한수지 51(4), 383-388, 2018 Original Article Korean J Fish Aquat Sci 51(4),383-388,2018 국내양식금붕어 (Carassius auratus) 와수입진주린 (Carassius auratus) 에서 Cyprinid Herpesvirus 2 (CyHV-2) 의검출 송해덕 박정수 권세련 선문대학교수산생명과학과 Molecular Evidence of Cyprinid Herpesvirus 2 (CyHV-2) in Domestic Goldfish Carassius auratus and Imported Pearlscale Goldfish Carassius auratus Hae Deok Song, Jeong Su Park and Se Ryun Kwon* Department of Aquatic life Medical Sciences, Sunmoon University, Asan 31460, Korea Goldfish hematopoietic necrosis (GHN) affects hematopoietic organs and causes high mortality in goldfish. The causative agent of GHN is cyprinid herpesvirus 2 (CyHV-2). The purpose of this study was to detect CyHV-2 in ornamental cyprinid fish. CyHV-2 monitoring was conducted on a monthly basis for 1 year using goldfish Carassius auratus and pearlscale goldfish Carassius auratus that had been cultured at a domestic aquafarm and imported from Singapore, respectively. The results of polymerase chain reaction (PCR) assays using specific primers for the CyHV-2 helicase gene showed that 27.8% of goldfish and 33.3% of pearlscale goldfish were positive for CyHV-2. No cytophatic effects were detected in koi fin or common carp brain cells inoculated with tissue homogenates from either fish. Our data provides the useful data for establishing future quarantine and disinfection policies. Key words: Goldfish, Pearlscale goldfish, Cyprinid herpesvirus 2, CyHV-2, Goldfish hematopoietic necrosis 서론 1983,. 5., (KMI, 2010). 2012 440 2016 640 (NFPQMS, 2016).., 3 (MOF, 2016)., (goldfish Carassius auratus) (pearlscale goldfish Carassius auratus). 1992 1993 (Jung and Miyazaki, 1995). GFHN (goldfish hematopoietic necrosis) HVHN (herpesviral hematopoietic necrosis), Herpesviridae cyprinid herpesvirus 2 (CyHV-2).,, CyHV-2 (Groff et al., 1998; Chang et al., 1999; Stephens et al., 2004; Goodwin et al., 2006; Jeffrey et al., 2007)., (Boitard et al., 2016; Ito et al., 2017). 15-25, 50-100% (Jung and Miyazaki, 1995; Jeffrey et al., 2007). https://doi.org/10.5657/kfas.2018.0383 Korean J Fish Aquat Sci 51(4) 383-388, August 2018 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 29 May 2018; Revised 20 June 2018; Accepted 4 July 2018 *Corresponding author: Tel: +82. 41.530. 2289 Fax: +82. 41. 530. 2917 E-mail address: srkwon@sunmoon.ac.kr Copyright 2018 The Korean Society of Fisheries and Aquatic Science 383 pissn:0374-8111, eissn:2287-8815
384 송해덕ㆍ박정수ㆍ권세련, (Groff et al., 1998).,. CyHV-2. 1. CyHV-2 primer,. 재료및방법 실험어 2016 12 2017 11.. 12 (4.62 0.60 cm, 8.47 2.54 g) 12 (3.4 0.14 cm, 5.98 0.52 g).., 4 pooling 3. -80. PCR 및염기서열분석, DNA. DNA DNeasy blood and tissue kit (QIAGEN Inc, Germany) -80.. CyHV-2 PCR (polymerase chain reaction) helicase gene primer CyHV2HelF: 5 - GGACTTGCGAAGAGTTTGATTTCTAC-3 CyHV2HelR: 5 -CCATAGTCACCATCGTCTCATC-3 (Waltzek et al., 2009). PCR Takara EX Taq (Takara Bio Inc., Japan) 95 2 60 45, 72 45, 95 30 35 72 2. PCR product 1.5% agarose gel, ethidium bromide gel doc UV illuminator (Bio-Rad). PCR Wizard SV gel and PCR clean-up system (Promega). CyHV-2 helicase gene Genebank 13. CyHV-2 BioEdit. CyHV-2 DNA helicase gene BioEdit program (BioEdit sequence alignment editor version 7.2.5) trimming clustalw mutiple alignment. 세포배양 PCR. 9 HBSS (Gibco) 4 4000 rpm 15 2.. KF-1 (koi fin) CCB (common carp brain) 10% fetal bovine serum x1 antibiotic-antimycotic (Gibco) minimum essential medium (MEM, Gibco) trypsin-edta (Gibco) MEM 24-well plate 1 ml 20. 100 µl 30. 1 ml 20 2 cytopathic effect (CPE). 결과및고찰 144 15. (Fig. 1). 1 3, 2 2, 3 2, 4 1 3, 5 1 3, 6 2, 7 1, 2, 3, 8 2, PCR 2 2, 6 2, 7 1, 2, 3 CyHV-2. PCR PCR band density. 144. Goodwin et al. (2009) CyHV-2 PCR.. Wang et al. (2012) CyHV-2
금붕어와진주린의 CyHV-2 모니터링 385 Fig. 1. Gross findings observed in pearlscale goldfish Carassius auratus. A, Bleeding in the gill and operculum; B, abnormal bleeding; C, splenomegaly; D, normal fish. 366bp (A) (B) Fig. 2. Agarose gel electrophoresis of PCR products generated from tissue samples of goldfish (A) and pearlscale goldfish Carassius auratus (B) using primer targeting helicase gene of cyprinid herpesvirus 2. Fish were sampled in January 2017. Number means each pooled sample consisting of 4 fish. M, marker; G, gill; S, spleen; K, kidney; N, negative control.., DNA PCR 366 bp (Fig. 2). PCR CyHV-2 helicase gene. 1 CyHV-2 Table 1. 1, 3, 4, 5, 8 9. 36 11, 11 14. 1, 3, 4, 5, 6 12. 36 11, 8 11. 33.3% 27.8%, 3. PCR 2017 4. 2017 4 GF 1704. 2017 5. 2017
386 송해덕ㆍ박정수ㆍ권세련 Fig. 3. Comparison of nucleotide sequences of helicase gene from 17 CyHV-2 isolates. GF, goldfish Carassius auratus; PG, pearlscale goldfish Carassius auratus. Table 1. Detection of cyprinid herpesvirus 2 (CyHV-2) by PCR assay Month Pearlscale goldfish Carassius auratus Goldfish Carassius auratus Gill Spleen Kidney Gill Spleen Kidney January + + + + + + + + + + + + + + February March + + April + + + + + + + + + + + + + + + + + + May + + + + + + + + + + June + + + + + + + + + July August + + + + + + + + + September + + October November December + + + +, PCR positive;, PCR negative.
금붕어와진주린의 CyHV-2 모니터링 387 5 PG 1705. 1 GF 1701 PG 1701. 4 (GF 1701, GF 1704, PG 1701 PG 1705) 13. GF 1701 PG 1701 helicase gene 11 100%, GF 1704, PG 1705, EU349287 JX164199 nucleotide 1-2 (Fig. 3). helicase GF 1704 tryptophane arginine, PG 1705 alanine aspartic acid. KF-1 CCB (data not shown). Groff et al. (1998) chinook salmon embryo (CHSE- 214), fathead minnow (FHM) epithelioma papulosum cyprinid (EPC) CPE. CyHV-2 GF-1 (goldfish fin) (Jung and Miyazaki, 1995), KF-1 (Wang et al., 2012). CyHV-2 KF-1, CPE (Jeffery et al., 2007). KF-1 CCB CyHV-2. Jeffery et al. (2007). 1 CyHV-2. CyHV-2 Ito et al. (2017) CyHV-2, CyHV-2.. Ryu (2015) 2014 4 6, CyHV-2.. CyHV-2 CyHV-2. 사사 2017 (NRF-2017R1D1A1A09000992). References Boitard PM, Baud M, Labrut S, De boisseson C, Jamin M and Bigarre L. 2016. First detection of Cyprinid Herpesvirus 2 (CyHV-2) in goldfish (Carassius auratus) in France. J Fish Dis 39, 673-680. http://dx.doi.org/10.1111/jfd.12400. Chang PH, Lee SH, Chiang HC and Jong MH. 1999. Epizootic of herpes-like virus infection in goldfish, Carassius auratus in Taiwan. Fish Pathol 34, 209-210. http://dx.doi. org/10.3147/jsfp.34.209. Goodwin AE, Khoo L, LaPatra SE, Bonar A, Key DW, Garner M, Lee MV and Hanson L. 2006. Goldfish haematopoietic necrosis herpesvirus (Cyprinid Herpesvirus 2) in the USA: molecular confirmation of isolates from diseased fish. J Aquat Anim Health 18, 11-18. http://dx.doi.org/10.1577/ H05-007.1. Goodwin AE, Sadler J, Merry GE and Marecaux EN. 2009. Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms. J Fish Dis 32, 271-278. http://dx.doi.org/10.1111/j.1365-2761.2008.0098.x. Groff JM, LaPatra SE, Munn RJ and Zinkl JG. 1998. A viral epizootic in cultured populations of juvenile goldfish due to a putative herpesvirus etiology. J Vet Diagn Invest 10, 375-378. http://dx.doi.org/10.1177/104063879801000415. Ito T, Kurita J and Haenen OLM. 2017. Importation of CyHV- 2-infected goldfish into the Netherlands. Dis Aquat Org 126, 51-62. http://dx.doi.org/10.3354/dao03157. Jeffrey KR, Bateman K, Bayley A, Feist SW, Hulland J, Longshaw C, Stone D, Woolford G and Way K. 2007. Isolation of a cyprinid herpesvirus 2 from goldfish, Carassius auratus (L.), in the UK. J Fish Dis 30, 649-656. http://dx.doi. org/10.1111/j.1365-2761.2007.00847.x. Jung SJ and Miyazaki T. 1995. Herpesviral haematopoietic necrosis of goldfish, Carassius auratus (L.). J Fish Dis 18, 211-220. http://dx.doi.org/10.1111/j.1365-2761.1995.tb00296.x. KMI (Korea Maritime Institute). 2010. Report of policy direction for a high value-added ornamental fish industry. KMI, Busan, Korea, 1-160. MOF (Ministry of Oceans and Fisheries). 2016. Rapid growth of ornamental fish market [Internet]. Retrieved from http:// www.mof.go.kr/article/view.do?menukey=376&boardkey
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