Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2)

Arathi Dharmaratnam; Raj Kumar; Basheer Saidmuhammed Valaparambil; Neeraj Sood; Pravata Kumar Pradhan; Sweta Das; T Raja Swaminathan

doi:10.7717/peerj.9373

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2)

PeerJ. 2020 Jul 14:8:e9373. doi: 10.7717/peerj.9373. eCollection 2020.

Authors

Arathi Dharmaratnam¹, Raj Kumar¹, Basheer Saidmuhammed Valaparambil¹, Neeraj Sood², Pravata Kumar Pradhan², Sweta Das¹, T Raja Swaminathan¹

Affiliations

¹ Peninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, India.
² ICAR National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, India.

Abstract

Background: Herpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line.

Methods: Caudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz's L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish.

Results: The cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30^th, 40^th and 56^th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 10^7.8±0.26 TCID₅₀/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2^nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10^th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Keywords: Caudal fin; Cell line; CyHV-2; Cyprinid herpesvirus-2; Goldfish.

Grants and funding

This research was carried out under the National Surveillance Program of Aquatic Animal Diseases, National Fisheries Development Board, Department of Animal Husbandry, Dairying and Fisheries (Grant Number: NFDB/Coord/NBFGR/ 2012-13/16720 dated 11.02.2013) and ICAR National Fellow Scheme of ICAR (ICAR letter no F.No Agri.edn.27/2/2015 HRD dated 13.02.2017), Department of Agricultural Research and Education, Ministry of Agriculture and Farmer’s Welfare, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.