Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido‐ and nodaviruses

Abstract

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.

Characterization of two grouper Epinephelus akaara cell lines: Application to studies of Singapore grouper iridovirus (SGIV) propagation and virus–host interaction

“…However, after 25 passages, the fibroblastic-like cells grew more rapidly and were predominant. In contrast, swim bladder cells derived from E. awoara are predominantly epithelial-like (Lai et al, 2003).…”

“…These two cell lines were well adapted to grow in L-15 medium supplemented with 10% FBS. L-15 medium was commonly used by different investigators for grouper cell cultures (Lai et al, 2003), while GSC cell lines derived from snout of grouper, E. akaara, was grown well in DMEM (Zhou et al, 2007). The suitable medium for EAGS and GAGSB cell cultures doesn't contain special requirements, such as NaCl addition, which is needed for other marine fish cells (Fernandez et al, 1993;Chew-Lim et al, 1994).…”

“…The varied chromosome numbers were also found in other marine fish cell lines derived from different organs in the same fish species. The previous investigations on the cell cultures in grouper, E. awoara, revealed that the number of chromosomes in the fish eye cells (GE), fin cells (GF), heart cells (GH), kidney cells (GK), liver cells (GL) and swim bladder cells (GSB) were 48, 66, 58, 60, 76 and 86, respectively (Lai et al, 2000(Lai et al, , 2003. The similar findings were also reported in the cell cultures in flounder, Paralichthys olivaceus.…”

“…Consequently, susceptible cell lines need to be established from marine fish for use in viral diagnostics. Recently, the tropical grouper (Epinephelus awoara and Epinephelus coioides) eye, fin, heart, swim bladder, kidney, snout, liver and spleen cell lines have been developed for virus isolation and for investigation of viral pathogenesis (Lai et al, 2000(Lai et al, , 2003Qin et al, 2006). However, to our knowledge, only one cell line was established from snout of red spotted grouper Epinephelus akaara, which is an important marine fish species cultured in southern China.…”

Development and characterization of two cell lines PDF and PDH from Puntius denisonii (Day 1865)

“…Initially, fibroblastic and epithelial cells were found in both the cell lines, but after several subcultures, fibroblastlike cells predominated in both the cell lines. Usually, a predomination of fibroblastic cells over epithelioid cells in cell cultures from fish has been reported (Bejar et al 1997;Lai et al 2003;Lakra et al 2006a). Ye et al (2006) developed a fibroblast-like cell line (LJH-2) from Lateolabrax japonicus.…”