Establishment and Characterization of a Novel Kidney-cell Line fromOrange-spotted Grouper, Epinephelus coioides, and its Susceptibility toGrouper Iridovirus

Abstract

A new continuous cell line, designated as GK-7, was developed from the kidney tissue of the marine grouper, Epinephelus coioides. The cell line grew well in Leibovitz’s L-15 medium supplemented with 15% fetal bovine serum at a range of temperatures from 20 to 32℃, with optimal growth at 25℃. Morphologically, the GK-7 cell line is spindleshaped epithelial-like cell comfirmed by immunophenotyping with cytokeratin antibody. Chromosome number analysis showed that GK-7 cells of the 50th and 150th cell passages had a modal diploid chromosome number of 48 and 66, respectively. Replication of GIV with the cell line showed that the maximum virus yield reached up 108.4 TCID50 mL-1 with the cells of the 50th passage. Electron micrographs showed abundant cytoplasmic icosahedral virions with a mean diameter of 200 nm in virus-infected cells. Negative staining of ultrathin sections of infected cells showed three-layered membrane enveloped mature viral particles with a diameter about 240 nm. Green fluorescent protein can be expressed in both cell lines at 48 hr after the cell lines were transfected with a green fluorescent reporter gene driven by a cytomegalovirus promoter. Our results showed that the GK-7 cell line provided valuable tools for the isolation and investigation of fish iridovirus and for vaccine production.