Establishment and characterization of a permanent heart cell line from largemouth bass Micropterus salmoides and its application to fish virology and immunology

Abstract

Largemouth bass (Micropterus salmoides) is among the most important cultured fish species in China and various viral diseases have greatly affected the largemouth bass industry. We established a permanent cell line from the heart of M. salmoides (MSH) that has been subcultured over 70 times and showed optimal growth at 27 °C using Medium 199 supplemented with 10% fetal bovine serum. MSH cells consisted predominantly of fibroblast-like cells and their karyotypes revealed diploid numbers for most cells at passage 60 with 2n = 64. Sequencing of cytochrome oxidase I and 16S rRNA genes confirmed that MSH cells were derived from M. salmoides. The cell line was tested for Mycoplasma contamination and found to be negative. MSH cells were successfully transfected with a GFP reporter gene indicating that these cells can be utilized for gene expression studies. The MSH cells showed susceptibility to M. salmoides rhabdovirus (MSRV), Largemouth bass ranavirus (LMBV), Infectious spleen and kidney necrosis virus(ISKNV), Spring viremia of carp virus (SVCV), Tilapia lake virus (TiLV) and Grass carp reovirus genotype I (GCRV-I) but were resistant to nervous necrosis virus (NNV). PCR assay and electron microscopy further confirmed that all the tested viruses except NNV can replicate in MSH cells. The replication efficiency of MSRV, LMBV, ISKNV, SVCV, TiLV and GCRV-I ranged from 107.24 to 108.82 TCID50/mL. In addition, type I interferon genes were induced with only NNV infection. In conclusion, we successfully generated an immortal cell line from the heart tissue of M. salmoides and the newly established MHS cell line was highly susceptible to many fish viruses that can be useful for future genetic, virological and immunological studies.