고수율의 이리도 바이러스 생산을 위한 IN VITRO 배양의 최적화

Abstract

Red sea bream iridovirus(RSIV) is a typical species of the genus Megalocytivirus in the family Iridoviridae. The genus Megalocytivirus, represented by red sea bream iridovirus (RSIV), the first identified and one of the best characterized megalocytiviruses, Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus, and numerous other isolates, is the newest genus within the family Iridoviridae. Megalocytiviruses are important emerging pathogens in both freshwater and marine finfish aquaculture. However, a limited number of piscine cell lines are persistently susceptible to these viruses, which greatly limits the study of megalocytiviruses. Cell lines are ideal tools for in vitro studies of cell-virus interactions, virus propagation, isolation and vaccine development. However, because of the issues related the sudden refraction of established cell lines against infection of iridovirus, we decided to start our experiments for culturing of iridovirus with primary culture from various organ(fry, fin, brain.) and fish species (rock bream, red sea bream, spotted parrot fish, black sea bream, olive flounder, Small yellow croaker fin, israel carp.). No morphological differences in progressing of subculture were detected depend upon the fish species. Following inoculation with IVS-1, Cytopathic effects (CPE) with round, enlarged, shiny cells and aggregated cells were observed in various primary cells within 3 days. And CPE was observed daily after virus inoculation and viral replication efficiency was determined for six viruses. In vitro challenge experiments in cell lines with megalocytivirus sachun-1(IVS-1) showed lower replication compared with primary cultured cells. Primary cultured cells displayed a cytopathic effect and increased virus copy number inoculation with three Megalocytivirus, namely IVS-1, FLIV-1, PGIV-K1. And the rock bream embryonic cell has especially good potential for the replication of various fish viruses such as Ranavirus, Betanodavirus, Novirhabdovirus, Vesiculovirus. Thus primary cultured cells may be useful for studying a wide range of fish viruses. But their susceptibility to Megalocytivirus cultured in vitro condition was progressively declined and reached almost similar level to that found in fish cell lines (PMF, GF, BF-2). The IRBF cells derived from the fin tissue of rock bream that infected IVS-1 is persistently infected with IVS-1 and continually reached high viral titre To improve quantification of very low levels of iridovirus in cell lines, we tested the ability of tissue homogenate and serum to enhance the sensitivity and replication of IVS-1. The virus titer of IVS-1 also enhanced by exposure of PMF, GF, BF-2 cells to 20µl/ml of tissue homogenate or serum from rock bream. But to added more than 20µl/ml of tissue homogenated was bad for cell condition as it decreases the virus propagation of IVS-1 in PMF, GF cells. The enhancing activity of tissue homogenate appeared to increase over time, indicating changes in viral copy population. It is suggested that the primary cultured fish cells could be used as a valuable tool for isolation and propagation of different iridoviruses. And these results suggest that addition tissue homogenate contribute to the replication and pathogenesis of IVS-1 to cell lines in vitvo.