Development of a new cell line from the snout of giant grouper, Epinephelus lanceolatus (Bloch), and its application in iridovirus and nodavirus pathogenesis

摘要

We developed and characterized a new marine fish cell line (ELGSN) from the snout of giant grouper, Epinephelus lanceolatus (Bloch). Two different phenotypes of cells, ELGSN(e), which consisted predominantly of epithelial-like cells, and ELGSN(f), which consisted predominantly of fibroblast-like cells, were isolated and cultured. Both ELGSN, and ELGSN(f) cells were subcultured for >80 passages and multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28 degrees C. Karyotyping analysis indicated that the modal chromosome numbers of ELGSN(e) and ELGSN(f) cells were 48 and 62, respectively. After transfection with EGFP-C1, bright green fluorescence was observed in these two cells and the transfection efficiency was 35% and 28%, respectively. Moreover, the two types of ELGSN cells both showed susceptibility to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), but not to soft-shelled turtle iridovirus (STIV) and spring viremia of carp virus (SVCV), indicated by the occurrence of severe cytopathic effect and increased viral titers. In SGIV-infected cells, paracrystalline arrays, different stages of virus particles and amorphous structures were observed under electron microscopy. In RGNNV-infected cells, numerous virus particles and circular virus-packed macropinosome-like structures were observed. All these data suggested that ELGSN cells could be used for effective virus propagation in vitro. In addition, we provided biochemical evidences that macropinocytosis was involved in RGNNV infection in ELGSN cells. Taken together, the new established ELGSN cell lines provided a useful tool for transgenic and genetic manipulation, as well as virus propagation and pathogenesis