Aquaculture International

, Volume 28, Issue 1, pp 349–362 | Cite as

Molecular detection of betanodavirus in orange-spotted grouper (Epinephelus coioides) broodstock maintained in recirculating aquaculture systems and sea cages

  • Sekar MegarajanEmail author
  • Ritesh Ranjan
  • Biji Xavier
  • Shubhadeep Ghosh
  • Narasimhulu Sadu
  • Chinnibabu Bathina
  • Achamveettil Gopalakrishnan


Wild collected orange-spotted grouper were domesticated in Recirculating Aquaculture Systems (RAS) and sea cages for fish breeding. While breeding, viral nervous necrosis (VNN) infection was observed in both the systems after 18 months of domestication. Out of 50 domesticated broodfish used in the study, 11 (22%) fishes were found positive for VNN examined by reverse transcriptase polymerase chain reaction (RT-PCR) method. Quantitative real-time PCR (qPCR) study also confirmed the presence of VNN in the suspected fish, and detectable amount of virus copy number was observed in the brain, optic nerve, caudal fin, muscle, and gills. Analysis of the isolated viral gene sequence from the fish was closely related to the red-spotted grouper nervous necrosis virus (RGNNV) genotype and suggests that the isolates are probably endemic to Indian coast. The study also indicated that the sub-adults of orange-spotted grouper infected in the wild and remained as carrier during domestication and subsequently disease outbreak has occurred when water temperature was increased in culture environment. This is the first report on VNN in orange-spotted grouper broodstock from India. The results of the study illustrated that large population of the wild marine fishes are sub-clinically infected with betanodavirus and therefore, early detection of the disease should be adopted as best management practices in breeding for sustainable aquaculture development of the species.


India Orange-spotted grouper Broodstock Betanodavirus RAS Cage 



The authors would like to thank Mr. Loveson L. Edward for productive input during the study.

Author contributions

There are seven authors who have contributed to accomplish the work with various levels. Sekar Megarajan has major contribution in planning and execution of the work. Ritesh Ranjan, Biji Xavier, Narasimhulu Sadu, and Chinnibabu Bathina had involved in sample examinations and further lab activities. Shubhadeep Ghosh and Achamveettil Gopalakrishnan had involved in overall management of the research program.

Funding information

The authors are grateful to the Director, ICAR-CMFRI, for providing financial support to carry out this work.

Compliance with ethical standards

Conflict of interest

This study was conducted under one of the institutional research project work under the major research area. Therefore, there is no separate fund allotted to carry out this particular research work. All the institutional research project work was funded by Indian Council of Agricultural Research, New Delhi. The authors declare that they have no conflict of interest.

Research involving animals

The study was carried out on vertebrate fish used our breeding program. During the study, samples were collected from both the dead and live fishes. When live fishes were used the fishes were properly anesthetized and then used for the sample collection and related research work. However, the established protocols for examining the dead and live fish by the institute were followed during the examination of the fish. Therefore, this article does not contain any studies that are against to ethics.


  1. Agnihotri K, Pease B, Chong R (2016) Molecular analysis of RNA1 and RNA2 sequences from a betanodavirus isolated from giant grouper (Epinephelus lanceolatus) in Australia. Virol Rep 6:25–31. CrossRefGoogle Scholar
  2. Azad IS, Shekhar MS, Thirunavukkarasu AR, Poornima M, Kailasam M, Rajan JJS, Ali SA, Mathew Abraham SA, Ravichandran P (2005) Nodavirus infection causes mass mortalities in hatchery produced larvae of Asian sea bass, Lates calcarifer: first report from India. Dis Aquat Org 63:113–118. CrossRefPubMedGoogle Scholar
  3. Binesh CP, Renuka K, Malaichami N, Greeshma C (2013) First report of viral nervousnecrosis-induced mass mortality in hatchery-reared larvae of clownfish, Amphiprion sebae Bleeker. J Fish Dis 36:1017–1020. CrossRefPubMedGoogle Scholar
  4. Burns KM, Restrepo V (2002) Survival of reef fish after rapid depressurization: field and laboratory studies. Am Fish Soc Symp 30:148–151. CrossRefGoogle Scholar
  5. Butcher PA, Broadhurst MK, Hall KC, Cullis BR, Raidal SR (2012) Assessing barotrauma among angled snapper (Pagrus auratus) and the utility of release methods. Fish Res 127:49–55 CrossRefGoogle Scholar
  6. Chi SC, Lo BJ, Lin SC (2001) Characterization of grouper nervous necrosis virus (GNNV). J Fish Dis 24:3–13CrossRefGoogle Scholar
  7. Frerichs GN, Tweedie A, Starkey WG, Richards RH (2000) Temperature, pH and electrolyte sensitivity, and heat, UV and disinfectant inactivation of sea bass (Dicentrarchus labrax) neuropathy nodavirus. Aquaculture 185:13–24CrossRefGoogle Scholar
  8. Fukuda Y, Nguyen HD, Furuhashi M, Nakai T (1996) Mass mortality of cultured seven-band grouper, Epinephelus septemfasciatus, associated with viral nervous necrosis. Fish Pathol 31:165–170. CrossRefGoogle Scholar
  9. Furusawa R, Okinaka Y, Toshihiro N (2006) Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes). J Gen Virol 87:2333–2339. CrossRefPubMedGoogle Scholar
  10. Gomez DK, Baeck GW, Kim JH, Choresca CH Jr, Park SC (2008) Molecular detection of betanodavirus in wild marine fish populations in Korea. J Vet Diagn Investig 20:38–44CrossRefGoogle Scholar
  11. Gomez DK, Sato J, Mushiake K, Isshiki T, Okinaka Y, Nakai T (2004) PCR-based detection of betanodaviruses from cultured and wild marine fish with no clinical signs. J Fish Dis 27:603–608. CrossRefPubMedGoogle Scholar
  12. Gopakumar G, Nazar AK, Tamilmani G, Sakthivel M, Kalidas C, Ramamoorthy N, Palanichamy S, Ashok MV, Rao KS, Rao GS (2011) Broodstock development and controlled breeding of cobia Rachycentron canadum (Linnaeus 1766) from Indian seas. Ind J Fish 58(4):27–32Google Scholar
  13. Jaramillo D, Hick P, Deece K, Tweedie A, Kirkland P, Arzey E (2016) Comparison of ELISA formats for detection of antibodies specific for nervous necrosis virus (Betanodavirus) in the serum of immunized barramundi Lates calcarifer and Australian bass Macquaria novemaculeata. Aquaculture 451:33–38CrossRefGoogle Scholar
  14. Jithendran KP, Vijayan KK, Alavandi SV, Kailasam M (2005) Benedenia epinepheli (Yamaguti, 1937), a monogenean parasite in captive broodstock of grouper, Epinephelus tauvina (Forskal). Asian Fish Sci 18(1):121–126. CrossRefGoogle Scholar
  15. Kiryu I, De la Pena LD, Maeno Y (2007) Distribution of nervous necrosis virus in orange spotted grouper Epinephelus coioides with asymptomatic infection. Fish pathology 42(3):163–165CrossRefGoogle Scholar
  16. Kuo HC, Wang TY, Chen PP, Chen YM, Chuang HC, Chen TY (2011) Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture. J Clin Microbiol 49(3):1090–1096. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Leong TS, Wong SY (1988) A comparative study of the parasite fauna of wild and cultured grouper (Epinephelus malabaricus Bloch & Schneider) in Malaysia. Aquaculture 68:203–207. CrossRefGoogle Scholar
  18. Mustafa S, Hajini MH, Senoo S, Seok Kian AY (2016) Conditioning of broodstock of tiger grouper, Epinephelus fuscoguttatus in a recirculating aquaculture system. Aquac Rep 2:117–119. CrossRefGoogle Scholar
  19. Nadia C, Amdouni F (2017) Nodaviruses in wild fish population collected around aquaculture cage sites from coastal areas of Tunisia fish. Aqua J 8(3):209. CrossRefGoogle Scholar
  20. Nguyen HD, Nakai T, Muroga K (1998) Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae. Dis Aquat Org 24:99–105CrossRefGoogle Scholar
  21. Oh MJ, Jung SJ, Kim SR, Rajendran KV, Kim YJ, Choi TJ, Kim HR, Kim JD (2002) A fish nodavirus associated with mass mortality in hatchery reared red drum, Sciaenops ocellatus. Aquaculture 211:1–7CrossRefGoogle Scholar
  22. Overgard AC, Nerland AH, Fiksdal IU, Patel S (2012) Atlantic halibut experimentally infected with nodavirus shows increased levels of T-cell marker and IFN transcripts. Dev Comp Immunol 37:139–150CrossRefGoogle Scholar
  23. Pomeroy R (2002) The status of grouper culture in Southeast Asia. SPC Live Reef Fish Inform Bull 10:22–26Google Scholar
  24. Ranjan R, Megarajan S, Xavier B, Dash B, Ghosh S, Menon M, Edward LL (2017) Conditioning, maturation and year-round natural spawning of orange-spotted grouper, Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system. Aquac Res 48(12):5864–5873. CrossRefGoogle Scholar
  25. Ranjan R, Xavier B, Santhosh B, Sekar M, Ghosh S (2018) Copepod parasite Lepeophtheirus kabatai (Ho & Dojiri, 1977) infestation in orange spotted grouper, Epinephelus coioides (Hamilton, 1822) and its control in captivity. Indian J Fish 65(3):122–125. CrossRefGoogle Scholar
  26. Sekar M, Ranjan R, Xavier B, Edward LL, Behera PR, Ghosh S (2014) Barotrauma in orange spotted grouper, (Epinephelus coioides) and it’s mitigation measures. In: Book of abstracts of 10th Indian Fisheries and Aquaculture Forum, NBFGR, Lucknow, 12–15 November 2014Google Scholar
  27. Sekar M, Ranjan R, Xavier B, Edward LL, Dash B, Ghosh S (2015) Grouper culture-a new venture for Indian aqua farmers. Fishing Chimes 35(9):46–49Google Scholar
  28. Simoes JM, Duarte Ine SG, Fonseca PJ, Turner GF (2008) Courtship and agonistic sounds by the cichlid fish Pseudotropheus zebra. J Acoust Soc Am 124:1332–1338. CrossRefGoogle Scholar
  29. Sharma SRK, Pradeep MA, Dube PN, Kumar TVA, Kumar R, Swaminathan TR (2018) Betanodavirus-associated mortality in Asian seabass (Lates calcarifer, Bloch) cultured in indoor tanks and sea cages. Aquac Int 27(1):279–286CrossRefGoogle Scholar
  30. Shetty M, Maiti B, Shivakumar SK, Venugopal MN, Karunasagar I (2012) Betanodavirus of marine and freshwater fish: distribution, genomic organization, diagnosis and control measures. Indian J Virol 23:114–123. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Toffan A, Pascoli F, Pretto T, Panzarin V, Abbadi M, Buratin A, Quartesan R, Gijon D, Oadros F (2017) Viral nervous necrosis in gilthead sea bream (Sparus aurata) caused by reassortant betanodavirus RGNNV/SJNNV: an emerging threat for Mediterranean aquaculture. Sci Rep 7(46755).
  32. Yong CY, Yeap SK, Omar AR, Tan WS (2017) Advances in the study of nodavirus. Peer J 5:e3841. CrossRefPubMedGoogle Scholar
  33. Whelan JA, Russell NB, Whelan MA (2003) A method for absolute quantification of cDNA using realtime PCR. J Immunol Methods 278(1–2):261–269CrossRefGoogle Scholar
  34. Zhang Q, Liu Q, Liu S, Yang H, Liu S, Zhu L, Yang B, Jin J, Ding L, Wang X, Liang Y, Wang Q, Huang J (2014) A new nodavirus is associated with covert mortality disease of shrimp. J Gen Virol 95:2700–2709. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.ICAR-Central Marine Fisheries Research InstituteRegional CentreVisakhapatnamIndia
  2. 2.ICAR-Central Marine Fisheries Research InstituteKochiIndia

Personalised recommendations