand histone's immune response at the protein level or in aquatic animals.