Establishment and characterization of a continuous marine fish fin cell line from Epinephelus fuscoguttatus and viral susceptibility to crustacean CMNV

Fish cell line provide a useful tool for studies in virology and molecular biology. To establish a novel continuous marine fish cell line (EFF) from caudal fin tissue of brown-marbled grouper Epinephelus fuscoguttatus, its susceptibility to the crustacean covert mortality nodavirus (CMNV) was evaluated. The primary cell cultures were initiated first by incomplete digestion of fin tissue blocks with dispase and collagenase, and then the explant was cultured in L-15 medium supplemented with 20% fetal bovine serum, 10% grouper muscle extract, and 20-ng/mL growth factors of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The EFF cells were continuously passaged beyond 50 times in fibroblast-like morphology, and they grew well in L-15 medium supplemented with a lower concentration fetal bovine serum (10%) at 28 °C after passage 10, without muscle extract and the growth factors. In addition, their grouper origin was confirmed by chromosome analysis and cytochrome oxidase 1 (CO1) gene analysis of these EFF cells. Transfection experiment via lipofectamine 8 000 indicated that the EFF cells had a high transfection potential with a transfection efficiency up to 32%. Cross-species viral susceptibility analysis showed that CMNV not only successfully infected the EFF cells as evidenced by obvious cytopathic effects like vacuolation, detachment and death of cells, but also multiplied in the EFF cells as indicated by the results of semi-quantitative RT-PCR in transmission electron microscopy. Therefore, the establishment of immortal EFF cell line provided a useful cell model for future works on the isolation, multiplication, and pathogenic mechanism of cross-species infection of CMNV as well as genetic manipulation.