Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Fish Sampling
2.2. Sequencing of Target Genes
2.3. Design and Verification of qPCR Markers
2.4. Optimization of qPCR Conditions
2.5. Standard Curves
2.6. Sensitivity Tests
2.7. Spike Tests Using Culture Water
2.8. Validation of the qPCR Diagnostic Method for Monitoring Viral Outbreaks in Eel Farms
3. Results
3.1. Phylogenetic Relationships of Target Genes
3.2. Optimization of Duplex qPCR
3.3. Standard Curve
3.4. Sensitivity of the qPCR Marker
3.5. Spike Tests on Culture Water
3.6. Validation of the qPCR Diagnostic Method for Monitoring Viral Outbreaks in Eel Farms
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
eDNA | environmental DNA |
cPCR qPCR | conventional PCR quantitative PCR |
JEECV | Japanese eel endothelial cells-infecting virus |
AnHV | Anguillid herpesvirus 1 |
VECNE | eel viral endothelial cell necrosis |
RASs | recirculating aquaculture systems |
Pol | DNA polymerase catalytic subunit |
Ltlg | polyomavirus large T-antigen-like protein gene |
References
- Ministry of Oceans and Fisheries Statistics System. Available online: https://www.mof.go.kr/statPortal/ (accessed on 2 May 2023). (In Korean).
- Lee, D.H.; Kim, J.D. Comparative study on growth of leafy vegetables grown in a hybrid BFT-aquaponics using Japanese eel, Anguilla japonica and hydroponics. Fish. Aquat. Sci. 2021, 24, 260–275. [Google Scholar] [CrossRef]
- Jang, M.H.; Lee, N.; Cho, M.; Song, J. 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. Korean J. Fish. Aquat. Sci. 2021, 54, 668–675. (In Korean) [Google Scholar] [CrossRef]
- Kim, H.J.; Yu, J.H.; Kim, D.W.; Kwon, S.R.; Park, S.W. Molecular evidence of anguillid herpesvirus-1 (AngHV-1) in the farmed Japanese eel, Anguilla japonica Temminck & Schlegel, in Korea. J. Fish Dis. 2012, 35, 315–319. [Google Scholar] [CrossRef] [PubMed]
- Kim, S.M.; Ko, S.M.; Jin, J.H.; Seo, J.S.; Lee, N.S.; Kim, Y.S.; Gu, J.H.; Bae, Y.R. Characteristics of viral endothelial cell necrosis of eel (VECNE) from culturing eel (Anguilla japonica, Anguilla bicolar) in Korea. Korean J. Ichthyol. 2018, 30, 185–193. (In Korean) [Google Scholar] [CrossRef]
- Park, S.W.; Jung, E.B.; Kim, D.W. Outbreak of anguillid herpesvirus-1 (AngHV-1) infection in cultured shortfin eel (Anguilla bicolor) in Korea. J. Fish Pathol. 2012, 25, 151–158. (In Korean) [Google Scholar] [CrossRef]
- Egusa, S.; Tanaka, M.; Ogami, H.; Oka, H. Histopathological observations on an intense congestion of the gills in cultured Japanese eel, Anguilla japonica. Fish Pathol. 1989, 24, 51–56. (In Japanese) [Google Scholar] [CrossRef]
- Mizutani, T.; Sayama, Y.; Nakanishi, A.; Ochiai, H.; Sakai, K.; Wakabayashi, K.; Tanaka, N.; Miura, E.; Oba, M.; Kurane, I.; et al. Novel DNA virus isolated from samples showing endothelial cell necrosis in the Japanese eel, Anguilla japonica. Virology 2011, 412, 179–187. [Google Scholar] [CrossRef]
- Sano, M.; Fukuda, H.; Sano, T. Isolation and characterization of a new herpesvirus from eel. Pathol. Mar. Sci. 1990, 15, 31. [Google Scholar]
- Chang, P.H.; Pan, Y.H.; Wu, C.M.; Kuo, S.T.; Chung, H.Y. Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan. Dis. Aquat. Organ. 2002, 50, 111–118. [Google Scholar] [CrossRef]
- Kobayashi, T.; Miyazaki, T. Characterization and pathogenicity of a herpesvirus isolated from cutaneous lesion in Japanese eel, Anguilla japonica. Fish Pathol. 1997, 32, 89–95. (In Japanese) [Google Scholar] [CrossRef]
- Lee, N.S.; Kobayashi, J.; Miyazaki, T. Gill filament necrosis in farmed Japanese eels, Anguilla japonica (Temminck & Schlegel), infected with herpesvirus anguillae. J. Fish Dis. 1999, 22, 457–463. [Google Scholar] [CrossRef]
- Ueno, Y.; Shi, J.W.; Yoshida, T.; Kitao, T.; Sakai, M.; Chen, S.N.; Kou, G.H. Biological and serological comparisons of eel herpesvirus in Formosa (EHVF) and herpesvirus anguillae (HVA). J. Appl. Ichthyol. 1996, 12, 49–51. [Google Scholar] [CrossRef]
- Rijsewijk, F.; Pritz-Verschuren, S.; Kerkhoff, S.; Botter, A.; Willemsen, M.; van Nieuwstadt, T.; Haenen, O. Development of a polymerase chain reaction for the detection of anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene. J. Virol. Methods 2005, 124, 87–94. [Google Scholar] [CrossRef]
- Davidse, A.; Haenen, O.L.M.; Dijkstra, S.G.; van Nieuwstadt, A.P.; van der Vorst, T.J.K.; Wagenaar, F.; Wellenberg, G.J. First isolation of herpesvirus of eel (herpesvirus anguillae) in diseased European eel (Anguilla anguilla L.) in Europe. Bull. Eur. Assoc. Fish Pathol. 1999, 19, 137–141. Available online: https://eafp.org/download/1999-Volume19/Issue%204/19%204%20137-141.pdf (accessed on 1 February 2025).
- Guo, R.; Zhang, Z.; He, T.; Li, M.; Zhuo, Y.; Yang, X.; Fan, H.; Chen, X. Isolation and identification of a new isolate of anguillid herpesvirus 1 from farmed American eels (Anguilla rostrata) in China. Viruses 2022, 14, 2722. [Google Scholar] [CrossRef] [PubMed]
- Panicz, R.; Eljasik, P.; Nguyen, T.T.; Vo Thi, K.T.; Hoang, D.V. First detection of herpesvirus anguillae (AngHV-1) associated with mortalities in farmed giant mottled eel (Anguilla marmorata) in Vietnam. J. Fish Dis. 2021, 44, 847–852. [Google Scholar] [CrossRef]
- van Beurden, S.J.; Engelsma, M.Y.; Roozenburg, I.; Voorbergen-Laarman, M.A.; van Tulden, P.W.; Kerkhoff, S.; van Nieuwstadt, A.P.; Davidse, A.; Haenen, O.L.M. Viral diseases of wild and farmed European eel Anguilla anguilla with particular reference to the Netherlands. Dis. Aquat. Organ. 2012, 101, 69–86. [Google Scholar] [CrossRef]
- Wen, C.M.; Liu, P.C.; Nan, F.H. Complete Genome Sequence of herpesvirus anguillae Strain HVA980811 Isolated in Chiayi, Taiwan. Genome Announc. 2017, 5, e01677-16. [Google Scholar] [CrossRef]
- Kole, S.; Kim, H.J.; Jung, S.J. Complete genome sequence of anguillid herpesvirus 1 isolated from imported Anguilla rostrata (American eel) from Canada. Microbiol. Resour. Announc. 2022, 11, e0082922. [Google Scholar] [CrossRef]
- van Beurden, S.J.; Bossers, A.; Voorbergen-Laarman, M.H.A.; Haenen, O.L.M.; Peters, S.; Abma-Henkens, M.H.C.; Peeters, B.P.H.; Rottier, P.J.M.; Engelsma, M.Y. Complete genome sequence and taxonomic position of anguillid herpesvirus 1. J. Gen. Virol. 2010, 91, 880–887. [Google Scholar] [CrossRef]
- Kim, H.J.; Bathige, S.D.N.K.; Jeon, H.B.; Kim, S.H.; Yu, C.; Kang, W.; Noh, K.; Lee, E.; Lim, B.S.; Shin, D.H. Development of a duplex qPCR assay for the detection of coinfection in eels by Japanese eel endothelial cell-infecting virus and anguillid herpesvirus-1. Aquaculture 2024, 578, 740091. [Google Scholar] [CrossRef]
- Haramoto, E.; Katayama, H.; Oguma, K.; Ohgaki, S. Recovery of naked viral genomes in water by virus concentration methods. J. Virol. Methods 2007, 142, 169–173. [Google Scholar] [CrossRef] [PubMed]
- Haramoto, E.; Kitajima, M.; Katayama, H.; Ohgaki, S. Detection of koi herpesvirus DNA in river water in Japan. J. Fish Dis. 2007, 30, 59–61. [Google Scholar] [CrossRef] [PubMed]
- Taengphu, S.; Kayansamruaj, P.; Kawato, Y.; Delamare-Deboutteville, J.; Mohan, C.V.; Dong, H.T.; Senapin, S. Concentration and quantification of tilapia tilapinevirus from water using a simple iron flocculation coupled with probe-based RT-qPCR. PeerJ 2022, 10, e13157. [Google Scholar] [CrossRef]
- Weli, S.C.; Bernhardt, L.V.; Qviller, L.; Myrmel, M.; Lillehaug, A. Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater. J. Virol. Methods 2021, 287, 113990. [Google Scholar] [CrossRef]
- Bohara, K.; Yadav, A.K.; Joshi, P. Detection of fish pathogens in freshwater aquaculture using eDNA methods. Diversity 2022, 14, 1015. [Google Scholar] [CrossRef]
Virus | Target Gene | PCR | Primer Name | Sequence (5′ → 3′) | Amplicon Size (bp) | Reference |
---|---|---|---|---|---|---|
AnHV | Pol | 1st | 2061f | CAAGCTGAAGGAGAACAGAT | 1045–1054 | This study |
3086r | GTTCGTGATCAGAGAGTTGT | This study | ||||
Nested | 2108f | CCATGTGCTTGACTGAAAAG | 825–846 | This study | ||
3016r | TCCTCAAAGAACCACGCTTT | This study | ||||
JEECV | Ltlg | 1st | 0343f | TTTGTGCAGACTGTACTGGT | 1033 | This study |
1455r | TACTCATTCATAGTGGCAATC | Modified from Mizutani et al. [8] | ||||
Nested | 0462f | AACAGGCATAGCAGTTGTGC | 723 | This study | ||
1219r | TCACCGTTCATGTTTAGGAC | Modified from Mizutani et al. [8] |
Target Virus | Primers and Probe | Sequence (5′–3′) |
---|---|---|
AnHV | AnHV-Pol-2173f | AAGGTGTTTCAGCCYACCAT |
AnHV-Pol-2587r | ATGAAGATCT GCGCCAACTC | |
AnHV-Pol-2260p | Cy5-AGCAACATGTGCGACGCCAA-BHQ2 | |
JEECV | JEECV-Ltlg-0546f | CAATGTGATGCAGGTAGCAA |
JEECV-Ltlg-0777r | TCTGTTGGTCGCTTCGACAT | |
JEECV-Ltlg-0662p | HEX-TGGGCTTTGACTACACGATGCT-BHQ1 |
Reagents | Amount |
---|---|
2× qPCR Master Mix | 10 μL |
Forward Primer (5–10 pmol) | 1–2 μL |
Reverse Primer (5–10 pmol) | 1–2 μL |
TaqMan Probe (5–10 pmol) | 1–2 μL |
ROX™ dye | 0.1 μL |
Template DNA | 2 μL |
DEPC-treated Water | up to 20 μL |
Total | 20 μL |
Step | Temperature | Time | No. of Cycles |
---|---|---|---|
Initial denaturation | 95 °C | 5 min | 1 |
Denaturation | 95 °C | 15 s | 40 |
Annealing/Extension | 63 °C | 1 min |
No. | Host | Area | Fish Size (cm) | AnHV | JEECV | ||
---|---|---|---|---|---|---|---|
cPCR | qPCR (Copies/mL) | cPCR 2 | qPCR (Copies/mL) | ||||
1 | A. japonica | Jeollabuk-do | 50.5 | ND 1 | ND | ND | ND |
2 | A. japonica | Jeollanam-do | 48.2 | ND | ND | ND | 1.1 |
3 | A. japonica | Jeollanam-do | 42.3 | Positive | 2308.9 | ND | 2.9 |
4 | A. marmorata | Gyeongsangbuk-do | 50.9 | ND | ND | ND | ND |
5 | A. marmorata | Gyeongsangbuk-do | 11.6 | Positive | 76,209.1 | ND | ND |
6 | A. bicolor | Gyeongsangnam-do | No data | Positive | 711.7 | ND | 0.1 |
7 | A. japonica | Jeollabuk-do | No data | ND | ND | ND | 2104.5 |
8 | A. japonica | Jeollabuk-do | 56.7 | ND | 11 | ND | 0.8 |
9 | A. australis | Jeollanam-do | 14.5 | ND | ND | ND | ND |
10 | A. australis | Jeollanam-do | 12.6 | ND | ND | ND | ND |
11 | A. australis | Jeollanam-do | 55.8 | ND | ND | ND | ND |
12 | A. japonica | Chungcheongnam-do | 53 | Positive | 1668.7 | ND | ND |
13 | A. japonica | Jeollanam-do | 24.6 | ND | 0.6 | ND | 0.5 |
14 | A. japonica | Jeollanam-do | 47 | ND | 28 | ND | ND |
15 | A. japonica | Jeollanam-do | 43.5 | ND | 0.3 | ND | ND |
16 | A. bicolor | Gyeongsangnam-do | 39.5 | ND | ND | ND | ND |
17 | A. japonica | Jeollanam-do | 46.5 | Positive | 167.6 | ND | ND |
18 | A. australis | Jeollanam-do | 13.9 | ND | 0.2 | ND | ND |
19 | A. japonica | Jeollanam-do | 59.5 | ND | ND | ND | ND |
20 | A. marmorata | Gyeongsangbuk-do | 52.8 | ND | ND | Positive | ND |
21 | A. bicolor | Gyeongsangnam-do | 42.2 | ND | ND | ND | ND |
22 | A. australis | Jeollanam-do | 48 | ND | ND | ND | ND |
23 | A. japonica | Jeollabuk-do | 53.4 | ND | ND | Positive | 2.5 |
24 | A. bicolor | Gyeongsangnam-do | No data | Positive | 464.9 | Positive | 0.3 |
25 | A. japonica | Jeollabuk-do | No data | ND | ND | Positive | 1182.9 |
26 | A. australis | Jeollanam-do | 55.8 | ND | ND | ND | ND |
27 | A. japonica | Chungcheongnam-do | 53 | Positive | 218.5 | ND | ND |
28 | A. japonica | Jeollanam-do | 24.6 | ND | 0.2 | ND | 0.3 |
29 | NTC-1 | - | ND | ND | ND | ND | |
30 | NTC-2 | - | ND | ND | ND | ND |
Sample Name | Sample Volume (L) | AnHV | JEECV | ||
---|---|---|---|---|---|
Cq Value | Copy No. | Cq Value | Copy No. | ||
Groundwater | 1 | ND | - | ND | - |
Groundwater | 2 | ND | - | ND | - |
Groundwater | 4 | ND | - | ND | - |
Groundwater | 10 | ND | - | ND | - |
Spiked groundwater | 1 | ND | - | 21.424 | 12,367 |
Spiked groundwater | 2 | ND | - | 19.606 | 44,250 |
Spiked groundwater | 4 | ND | - | 17.662 | 173,072 |
Spiked groundwater | 10 | ND | - | 16.556 | 375,871 |
Farm | Samples | 1st | 2nd | 3rd | 4th | 5th | 6th |
---|---|---|---|---|---|---|---|
A (A. japonica) | Fin | ND | ND | JEECV 2 × 102 | JEECV 9 × 10 | ND | ND |
Gill | ND | ND | JEECV 4 × 103 | JEECV 2 × 102 | JEECV 2 × 10 | JEECV 3 × 100 | |
Spleen | ND | ND | JEECV 1 × 102 | JEECV 6 × 100 | JEECV 2 × 10 | JEECV 1 × 10 | |
Kidney | ND | ND | JEECV 6 × 102 | JEECV 2 × 10 | JEECV 8 × 100 | JEECV 5 × 100 | |
eDNA | ND | JEECV 1 × 10 | JEECV 4 × 10 | JEECV 1 × 102 | ND | ND | |
Status | S | S | M | M | M | M | |
Detection of virus from fish | |||||||
Detection of virus from fish | |||||||
Fish mortalities | |||||||
Farm | Samples | 1st | 2nd | 3rd | 4th | 5th | 6th |
B (A. marmorata) | Fin | ND | AnHV 1 × 104 | ND | AnHV 3 × 103 | ND | ND |
Gill | ND | AnHV 2 × 100 | ND | AnHV 4 × 105 | AnHV (D) | AnHV 4 × 105 | |
Spleen | ND | AnHV 1 × 100 | ND | AnHV 1 × 105 | AnHV (D) | ND | |
Kidney | ND | AnHV 4 × 10 | ND | AnHV (D) | AnHV (D) | AnHV 2 × 105 | |
eDNA | AnHV (D) | ND | ND | AnHV 1 × 10 | ND | ND | |
Status | S | S | W | M | M | M | |
Detection of virus from fish | |||||||
Detection of virus from eDNA | |||||||
Fish mortalities | |||||||
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Song, J.-Y.; Kim, K.-Y.; Kim, A. Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus. Biology 2025, 14, 264. https://doi.org/10.3390/biology14030264
Song J-Y, Kim K-Y, Kim A. Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus. Biology. 2025; 14(3):264. https://doi.org/10.3390/biology14030264
Chicago/Turabian StyleSong, Jun-Young, Keun-Yong Kim, and Ahran Kim. 2025. "Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus" Biology 14, no. 3: 264. https://doi.org/10.3390/biology14030264
APA StyleSong, J.-Y., Kim, K.-Y., & Kim, A. (2025). Highly Sensitive Duplex Quantitative PCR Assay for Simultaneous Detection of Two Japanese Eel Viruses, Anguillid Herpesvirus 1 and Japanese Eel Endothelial Cells-Infecting Virus. Biology, 14(3), 264. https://doi.org/10.3390/biology14030264