한수지 50(5), , 2017 Original Article Korean J Fish Aquat Sci 50(5), ,2017 신경괴사증바이러스 (Nervous Necrosis Virus, NNV) 모니터링을통한무감염능성어 (Hyporthodu

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1 한수지 50(5), , 2017 Original Article Korean J Fish Aquat Sci 50(5), ,2017 신경괴사증바이러스 (Nervous Necrosis Virus, NNV) 모니터링을통한무감염능성어 (Hyporthodus septemfasciatus) 친어의선발 김시우 1,3 김위식 1 서한길 2 김경민 3 오명주 1 * 1 전남대학교수산생명의학과, 2 국립수산과학원병리연구과, 3 국립수산과학원남해연구소양식산업과 Monitoring of Nervous Necrosis Virus (NNV) in the Broodstock of Seven Band Grouper Hyporthodus septemfasciatus Si-Woo Kim 1,3, Wi-Sik Kim 1, Han-Gill Seo 2, Kyong Min Kim 3 and Myung-Joo Oh 1 * 1 Department of Aqualife medicine, Chonnam National University, Yeosu , Korea 2 Pathology Research Division, National Fisheries Research and Development Institute, Busan 46083, Korea 3 South Sea Fisheries Research Institute, National Institute of Fisheries Science, Yeosu 59780, Korea 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 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 Additionally, 4 of 34 broodstocks (11%) investigated in 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. Key words: Sevenband grouper, BroodStock, Nervous necrosis virus, ELISA, Vertical transmission 서론 (Hyporthodus septemfasciatus),.,,. 2003, , , (statistics Korea, 2017).,, (Hong et al., 2015; Kim and Kim, 2015). (viral nervous necrosis, VNN). VNN (Munday et al., 2002). VNN,,,,, (Pseudocaranx dentex), (Paralichthys olivaceus), (Dicentrarchus labrax), (Sciaenops ocellatus), (Sebastes oblongus) Korean J Fish Aquat Sci 50(5) , October 2017 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 25 September 2017; Revised 12 October 2017; Accepted 23 October 2017 *Corresponding author: Tel: Fax: address: ohmj@chonnam.ac.kr Copyright 2017 The Korean Society of Fisheries and Aquatic Science 527 pissn: , eissn:

2 528 김시우ㆍ김위식ㆍ서한길ㆍ김경민ㆍ오명주 (Munday et al., 2002). 1989,, 80% (Sohn et al., 1991).,,, (Nguyen et al., 1996; Nopadon et al., 2009). VNN Nodaviridae nervous necrosis virus (NNV),, positive sense single-stranded RNA. Virion nm, 20 (Munday et al., 2002). NNV coat protein gene nucleotide sequence striped jacked nervous necrosis virus (SJNNV), tiger puffer nervous necrosis virus (TPNNV), barfin flounder nervous necrosis virus (BFNNV), red-spotted grouper nervous necrosis virus (RGNNV) turbot nervous necrosis virus (TNV) 5 group genotype (Johansen et al., 2004; Nishizawa et al., 1997; Nishizawa et al., 1995). RGNNV SJNNV (Oh et al., 2005). NNV (P. dentex), (D. labrax), (V. moseri) (E. tukula) (Nguyen et al., 1996; Peducasse et al., 1999; Breuil et al., 2000; Watanabe et al., 2000; Breuil et al., 2002; Kai et al., 2010). NNV, (Mushiake et al., 1994; Arimoto et al., 1996; Grotmol and Totland, 2000; Watanabe et al., 2000)., RGNNV, , enzyme-linked immunosorbent assay (ELISA) NNV nested polymerase chain reaction (PCR) NNV, NNV. Fig 1. Broodstock sampling location in Korea (a)geomun island, (b)yeosu, (c) Tongyeong. 재료및방법 능성어친어샘플링 ,, 4-6 ( kg / cm), (Fig. 1) , , (Table 1). 바이러스 NNV Table. 1. List of experiment done in , and result Year Sampling location Number of tested broodstock PCR ELISA in vitro cultivation 2012 Geomun island 168 N.T 45/166 (27 %) 1/ Yeosu 3 2/3 (66%) 1 N.T 0/ Tongyeong 31 2/31 (6.5%) 1 14/31 (45%) 0/19 1 Positive sample/total sample (positive percentage). N.T, not tested.

3 NNV 무감염능성어친어의선발 NNV Yeosu08 isolate (RGNNV) (Kim et al., 2012). NNV Striped Snakehead (SSN-1). 10% (V/V) fetal bovine serum (FBS), 100 IU/ ml penicillin G, 100 ug/ml streptomycin Leibovitz`s L-15 medium cm 2 (Corning, USA) SSN-1 cell NNV Yeosu08, 25 (cytopathic effect, CPE). 7, 8,000 rpm ml tube 200 ul -80. NNV 검출을위한 RT-PCR 및 nested RT-PCR Total RNA mirneasy MiNi Kit (Qiagen, USA), RNA. RNA, -80. total RNA cdna M-MLV reverse transcriptase (Bioneer, Korea). RNA 5 ul R3 (5`-CGA GTC AAC ACG GGT GAA GA-3`) reverse primer (10 pmol) 2 ul, RNasefree water 4 ul RNA primer denaturation. RNA primer reaction buffer 4 ul, 10 mm mixed dntp 2 ul, 100 mm DTT 2 ul, RNase inhibitor (40 U/uL) 0.5 ul, M- MLV reverse transcriptase (200 U/uL) 0.5 ul ul volume reaction mixture MyGenie 32 Thermal Block (Bioneer, Korea) 37 1, 94 5, 4 3. RT-PCR primer RGNNV RNA2 T4 forward primer F2 (5`-CGT GTC AGT CAT GTG TCG CT-3`) reverse primer R3 (5`-CGA GTC AAC ACG GGT GAA GA-3`) (Nishizawa et al., 1994). PCR pre-incubation 72 10, pre-denaturation 95 2, denaturation 95 40, annealing 50 40, extension cycle post-extension PCR. Nested RT-PCR primer forward primer NF2 (5`-GTT CCC TGT ACA ACG ATT CC-3`) reverse primer NR3 (5`-GGA TTT GAC GGG GCT GCT CA-3`) (Thiery et al., 1999). PCR pre-denaturation 94 2, denaturation 94 40, annealing 50 40, extension cycle post-extension PCR 5 ul 1% agarose gel, 100 Volt 25 UV transilluminator (Bio- Rad, USA). 배양세포주를이용한 NNV 바이러스분리배양 hanks balanced salt solution (HBSS, Gibco, USA) 1:9, 0.45 µm syringe filter (Pall corporation, USA), SSN-1 25 CPE. 혈액중 NNV 특이항체검출 ELISA (6,000 rpm, 4, 20 min) ELSIA Kim et al. (2007) TCID 50 /ml Yeosu08 isolate (RGNNV) 320 ELISA plate 50 ul, 37 overnight. Tween % PBS (T-PBS) 3 5% skim milk 380 L % skim milk (40 ) 1, 2 well 50 L, 2 5% skim milk 500 IgM 50 L, 3 5% skim milk 1000 peroxidase conjugated goat anti rabbit immunoglobulin antibody (DakoCytomation, Denmark) 50 L T-PBS 5 ELISA (100 mm Na 2 HPO 4, 50 mm citric acid, 1 mg/ 1 ml o-phenylenediamine, 0.03% H 2 O 2 ) well 50 L well 2N H 2 SO 4 50 L ELISA plate reader (Spectra max 340, USA) 492 nm O.D (optical density). 결과 NNV 바이러스분리배양 NNV,, SSN-1 25 (CPE) NNV (Fig. 3), CPE NNV RT- PCR NNV

530 김시우ㆍ김위식ㆍ서한길ㆍ김경민ㆍ오명주 혈액 중 NNV 특이항체 검출 ELISA 결과 능성어 친어의 NNV 감염이력과 Anti-NNV 특이항체 보유유 무를 확인하기 위해, 2012-2014년 동안 산란기에 앞서 능성어 친어의 혈액을 이용하여 ELISA실험을 실시하였다(Table 1).

4 530 김시우ㆍ김위식ㆍ서한길ㆍ김경민ㆍ오명주 혈액 중 NNV 특이항체 검출 ELISA 결과 능성어 친어의 NNV 감염이력과 Anti-NNV 특이항체 보유유 무를 확인하기 위해, 년 동안 산란기에 앞서 능성어 친어의 혈액을 이용하여 ELISA실험을 실시하였다(Table 1). 2012년 총 166마리의 능성어 친어를 대상으로 ELISA를 실시 한 결과 흡광도(optical density, O.D) 값 0.1이상을 나타내는 45 마리에서 NNV 특이항체가 존재하는 것을 확인할 수 있었다. 2014년 능성어 친어 ELISA의 결과는 31마리의 능성어 친어 중 14마리에서 O.D 값 0.1이상을 확인하여, 검사 친어 중 14마리 에서 NNV 특이항체가 존재함을 확인하였다(Fig. 2). 종묘생산 Fig. 3. SSN-1 cell, inoculated with homogenized egg supernatant, showed typical NNV CPE. NNV, nervous necrosis virus; CPE, cytopathic effect. 확인할 수 있었다. 2013년 1마리, 2014년 19마리의 친어에서 취한 난을 이용하여 NNV의 분리배양을 실시한 결과 전 시료에 서 CPE가 나타나지 않아 음성임을 확인하였다. NNV 바이러스 특정유전자의 검출 능성어 친어 어체내의 NNV 보균유무를 확인하기 위해, 산 란기에 앞서 능성어 친어의 난과 정자시료를 채집하여, nested RT-PCR법 적용한 NNV 검출을 실시하였다(Table 1). 2013년 3마리의 친어에서 채집한 난과, 14년 31마리(암컷 22마리, 수 컷 9마리)의 친어에서 채집한 난과 정자를 대상으로 실시한 RTPCR 결과, 2013년 2개의 난 시료와 2014년 2개의 정자 시료에 서 양성의 결과를 확인할 수 있었다. 2014년 경남 통영의 양식장에서 ELISA와 RT-PCR의 결과 모두에서 음성이 확인된 암 수 능성어 친어를 선별하여 종묘생 산에 사용하였다. 수컷 2마리와 암컷 6마리에서 총 4,500 ml 의 수정난을 취하였고, 침전난을 제거한 2,700 ml의 부상난 중 1,000 ml에서 약 1,000,000마리의 치어를 부화시켰다. 종묘 생산에 앞서 종묘생산장의 사육수조와 기구의 소독이 이루어 졌으며, 오염된 사육수를 통한 NNV감염을 차단하기 위하여 UV멸균 해수를 사용하였다. 아울러 초기 먹이생물로 공급되 는 로티퍼와 알테미아 유생의 NNV 오염유무를 먹이공급에 앞 서 RT-PCR을 실시하여 검사하였고, 음성의 결과를 확인 후 공 급하였다. 이후 정기적인 샘플링을 실시하여 건강한 능성어 치 어, 폐사어, 바닥의 침전물(배설물, 먹이잔여물)에 대해서 NNV 검출 RT-PCR을 하였으며, 종묘생산 기간 중 전 시료에서 NNV 는 검출되지 않았다. 14년 10월경 종묘로 판매가능한 8-10 cm (14.7±1.4 g) 크기로 성장한 능성어 치어 약 3만마리를 생산하 였다. 친어선별을 통한 NNV 수직감염 예방과 종묘생산기간 중 외부로부터의 NNV 수평감염 차단의 결과, 능성어 자치어 시 기의 NNV로 인한 대량폐사 없이 건강한 능성어 치어를 생산 할 수 있었다. ELISA OD value (492 nm) P.C N.C Fig. 2. Year of 2012 & 2014, bloodstock antibody detection ELISA result. Each dot indicate individual broodstocks. 59 samples out of 197 samples (30%) showing positive values (OD 0.1). P.C, positive control; N.C, negative control.

5 NNV 무감염능성어친어의선발 531 고찰 NNV (Peducasse et al., 1999; Breuil et al., 2000; Watanabe et al., 2000; Breuil et al., 2002; Kai et al., 2010), NNV (Mushiake et al., 1994; Arimoto et al., 1996; Grotmol and Totland, 2000; Watanabe et al., 2000). NNV ELISA RT-PCR NNV (Kim and Kim., 2015). NNV, VNN,,,, NNV, (Munday et al., 2002). 7-15,. NNV RT- PCR. NNV, nested RT-PCR 34 4 ( 2, 2) 22 NNV 1 NNV. NNV, (Nishizawa et al., 1997; Watanabe et al., 2000; Janina and Kim, 2016). (, carrier stage), (Agius et al., 1982; Munro et al., 2004).,,. NNV NNV ELISA, NNV , NNV (30%) NNV, NNV. NNV. ELISA nested RT-PCR NNV, SPF , NNV NNV. nested RT-PCR NNV, ELISA NNV. VNN.,, (Cano et al., 2012; Zhang et al., 2006). 사사 2017 (R ). References Agius C, Mangunwiryo H, Johnson H and Smail DA A more sensitive technique for isolating infectious pancreatic necrosis virus from asymptomatic carrier rainbow trout, Salmo gairdneri Richardson. J Fish Dis 5, / j tb00484.x. Arimoto M, Sato J, Maruyama K, Mimura G and Furusawa I Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV). Aquaculture 143, (96) Breuil G, Pepin J, Boscher S and Thiery R Experimental vertical transmission of nodavirus from brood fish to

6 532 김시우ㆍ김위식ㆍ서한길ㆍ김경민ㆍ오명주 eggs and larvae of the sea bass, Dicentrarchus labrax (L.). J Fish Dis 25, Breuil G, Pepin J, Castric J, Fauvel C and Thiery R Detection of serum antibodies against nodavirus in wild and farmed adult sea bass: application to the screening of brood stock in sea bass hatcheries. B Eur Assoc Fish 20, Cano I, Valverde EJ, Garcia-Rosado E, Alonso MC, Lopez- Jimena B, Ortiz-Delgado JB, Borrego JJ, Sarasquete C and Castro D Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae. J Fish Dis 20, Grotmol S and Totland G Surface disinfection of Atlantic halibut Hippoglossus hippoglossus eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae. Dis Aquat Organ 39, dao Hong CG, Cho JK, Park JY, Son MH, Park JM, Han KH and Kang HW Ovulation Induction Effect of Sevenband Grouper, Epinephelus septemfasciatus by Treating Hormones. J Fish Mar Sci Educa 27, org/ /jfmse Huang B, Tan C, Chang SF, Munday B, Mathew JA, Ngoh GH and Kwang J Detection of nodavirus in barramundi, Lates calcarifer (Bloch), using recombinant coat proteinbased ELISA and RT-PCR. J Fish Dis 24, doi.org/ /j x. Janina ZC and Kim DT Understanding the interaction between Betanodavirus and its host for the development of prophylactic measures for viral encephalopathyand retinopathy. Fish Shellfish Immunol 53, org/ /j.fsi Johansen R, Sommerset I, Tørud B, Korsnes K, Hjortaas MJ, Nilsen F, Nerland AH and Dannevig BH Characterization of nodavirus and viral encephalopathy and retinopathy in farmed turbot, Scophthalmus maximus (L.). J Fish Dis 27, Kai YH, Su HM, Tai KT and Chi SC Vaccination of grouper broodfish (Epinephelus tukula) reduces the risk of vertical transmission by nervous necrosis virus. Vaccine 28, Kim CS, Kim WS, Nishizawa T and Oh MJ Prevalence of viral nervous necrosis (VNN) in sevenband grouper Epinephelus septemfasciatus farms. J Fish Pathol 25, Kim WS and Kim JO Prevention Strategies for Viral Nervous Necrosis (VNN) in Sevenband Grouper Epinephelus septemfasciatus Aquaculture Farms. Korean J Fish Aquat Sci 48, Kim WS, Nishizawa T and Yoshimizu M Non-speci c adsorption of sh immunoglobulin M (IgM) to blocking reagents on ELISA plate wells. Dis Aquat Org 78, Munday BL and Nakai T Nodaviruses as pathogens in larval and juvenile marine finfish. World J Microbiol Biotechnol 13, Munro ES, Gahlawat SK and Ellis AE A sensitive nondestructive method for detecting IPNV carrier Atlantic salmon, Salmo salar L., by culture of virus from plastic adherent blood leucocytes. J Fish Dis 27, /j x. Mushiake K, Nishizawa T, Nakai T, Furusawa I and Muroga K Control of VNN in striped jack-selection of spawners based on the detection of SJNNV gene by polymerase chain-reaction (PCR). Fish Pathol 29, org/ /jsfp Nguyen HD, Nakai T and Muroga K Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae. Dis Aquat Organ 24, doi.org/ /dao Nishizawa T, Furuhashi M, Nagai T, Nakai T and Muroga K Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl Environ Microbiol 64, Nishizawa T, Mori KI, Furuhashi M, Nakai T, Furusawa I and Muroga K Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish. J Gen Virol 76, doi.org/ / Nishizawa T, Mori KI, Nakai T, Furusawa I and Muroga K Polymerase chain reaction (PCR) amplification of RNA of striped jack nervous necrosis (SJNNV). Dis Aquat Organ 18, Nopadon P, Aranya P, Tipaporn T, Toshihiro N, Takayuki K, Masashi M and Makoto E Nodavirus associated with pathological changes in adult spotted coralgroupers (Plectropomus maculatus) in Thailand with viral nervous necrosis. Res Vet Sci 87, rvsc Oh MJ, Jung SJ and Kitamura SI Comparison of the coat protein gene of nervous necrosis virus (NNV) detected from marine fishes in Korea. J World Aquac Soc 36, Peducasse S, Castric J, Thiery R, Jeffroy J, Le Ven A and Baudin Laurencin F Comparative study of viral encephalopathy and retinopathy in juvenile sea bass Dicentrarchus labrax infected in different ways. Dis Aquat Organ 36, Sohn SG, Park MA, Lee SD and Chun SK Studies on the mass mortality of the cultured grouper, Epinephelus septemfasciatus. J Fish Pathol 4, Statistics Korea Fish farm trends survey. Retrieved from

7 on Feb 11, Thiery R, Raymond JC and Castric J Natural outbreak of viral encephalopathy and retinopathy in juvenile sea bass, Dicentrarchus labrax: study by nested reverse transcriptase polymerase chain reaction. Virus Res 63, doi.org/ /s (99) Watanabe KI, Nishizawa T and Yoshimizu M Selection of brood stock candidates of barfin flounder using an ELISA system with recombinant protein of barfin flounder nervous necrosis virus. Dis Aquat Organ 41, org/ /dao Zhang JS, Dong SL, Tian XL, Dong YW, Liu XY and Yan DC Studies on the rotifer (Brachionus urceus Linnaeus, 1758) as a vector in white spot syndrome virus (WSSV) transmission. Aquaculture 261, org/ /j.aquaculture NNV 무감염능성어친어의선발 533

170 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주, (LaPatra, 1996; Watanabe et al., 1998; 2000; Kibenge et al., 2002). ELISA, 2 IgM. ELISA IgM (monoclonal antibody, MAb). IgM manna

170 김위식ㆍ김기홍ㆍ김춘섭ㆍ오명주, (LaPatra, 1996; Watanabe et al., 1998; 2000; Kibenge et al., 2002). ELISA, 2 IgM. ELISA IgM (monoclonal antibody, MAb). IgM manna 한수지 50(2), 169-174, 2017 Original Article Korean J Fish Aquat Sci 50(2),169-174,2017 넙치 (Paralichthys olivaceus) 의 immunoglobulin M 에대한단클론항체생산 김위식 김기홍 김춘섭 1 오명주 * 전남대학교수산생명의학과, 1 엔바이로젠 Production of Monoclonal