Megalocytivirus 연속 생산을 위한 PMF 주화세포의 활용

Abstract

Megalocytivirus belongs to the Iridoviridae family is known to agent of Red Sea Bream Iridovirus (RSIV) in red sea bream (Pagrus major) and other farmed fish species. Fish belonging to the genus Oplegnathus were more sensitive to RSIV infection than other species, including red sea bream (Pagrus major). Megalocytivirus sachun-1 (IVS-1) strain has been isolated from rock bream, an economic fish species in Korea. IVS-1 is causative agent of provoking high mortality of rock bream. In this study, the Pagrus major fin (PMF) cell line persistently infected with Megalocytivirus (PI-PMF cell) was established experimentally by subculturing method after Megalocytivirus infection and characterized. It came out that PMF cell line was suitable for induction of persistence with Megalocytivirus rather than GF cell line. After several subculture the infected cell, Megalocytivirus was maintained or increased up to 109 copies/㎖ in PMF cell line, while Megalocytivirus was decreased or cleaned up in GF cell line. So we were able to increase the concentration of virus from the infected PMF cell line at a low concentration, therefore we can harvest high concentration of Iridovirus from PI-PMF cell efficiently and persistently. The PI-PMF cells were morphologically distinguishable PMF cell line with CPE and continued to grow. But, moiety of GF cell line was degraded after subculture. The cytopathic effects (CPE) with round, enlarged, shiny cells were continuously observed in PI-PMF cell. The PI-PMF cell has been subcultured >35 times. The persistence of Iridovirus designated as Megalocytivirus in the PI-PMF cell was demonstrated by; (1) the detection of the infectious virus in the culture supernatant, (2) the detection of Iridovirus nucleic acids extracted from the PI-PMF cell. Optimum condition of PI-PMF cell was culture with 5% FBS supplemented L-15 at 25 ℃. Culture medium exchange to fresh 5% L-15 provided nutrients to the PI-PMF cell for virus replication and cell growth. But Serum Free Medium (SFM) treatment gave the damages to the PI-PMF cell and viral copy number of Iridovirus also was decreased as compare with SFM non-treatment. IVS-1 from PI-PMF cell was still susceptible to rock bream (Oplegnathus fasciatus) with 100% mortality. This study will help to study mechanism of viral persistence in vitro and increase viral copy number of Iridovirus or other viruses in other cell lines with low susceptibility.