Establishing the optimal conditions to support the propagation of red seabream iridovirus (RSIV) in grunt fin (GF) and dwarf gourami fin (DGF) cells

Abstract

Red sea bream iridovirus (RSIV), classified within the genus Megalocytivirus, is known to affect various fish species, leading to significant economic losses in the aquaculture industry. This study aims to elucidate the optimal conditions for RSIV replication in two different cell lines, namely grunt fin (GF) and dwarf gourami fin (DGF) cells. The effects of different incubation temperatures (23°C, 25°C, and 28°C) and varying concentrations of fetal bovine serum (FBS) (0%, 2%, 5%, and 10%) on the replication of RSIV were explored. Throughout the 10-day observation period, the morphological integrity of both GF and DGF cells remained consistent across all tested conditions. Among the experimental conditions, the most favorable conditions for cell viability and proliferation were identified at 28°C with a 10% FBS-supplemented medium. In the comparison of cytopathic effects following RSIV inoculation at different incubation temperatures, distinct observations were revealed. In the RSIV-inoculated DGF cells, an apparent CPE was observed from 5 days post-inoculation (dpi) at 28 °C, and all CPE was identified in all experimental conditions at 7 and 10 dpi. In contrast, RSIV-inoculated GF cells slightly displayed CPE at 25°C and 28°C. Notably, the initial RSIV replication appeared at 28°C in both cells. Moreover, at the different initial concentrations of RSIV inoculations (high dose as 106 genome copies/mL and low dose as 104 genome copies/mL), even though DGF cells exhibited an apparent appearance of CPE in both conditions, GF showed CPE when exposed to the high dose of RSIV inoculation. Notably, RSIVs propagated from GF and DGF cells under varying FBS concentrations exhibited a striking contrast in viral copy numbers. In all experimental conditions, RSIV propagated from GF cells yielded about 104 copies/mL, whereas RSIV from DGF cells exhibited a significantly higher about 107-8 copies/mL at 10 dpi. From the disparity in viral replication, the optimal FBS concentrations for RSIV propagation on DGF cells were determined to be 5%, respectively. Furthermore, through an assessment of the correlation between TCID50 and viral copies determined in the GF and DGF cells, the minimum infectious dose for RSIV in DGF cells is over 100 times higher compared to GF cells (104 genome copies/mL in DGF cells compared to 106 genome copies/mL in GF cells). These findings indicate the importance of tailoring the cell culture conditions (temperature and FBS supplementation) to maximize RSIV replication in specific host cell types and support that DGF cells are highly permissive cells for RSIV replication.