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)
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 ti...
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PeerJ Inc.
2020-07-01
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| author | Arathi Dharmaratnam Raj Kumar Basheer Saidmuhammed Valaparambil Neeraj Sood Pravata Kumar Pradhan Sweta Das T. Raja Swaminathan |
| author_facet | Arathi Dharmaratnam Raj Kumar Basheer Saidmuhammed Valaparambil Neeraj Sood Pravata Kumar Pradhan Sweta Das T. Raja Swaminathan |
| author_sort | Arathi Dharmaratnam |
| collection | DOAJ |
| description | 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 30th, 40th and 56th 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 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th 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. |
| first_indexed | 2024-03-09T06:54:40Z |
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| spelling | doaj.art-f0ca29f4c90e423c86cf4a1d297b49702023-12-03T10:14:09ZengPeerJ Inc.PeerJ2167-83592020-07-018e937310.7717/peerj.9373Establishment 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 Dharmaratnam0Raj Kumar1Basheer Saidmuhammed Valaparambil2Neeraj Sood3Pravata Kumar Pradhan4Sweta Das5T. Raja Swaminathan6Peninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, IndiaPeninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, IndiaPeninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, IndiaICAR National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, IndiaICAR National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, IndiaPeninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, IndiaPeninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, IndiaBackground 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 30th, 40th and 56th 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 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th 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.https://peerj.com/articles/9373.pdfGoldfishCyprinid herpesvirus-2CyHV-2Caudal finCell line |
| spellingShingle | Arathi Dharmaratnam Raj Kumar Basheer Saidmuhammed Valaparambil Neeraj Sood Pravata Kumar Pradhan Sweta Das T. Raja Swaminathan 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 Goldfish Cyprinid herpesvirus-2 CyHV-2 Caudal fin Cell line |
| title | 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) |
| title_full | 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) |
| title_fullStr | 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) |
| title_full_unstemmed | 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) |
| title_short | 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) |
| title_sort | 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 |
| topic | Goldfish Cyprinid herpesvirus-2 CyHV-2 Caudal fin Cell line |
| url | https://peerj.com/articles/9373.pdf |
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