Simple SummaryFish-derived primary cells are highly valuable for investigating cell–virus interactions, virus isolation, diagnosis, and vaccine development because of their rapid growth rate and high susceptibility to viruses. We established a new cell line, the dwarf gourami fin (DGF), from the caudal fin of the dwarf gourami (Trichogaster lalius). We analyzed the optimal growth conditions, including media, temperature, and fetal bovine serum concentration, modal chromosome number, and transfection efficiency. The susceptibility of DGF cells to different viruses, including red sea bream iridovirus, infectious spleen and kidney necrosis virus, viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus was evaluated.AbstractThe detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz’s L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n= 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.