Establishment of a cell line from the brain of grouper (Epinephelus akaara) for cytotoxicity testing and virus pathogenesis

Abstract

We established and characterized a continuous cell line (EAGB) from the brain of grouper (Epinephelus akaara). EAGB cells multiplied well in MEM that was supplemented with 10% fetal bovine serum at 30°C. The EAGB cell line consisted predominantly of fibroblast-like cells from grouper, which was confirmed by the sequence of mitochondrial 18s rRNA. Karyotyping indicated that the modal chromosome number was 60. The transfection efficiency of pEGFP-N3 into EAGB cells reached 34%. Bacterial cytotoxicity studies showed that the extracellular products from Vibrio alginolyticus or Vibrio anguillarum were toxic to EAGB cells. In addition, EAGB cells were susceptible to Singapore grouper iridovirus (SGIV) and viral nervous necrosis virus (VNNV), which was demonstrated by the presence of a severe cytopathic effect (CPE), abundant virus particles, and increased viral titers. Although the CPE was observed in soft-shelled turtle iridovirus (STIV)-infected EAGB cells, the virus titer was not increased and virus particles were not observed by electron microscopy. Nuclear morphological observations showed that STIV infection induced typical apoptotic bodies, whereas SGIV infection evoked non-apoptotic death in EAGB cells. The newly established EAGB cell line provided a useful tool for genetic manipulation, cytotoxicity testing, and virus pathogenesis.