ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow, Uttar Pradesh, 226 002, India.
ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow, Uttar Pradesh, 226 002, India; Tumbes National University, Tumbes, 24001, Peru.
J Virol Methods. 2021 Jan;287:113989. doi: 10.1016/j.jviromet.2020.113989. Epub 2020 Oct 6.
In the present study, we have developed a continuous cell line from the heart tissue of the Oreochromis niloticus and used for studying susceptibility to tilapia lake virus (TiLV). The cell line, designated as OnH, has been subcultured up to 82 passages. The optimal growth of OnH cells was observed at 28-32 °C in iL-15 medium supplemented with 20 % fetal bovine serum. Karyotype analysis revealed that the modal chromosome number of OnH cells was 44. Partial amplification and sequencing of 16S rRNA gene confirmed the origin of OnH cell line from O. niloticus. Immunophenotyping revealed that OnH cells were of epithelial origin. These cells were successfully transfected with pAcGFP1-N1 mammalian expression vector. OnH cells showed cytopathic effects following inoculation with TiLV. The virus titration study indicated that the cells were highly susceptible to TiLV with TCID value of 10/mL. The qRT-PCR studies revealed that the optimal temperature for TiLV replication in OnH cells was 28 °C. Further, transmission electron microscopy of TiLV-infected OnH cells showed a number of electron-dense virus particles measuring 60-90 nm diameter, which were enclosed in the vesicles in the cytoplasm. Therefore, the newly established OnH cell line provides a valuable tool for isolation of viruses from disease cases suspected to be of viral etiology in this candidate species' and also for transgenic and genetic manipulation studies.
在本研究中，我们从奥利亚罗非鱼的心脏组织中开发了一种连续细胞系，并用于研究对罗非鱼湖病毒（TiLV）的易感性。该细胞系被命名为 OnH，已传代培养至 82 代。OnH 细胞的最佳生长温度为 28-32°C，在补充有 20%胎牛血清的 iL-15 培养基中。染色体核型分析显示，OnH 细胞的模式染色体数为 44。16S rRNA 基因的部分扩增和测序证实了 OnH 细胞系源自奥利亚罗非鱼。免疫表型分析显示 OnH 细胞来源于上皮细胞。这些细胞成功地转染了 pAcGFP1-N1 哺乳动物表达载体。用 TiLV 接种后，OnH 细胞出现细胞病变效应。病毒滴定研究表明，细胞对 TiLV 高度敏感，TCID 值为 10/mL。qRT-PCR 研究表明，TiLV 在 OnH 细胞中的最佳复制温度为 28°C。进一步，用 TiLV 感染的 OnH 细胞的透射电子显微镜显示了许多电子致密的病毒颗粒，直径为 60-90nm，这些病毒颗粒被包裹在细胞质中的小泡中。因此，新建立的 OnH 细胞系为从疑似病毒性病因的疾病病例中分离病毒提供了一种有价值的工具，也为转基因和遗传操作研究提供了一种工具。
