Dharmaratnam Arathi, Kumar Raj, Valaparambil Basheer Saidmuhammed, Sood Neeraj, Pradhan Pravata Kumar, Das Sweta, Swaminathan T Raja
Peninsular and Marine Fish Genetic Resources Centre, ICAR National Bureau of Fish Genetic Resources, Kochi, Kerala, India.
ICAR National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, India.
PeerJ. 2020 Jul 14;8:e9373. doi: 10.7717/peerj.9373. eCollection 2020.
Herpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, . 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.
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.
The cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30 40 and 56 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 TCID/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2 day post-inoculation (dpi) and complete destruction of cells was observed by the 10 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.
由鲤疱疹病毒2型（CyHV-2）引起的疱疹病毒性造血坏死病，导致金鱼养殖业大量死亡。通过牺牲鱼类，已从各种金鱼组织中建立了用于CyHV-2分离和增殖的允许细胞系。在此，我们报告了一种使用非致死采样从金鱼尾鳍建立的细胞系FtGF（扇尾金鱼鳍细胞系）。我们还描述了一种简单的方案，用于通过外植体方法成功建立和表征允许细胞系，并使用该细胞系以高病毒滴度连续传代CyHV-2。
从金鱼采集尾鳍组织样本而不杀死鱼。使用含有20%胎牛血清（FBS）和1倍浓度抗生素抗真菌溶液的Leibovitz's L-15（L-15）培养基进行金鱼尾鳍细胞的细胞培养，在28°C下孵育。通过对16S rRNA和COI基因片段进行测序对细胞进行表征并确认细胞来源。CyHV-2在FtGF细胞中生长并连续传代20次。通过对未感染的金鱼进行实验性感染，确认了使用FtGF细胞分离的CyHV-2的感染性。
该细胞系在含有10% FBS的L-15培养基中已传代达56次。在第30、40和56代对FtGF细胞进行核型分析表明，众数染色体数为2n = 104。通过对16S rRNA和COI基因进行测序对FtGF进行物种鉴定。该细胞系用于CyHV-2的连续传代超过20代，病毒滴度高达10 TCID/mL。接种CyHV-2阳性组织匀浆后，FtGF细胞在接种后2天（dpi）出现细胞病变效应，在10 dpi时观察到细胞完全破坏。使用来自感染FtGF细胞的上清液对未感染的金鱼进行实验性感染导致100%死亡，通过DNA聚合酶基因扩增、组织病理学和透射电子显微镜确认了受攻击鱼中的CyHV-2感染。这些发现证实FtGF细胞系对CyHV-2的增殖高度敏感。
