Development and Characterisation of an Immortal Cell Line From Largemouth Bass (Micropterus salmoides) for Viral Studies.

Abstract

Largemouth bass (Micropterus salmoides), a freshwater fish species, is cultivated widely across China. The industry has been greatly affected by various viral diseases. We generated a new immortal cell line from the fin of M. salmoides (MSF). It has been successfully maintained in continuous culture for over 80 passages. Cells multiply well at 28°C using Medium 199 or Leibovitz's L-15 supplemented with 10%-20% fetal bovine serum. MSF cells comprise a mass of epithelialoid cells. Chromosome analysis showed that ploidy at passage 55 was 2n = 58. Amplification and analysis of the sequence of cytochrome oxidase I gene ascertained the origin of MSF cells from M. salmoides. The cell line is devoid of contamination with mycoplasma. Successful transfection with a GFP reporter gene indicated that the MSF cell line can be a useful tool for further gene expression studies. MSF cells exhibited susceptibility to Largemouth bass ranavirus (LMBV), Infectious spleen and kidney necrosis virus (ISKNV), and Siniperca chuatsi rhabdovirus (SCRV). Transmission electron microscopy (TEM), and immunofluorescence confirmed that the MSF cell line showed high susceptibility to all the tested viruses. The titre values of MSF after infection with LMBV, ISKNV, and SCRV reached 10^7.4 TCID₅₀/mL, 10^6.15 TCID₅₀/mL, and 10^6.35 TCID₅₀/mL at 7dpi, respectively. In conclusion, an immortal cell line derived from the fin tissue of M. salmoides was successfully developed and certain fish viruses can effectively propagated in the newly established MSF cell line that can be helpful for future gene manipulation and infectious viral mechanistic studies in the future.