{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T01:10:58Z","timestamp":1776906658479,"version":"3.51.2"},"reference-count":27,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,3,5]],"date-time":"2025-03-05T00:00:00Z","timestamp":1741132800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Institute of Fisheries Science","award":["R2025052"],"award-info":[{"award-number":["R2025052"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Japanese eel endothelial cells-infecting virus (JEECV) and Anguillid herpesvirus 1 (AnHV) are major pathogens in farmed eels. JEECV causes eel viral endothelial cell necrosis (VECNE), while AnHV leads to symptoms such as head erythema and gill necrosis. Both viruses cause severe mortality alone or in combination, necessitating rapid and early detection of their presences. In this study, we developed a highly efficient duplex quantitative PCR method (r2 = 0.999) using hydrolysis probes for the rapid and simultaneous detections of AnHV and JEECV. This new diagnostic method demonstrated a 1.7-fold higher detection rate for AnHV and a 2.5-fold higher detection rate for JEECV than conventional PCR. Quantitative analysis of water and eel tissue samples from aquaculture facilities revealed that the two viruses could be detected in water 1\u20133 months prior to mortality, enabling their early identification of infections through water testing alone. Notably, the method reliably detected low viral loads (&lt; 1 copy) in both water and tissue samples, facilitating preclinical detection and proactive disease management. This approach reduces the risk of mass mortality and economic losses in eel farming. This study underscores the critical role of advanced molecular diagnostic technologies in enhancing health management in aquaculture.<\/jats:p>","DOI":"10.3390\/biology14030264","type":"journal-article","created":{"date-parts":[[2025,3,5]],"date-time":"2025-03-05T09:37:52Z","timestamp":1741167472000},"page":"264","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2433-3665","authenticated-orcid":false,"given":"Jun-Young","family":"Song","sequence":"first","affiliation":[{"name":"Department of Aquatic Life Medical Sciences, Sunmoon University, Asan 31460, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Keun-Yong","family":"Kim","sequence":"additional","affiliation":[{"name":"Genetic Analysis Team, AquaGenTech Co., Ltd., Busan 48228, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ahran","family":"Kim","sequence":"additional","affiliation":[{"name":"Pathology Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,5]]},"reference":[{"key":"ref_1","unstructured":"(2023, May 02). Ministry of Oceans and Fisheries Statistics System. (In Korean)."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"260","DOI":"10.47853\/FAS.2021.e26","article-title":"Comparative study on growth of leafy vegetables grown in a hybrid BFT-aquaponics using Japanese eel, Anguilla japonica and hydroponics","volume":"24","author":"Lee","year":"2021","journal-title":"Fish. Aquat. Sci."},{"key":"ref_3","first-page":"668","article-title":"Status and characteristics of JEECV (Japanese eel endothelial cell-infecting virus) and AnHV (anguillid herpesvirus 1) infections in domestic farmed eels Anguilla japonica, Anguilla bicolor and Anguilla marmorata","volume":"54","author":"Jang","year":"2021","journal-title":"Korean J. Fish. Aquat. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1111\/j.1365-2761.2012.01344.x","article-title":"Molecular evidence of anguillid herpesvirus-1 (AngHV-1) in the farmed Japanese eel, Anguilla japonica Temminck & Schlegel, in Korea","volume":"35","author":"Kim","year":"2012","journal-title":"J. Fish Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"185","DOI":"10.35399\/ISK.30.4.1","article-title":"Characteristics of viral endothelial cell necrosis of eel (VECNE) from culturing eel (Anguilla japonica, Anguilla bicolar) in Korea","volume":"30","author":"Kim","year":"2018","journal-title":"Korean J. Ichthyol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"151","DOI":"10.7847\/jfp.2012.25.3.151","article-title":"Outbreak of anguillid herpesvirus-1 (AngHV-1) infection in cultured shortfin eel (Anguilla bicolor) in Korea","volume":"25","author":"Park","year":"2012","journal-title":"J. Fish Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"51","DOI":"10.3147\/jsfp.24.51","article-title":"Histopathological observations on an intense congestion of the gills in cultured Japanese eel, Anguilla japonica","volume":"24","author":"Egusa","year":"1989","journal-title":"Fish Pathol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.virol.2010.12.057","article-title":"Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica","volume":"412","author":"Mizutani","year":"2011","journal-title":"Virology"},{"key":"ref_9","first-page":"31","article-title":"Isolation and characterization of a new herpesvirus from eel","volume":"15","author":"Sano","year":"1990","journal-title":"Pathol. Mar. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"111","DOI":"10.3354\/dao050111","article-title":"Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan","volume":"50","author":"Chang","year":"2002","journal-title":"Dis. Aquat. Organ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"89","DOI":"10.3147\/jsfp.32.89","article-title":"Characterization and pathogenicity of a herpesvirus isolated from cutaneous lesion in Japanese eel, Anguilla japonica","volume":"32","author":"Kobayashi","year":"1997","journal-title":"Fish Pathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1046\/j.1365-2761.1999.00197.x","article-title":"Gill filament necrosis in farmed Japanese eels, Anguilla japonica (Temminck & Schlegel), infected with herpesvirus anguillae","volume":"22","author":"Lee","year":"1999","journal-title":"J. Fish Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1111\/j.1439-0426.1996.tb00059.x","article-title":"Biological and serological comparisons of eel herpesvirus in Formosa (EHVF) and herpesvirus anguillae (HVA)","volume":"12","author":"Ueno","year":"1996","journal-title":"J. Appl. Ichthyol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.jviromet.2004.11.007","article-title":"Development of a polymerase chain reaction for the detection of anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene","volume":"124","author":"Rijsewijk","year":"2005","journal-title":"J. Virol. Methods"},{"key":"ref_15","first-page":"137","article-title":"First isolation of herpesvirus of eel (herpesvirus anguillae) in diseased European eel (Anguilla anguilla L.) in Europe","volume":"19","author":"Davidse","year":"1999","journal-title":"Bull. Eur. Assoc. Fish Pathol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Guo, R., Zhang, Z., He, T., Li, M., Zhuo, Y., Yang, X., Fan, H., and Chen, X. (2022). Isolation and identification of a new isolate of anguillid herpesvirus 1 from farmed American eels (Anguilla rostrata) in China. Viruses, 14.","DOI":"10.3390\/v14122722"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"847","DOI":"10.1111\/jfd.13350","article-title":"First detection of herpesvirus anguillae (AngHV-1) associated with mortalities in farmed giant mottled eel (Anguilla marmorata) in Vietnam","volume":"44","author":"Panicz","year":"2021","journal-title":"J. Fish Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"69","DOI":"10.3354\/dao02501","article-title":"Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands","volume":"101","author":"Engelsma","year":"2012","journal-title":"Dis. Aquat. Organ."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e01677-16","DOI":"10.1128\/genomeA.01677-16","article-title":"Complete Genome Sequence of herpesvirus anguillae Strain HVA980811 Isolated in Chiayi, Taiwan","volume":"5","author":"Wen","year":"2017","journal-title":"Genome Announc."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Kole, S., Kim, H.J., and Jung, S.J. (2022). Complete genome sequence of anguillid herpesvirus 1 isolated from imported Anguilla rostrata (American eel) from Canada. Microbiol. Resour. Announc., 11.","DOI":"10.1128\/mra.00829-22"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"880","DOI":"10.1099\/vir.0.016261-0","article-title":"Complete genome sequence and taxonomic position of anguillid herpesvirus 1","volume":"91","author":"Bossers","year":"2010","journal-title":"J. Gen. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"740091","DOI":"10.1016\/j.aquaculture.2023.740091","article-title":"Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and anguillid herpesvirus-1","volume":"578","author":"Kim","year":"2024","journal-title":"Aquaculture"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.jviromet.2007.01.024","article-title":"Recovery of naked viral genomes in water by virus concentration methods","volume":"142","author":"Haramoto","year":"2007","journal-title":"J. Virol. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1111\/j.1365-2761.2007.00778.x","article-title":"Detection of koi herpesvirus DNA in river water in Japan","volume":"30","author":"Haramoto","year":"2007","journal-title":"J. Fish Dis."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e13157","DOI":"10.7717\/peerj.13157","article-title":"Concentration and quantification of tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR","volume":"10","author":"Taengphu","year":"2022","journal-title":"PeerJ"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"113990","DOI":"10.1016\/j.jviromet.2020.113990","article-title":"Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater","volume":"287","author":"Weli","year":"2021","journal-title":"J. Virol. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Bohara, K., Yadav, A.K., and Joshi, P. (2022). Detection of fish pathogens in freshwater aquaculture using eDNA methods. Diversity, 14.","DOI":"10.20944\/preprints202210.0291.v1"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/3\/264\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:47:43Z","timestamp":1760028463000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/14\/3\/264"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,5]]},"references-count":27,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["biology14030264"],"URL":"https:\/\/doi.org\/10.3390\/biology14030264","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,5]]}}}