Development and application of quantitative detection method for nervous necrosis virus （NNV） isolated from sevenband grouper Hyporthodus septemfasciatus

Jong-Oh Kim， Jae-Ok Kim， Wi-Sik Kim， Myung-Joo Oh

Department of Aqualife Medicine， College of Fisheries and Ocean Science， Chonnam National University， Yeosu 550-749， Korea

Development and application of quantitative detection method for nervous necrosis virus （NNV） isolated from sevenband grouper Hyporthodus septemfasciatus

Jong-Oh Kim， Jae-Ok Kim， Wi-Sik Kim， Myung-Joo Oh?

Department of Aqualife Medicine， College of Fisheries and Ocean Science， Chonnam National University， Yeosu 550-749， Korea

ARTICLE INFO

Article history：

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 15 July 2016

Available online 20 August 2016

Nervous necrosis virus （NNV）

Objective： To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus. Methods： The viral genes of the NNV （SGYeosu08） isolated from sevenband grouper were phylogenetically analyzed. In addition， novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method （TCID50） using in vitro and in vivo samples. Results： The phylogenetic analysis of viral genes demonstrated the relationship of SGYeosu08 with members of red-spotted grouper nervous necrosis virus（RGNNV）. The qNNV_R1 primer set （R1_F and R1_R） and the qNNV_R2 primer set （R2_ F and R2_R） revealed 93% primer efficiency （regression： y=-0.2861x + 9.9401， R2= 0.9976）and the revealed 108% primer efficiency （regression： y=-0.3172x + 10.0611， R2= 0.9982），respectively. Its comparison with viral infectivity calculated by TCID50method showed similar kinetic pattern at in vitro and NNV challenged fish （in vivo） samples. Conclusions： Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method （TCID50）.

1. Introduction

Viral nervous necrosis （VNN）， also known as viral encephalopathy and retinopathy （VER） is a severe problematic disease in the world aquaculture industry ［1-3］. It has been reported from a variety of marine aquaculture species over 20 kinds including rock bream（Oplegnathus fasciatus）， olive flounder （Paralichthys olivaceus），barramundi （Lates calcarifer）， turbot （Scophthalmus maximus），sevenband grouper （Hyporthodus septemfasciatus） since its first reportfrom bigeye trevally （Caranx sexfasciatus） at 1980s ［1-3］. Normally，the symptoms of diseased fish appear with vacuolization and necrosis of the central nervous system and the retina and showing abnormal swimming ［1］. The high mortality rate over 80% was reported from various fish species at larvae and juveniles stages［1］. Nervous necrosis virus （NNV）， the causative agent of VNN，is a small non-enveloped icosahedral virus containing bisegmented single strand positive RNA as genetic materials. RNA1（approximately 3.1 kb in length） encodes a RNA dependent RNA polymerase for viral replication while RNA2 （1.4 kb） encrypts a viral capsid protein. It belongs to the family Nodaviridae and the genus Betanodavirus. Betanodaviruses has 4 genogroups based on the T4 region sequence of RNA2 as barfin flounder nervous necrosis virus （BFNNV）， red-spotted grouper nervous necrosis virus （RGNNV）， striped jack nervous necrosis virus （SJNNV），tiger puffer nervous necrosis virus （TPNNV） ［4］. Besides， Johansen and colleagues ［5］ suggested new nodavirus isolates from turbot，Scophthalmus maximus， as a fifth genotype.

In Korea， mass mortalities caused by VNN were reported from various cultured marine fish such as sevenband grouper （Hyporthodus septemfasciatus）， rock bream （Oplegnathus fasciatus）， red drum（Sciaenops ocellatus） and olive flounder （Paralichthys olivaceus） since 1990 ［6-8］. Furthermore， NNV has been detected from wild marine fishes in seaside of the Korea peninsula ［9］.

Sevenband grouper is one of the most valuable cultured fish in Korea. However， recently the outbreaks of the VNN were observed in aqua-farms of sevenband groupers during the summer and have made huge economic losses ［10］. It is necessary to develop sensitive and accurate diagnostic method to minimize huge losses caused by NNV infection. The World Organization for Animal Health Office International des Epizooties （OIE） has recommended methods to diagnosis NNV infection such as PCR， antibody-based assay，50% tissue culture infectivity dose （TCID50） and so on. Recently，quantitative RT-PCR （qRT-PCR） based on the SYBR Green assay was also established with faster than traditional bio assay method（TCID50） ［11］. However， the primers were not validated for all genogroups of NNV strains ［12］. Thus， in this aspect it is necessary to develop specific quantitative detection tool for Korean NNV isolate based on the complete understanding of genetic information.

In this study， we analyzed viral genes of the NNV （SGYeosu08）isolated from sevenband grouper in Korea and figured out phylogenetic relationships with previously reported strains. In addition， we newly developed a quantitative detection method for NNV based on the Korean isolate and compared it with the conventional bio-assay method （TCID50） using in vitro and in vivo samples.

2. Materials and methods

2.1. Virus preparation

The NNV used in this study was isolated from sevenband grouper aquafarm in Yeosu， Korea in 2008 and propagated in the striped snakehead（SSN-1） cell line. SSN-1 cells were grown at 25 ℃ in Leibovitz L-15 medium （Sigma Aldrich， St. Louis， MO， USA） containing 10% fetal bovine serum （Gibco， Gland Island， NY， USA）， 150 U/mL penicillin G， and 100 μg/mL streptomycin. NNV was inoculated on a confluent SSN-1 cell monolayer and incubated at 25 ℃ to replicate the virus. Viral samples were aliquot in small volumes and stored at 80 ℃ until use.

2.2. Cloning and sequencing analysis of viral genes

Viral RNA was extracted using miRNeasy Mini Kit （Qiagen， Germany） and cDNA was synthesized using ReverTra Ace qPCR RT Kit （Toyobo， Japan） following the manufacturer’s protocols. The synthesized cDNA was amplified in 20 μL of PCR mixture containing 5 μL of 10 Ex Taq buffer， 4 μL of 2.5 mM dNTP mixture（each）， 0.5 μL of Ex Taq （5 U/μL）， and 20 pmol of open reading frame （ORF） primer sets in Table 1. A primer set used in this study were designed by Primer3Plus ［13］ based on the NNV SGYeosu08 genome sequence ［14］. PCR condition was pre-denaturation at 95 ℃for 5 min， 30 cycles of 1 min denaturation at 95 ℃， 1 min annealing at 58 ℃， and 3 min for RNA1 or 1 min for RNA2 extension at 72 ℃，followed by a 5 min final extension at 72 ℃. The amplified products were purified using QIAquick Gel Extraction Kit （QIAGEN） and cloned into pCR2.1-Topo vector （Invitrogen， USA）. Plasmid DNA was extracted with an Accuprep Plasmid Mini Extraction Kit（Bioneer， Korea） and analyzed ORF sequence by ABI 3730 XL DNA sequencer.

Table 1PCR primers used in this study.

2.3. Phylogenetic analysis

All sequences of other nodaviruses were obtained from GenBank database of the National Center for Biotechnology Information web site （http：//www.ncbi.nlm.nih.gov/GenBank/）. Sequence similarity analysis was conducted with Basic Local Alignment Search Tool （BLAST， http：//www.ncbi.nlm.nih.gov/blast/） of NCBI and Pairwise Sequence Alignment Tool （http：//www.ebi.ac.uk/ Tools/psa/） of EMBL-EBI （the European Bioinformatics Institute）and organized by manual. The multiple sequence alignments were performed with the Clustalw2 web service （http：//www.ebi.ac.uk/ Tools/msa/clustalw2/）. Phylogenetic analyses were conducted using the MEGA6 software using a Neighbor-Joining method with 1 000bootstrap replicates ［15］.

2.4. Standard and primer efficiency

Primer efficiency was examined by the quantitative PCR with 10-fold diluted plasmid DNA. The quantitative PCR was carried out in an Exicycler 96 Real-Time Quantitative Thermal Block （Bioneer）followed by manufacturer`s instructions using SYBR green mixture，AccuPower Greenstar quantitative PCR premix （Bioneer）. Briefly，a 10 min pre-denaturation cycle at 95 ℃， 40 cycles of 20 sec denaturation at 95 ℃， and a 40 sec extension at 58 ℃ were used. The specification of the quantitative PCR was analyzed through melting curve analysis， and the baseline was determined automatically by the Exicycler Analysis Software （Bioneer）.

2.5. qRT-PCR and TCID50method （in vitro）

One hundred microliter of 105.8TCID50/mL NNV was inoculated into fourteen of 25 cm2culture flasks containing SSN-1 cells. The supernatant and cells were sampled immediately after virus inoculation （0h）， at 3， 6， 12， 24 h， and at 2 and 3 days from each of two culture flasks to compare the TCID50method and the qRTPCR method. To measure the viral infectivity， SSN-1 cells （about 105cells/well） were cultured in 96-well plates， and 50 μL of 10-fold diluted virus （10-1to 10-8） was inoculated onto the 96-well plates. TCID50value was calculated 14 days after inoculation using Reed and Muench method ［16］. All samples were statistically analyzed and all the data were represented as the mean ± the standard error.

2.6. Challenging experiment （in vivo）

Sevenband groupers were purchased from aqua-farm which has no history of VNN occurrence. Prior to experiments， 10 fish were randomly sampled from fish stock and the brain samples were examined for betanodavirus by RT-PCR according to a previous report ［4］. Total thirty five sevenband groupers ［（average weight= 30.9 ± 8.2） g］ were reared in aquaria. Ten of thirty five fish were cultivated separately to calculate cumulative mortality. NNV at dose of 103.8TCID50/100 μL/fish was injected intramuscularly into the fish in aquaria and the challenged fish were daily observed for two weeks. Brain tissues of three challenged fish were collected on the 24， 48， 72， 84， 90 and 96 hours after injection for measuring titration of NNV and viral copy number. The obtained tissues were homogenized with nine volumes of L-15 medium and centrifuged at 6 000 g for 30 min （4 ℃）， and then the supernatant was used for qRT-PCR and TCID50.

3. Results

3.1. Comparison with other NNV isolates and phylogenetic analysis

The percent of deduced amino acid sequence identities of NNV SGYeosu08 isolate with published genome sequence of NNV were determined by EMBOSS Needle pairwise sequence alignment tool（http：//www.ebi.ac.uk/Tools/psa/emboss_needle/） and the results are shown in Table 2. In the result of deduced protein （RNA dependent RNA polymerase， RdRp） from RNA 1， NNV SGYeosu08 showed over 98.0% （98.0% - 99.4%） of identity with other RGNNV， 88.3%-88.7% of BFNNV， 88.6% with TPNNV， 87.9% with SJNNV，respectively. In case of RNA2， NNV SGYeosu08 showed over 99.1%（99.1%-100.0%） of identity with other RGNNV， 85.5%- 87.0% of BFNNV， 81.5% with TPNNV， 81.5% with SJNNV， 78.5% with TNV， respectively. RdRp encoded by RNA1 was higher conserved protein （87.9%- 99.4% identities） while coat protein from RNA2 was more divergent protein that showed 78.5%-100.0% identities（Table 2）. Phylogenetic analyses of deduced protein from both viral genomes were performed to determine the relationships among betanodaviruses from various hosts. In phylogenetic analysis of deduced protein， SGYeosu08 was included in a branch of RGNNV and RGNNV isolated from Golden pompano in Malaysia and Sevenband grouper in Japan were closer to SGYeosu08 （Figure 1）.

Figure 1. Phylogenetic analysis of RNA1 （A） and RNA2 （B） of NNV SGYeosu08 isolate. Phylogenetic analysis was conducted by the neighborjoining method （1 000 bootstrap） by MEGA 6.0 ［15］.

Table 2Comparison of nucleotide or deduced amino acid sequence within betanodavirus.

3.2. Standard and primer efficiency

The serial 10-fold dilutions of the cloned plasmid were amplified in duplicate by quantitative PCR to determine the sensitivity of the assay. The slope and R2 of the RNA 1 primers set were -0.2861（93% effic iency） and 0.9976， respectively. Moreover， the slope and R2of the RNA 2 primers set were -0.3172 （108% efficiency） and 0.9982， respectively （Figure 2）. The primer set showed an equivalent efficiency and satisfactory coefficient of determination （R2） values compared to other studies ［17，18］.

3.3. qRT-PCR and TCID50method （in vitro）

Figure 3 exposes both replication curves in NNV infectivity by the TCID50/mL value （gray bar） and viral copy numbers calculated by qRT-PCR （black bar）. In case of cell， infectivity was lower than 103.1TCID50/mL until 6 h after infection and then virus replication increased rapidly after 12 h （104.0TCID50/mL）， 24 h （106.1TCID50/mL），48 h （107.5TCID50/mL） and 72 h （108.8TCID50/mL）. Similarly， the viral copy number remained almost unchanged until 6 h （under 102.2copies/mL） and then gradually increased until 48 h. Then，About 106.5copies/mL were maintained until 72 h （Figure 3A）. In supernatant， meanwhile， infectivity was lower than 104.1TCID50/mL until 24 h after infection and then virus radically increased after 48 h（105.8TCID50/mL）， 72 h （107.9TCID50/mL）. Likewise， the viral copy number maintained under 102.9copies/mL till 24 h and then deeply improved until 48 h （104.1copies/mL）， 72 h （106.0copies/mL） （Figure 3B）. This comparison of the change in NNV infectivity and its gene copy numbers in vitro showed similar change patterns.

Figure 2. Quantitative PCR standard curve of RNA1 （A）， RNA2 （B） and melting curve （C）. The plasmid DNA harboring NNV genes were diluted by 10-fold and amplified in duplicate by quantitative PCR.

Figure 3. Comparison of NNV titer （TCID50/mL） and copy number in SSN-1 cell （A） and supernatant （B） （in vitro）. Black bar indicates log values of the copy number and gray bar indicates log values of NNV titer （TCID50/mL）.

3.4. qRT-PCR and TCID50method with NNV challenged sevenband grouper （in vivo）

The challenged fish died from day 3 after infection and cumulative mortality was estimated as 100% （Figure 4A）. Entire diseased fish showed abnormal swimming behavior and darkening body color. A total of 18 fish samples （three fish from each sampling time） were used to determine average viral copy numbers/mL and TCID50/mL and these results are in Figure 4b. NNV was under detection limit on the 24 hours after injection （H.A.I.）， but the virus rapidly multiplied on the 48 H.A.I. The NNV infectivity on the 72， 84 and 90 H.A.I were 105.0， 104.0， 105.8TCID50/mL， respectively. Meanwhile， two fish were died on the 96 H.A.I. thus it recorded the highest titer of 108.6TCID50/mL. Equally， the viral copy number was not detected on the 24 H.A.I. but went up to 104.0copies/mL on the 48 H.A.I. The NNV copy numbers on the 72， 84， 90 and 96 H.A.I were 104.0， 104.0，104.9and 107.2copies/mL， respectively. Figure 4c represents the copy numbers of NNV versus the infectivity （TCID50/mL） for individualsamples. From these results， the changes of NNV infectivity in challenged fish were comparable to that of the copy numbers.

Figure 4. NNV challenge test and comparison of NNV titer （TCID50/mL）and copy number （in vivo）. （A） Mortality of challenged fish； （B） Black bar indicates average log values of the NNV copy number and gray bar indicates average log values of NNV titer （TCID50/mL）； （C） The copy numbers of NNV determined from qRT-PCR versus those from NNV titer （TCID50/mL）for individual samples.

4. Discussion

In this study， we have shown that the NNV SGYeosu08 isolated from Korea was included in RGNNV group from both viral gene（RNA1 and RNA2）. Since Panzarin et al. demonstrated the presence of the RGNNV/SJNNV reassortants harboring the RNA1 of the RGNNV and the RNA2 of SJNNV， or opposite combination in Southern Europe ［19］ it is necessary to analyze both NNV genes. Based on the complete genome sequence， the deduced coat protein sequence was more diverse than RNA dependent RNA polymerase compared to other betanodaviruses. This is similar result that of viral hemorrhagic septicemia virus （VHSV）， RNA polymerase protein is the highest conserved protein among six encoding proteins even it is the largest gene ［20］. Therefore， RdRp gene might be more appropriate target gene to develop diagnostic tool for certifying all genotypes whereas previous studies were developed mainly using a coat protein gene ［18， 21］.

The greater the damage of sevenband grouper industry， more sensitive and accurate detection method depending on the genotype is required to minimize enormous damages caused by NNV infection. We developed new quantitative RT-PCR tool based on its genetic information， moreover applied it to virus replication kinetics and demonstrated that it is comparable to traditional bio-assay method （TCID50）. This is the first evaluation study of the changes in NNV replication kinetics and its gene copy numbers in vitro and in vivo. Interestingly， in in vitro experiment， inside of SSN-1 cell the virus replicated gradually from the beginning of experiment while the virus kept start level until 24 h then rapidly rising at 48 h in the supernatant. This result gives a simple hint that the NNV was released out from cells after 24 h post infection. By the way，minimal viral replication times were approximately 13 min for T7 bacteriophages ［22］， 1.2 days for HIV ［23］， and 30 h for duck hepatitis B virus ［24］.

In the challenged experiment， the NNV SGYeosu08 isolate showed high mortality up to 100 % within 6 day. The viral infectivity and copy numbers in the brain tissue were increased along to the mortality rate. Although the morality rate was 50% at 96 H.A.I （4th day）， the viral replication was reached approximately maximum value in fish.

In case of enveloped virus， it shows more high value of the infectivity compare to the copy numbers in in vitro experiments while the copy numbers was higher than that of the infectivity in in vivo trials ［25］. In contrast， there was no significant difference on the results between in vitro and in vivo experiments in this study.

Hick and Whittington also estimated both viral copy number and viral infectivity （TCID50） with NNV diluted in cell culture media. In their study， the number of viral copies was strongly correlated with the number of TCID50［18］. Similarly， in case of brain tissue from our study， most samples were correlated intensely. However， few samples were out of regression. For example， one sample had about6 Log TCID50/mL but it showed different copies. Thus， extra studies with more field samples are required to find accurate reasons what causes the differences between in vivo results.

In conclusion， the qRT-PCR assay developed in this study has great advantages such as high sensitivity and low-time consuming work to detect NNV in vitro and in vivo. This tool will be very useful for rapid detection of NNV in fish of aqua-farms and researches to understand the relationship between virus replication and occurrence of NNV.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

This research was a part of the project titled 'Production of diagnostic antibodies for viral diseases in aquatic animals' （Project No. 20150259）， funded by the Ministry of Oceans and Fisheries，Korea.

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10.1016/j.apjtm.2016.06.014

Jong-Oh Kim， Department of Aqualife Medicine， College of Fisheries and Ocean Science， Chonnam National University， Yeosu 550-749， Korea.

Tel： +82-61-659-6947

E-mail： jongoh.kim77@gmail.com

?Corresponding author： Myung-Joo Oh， Department of Aqualife Medicine， College of Fisheries and Ocean Science， Chonnam National University， Yeosu 550-749， Korea.

Tel： 061-659-7173

Fax： 061-659-7173

E-mail： ohmj@jnu.ac.kr

This research was a part of the project titled 'Production of diagnostic antibodies for viral diseases in aquatic animals' （Project No. 20150259）， funded by the Ministry of Oceans and Fisheries， Korea.

Quantitative detection

Diagnostic

Sevenband grouper