Detection Of Lymphocystis Disease Virus Which Infects Flounder Gill Cell Line

Lymphocystis disease is caused by lymphocystis disease virus (LCDV), which has been observed among many species of fish. Flounder is economically important fish species however most of the fish infected LCDV lose the commercial value because of terrible outlook. So, it's very important to isolate the virus by developing a host cell line in order to make neutralizing monoclonal antibodies and vaccine. In this study, selecting a cell line in which LCDV could replicate is the basic. In this paper, flounder gill cell line (FG-9307) is confirmed susceptive to LCDV by several methods.In the first chapter, the conditions of flounder gill cells growth are studied. The best consistency of inoculated cells is 3-4 X 105cells/ml. When FG cells are cultured in the plate, they should be put in 2% COa. The MEM medium with 2% serum is used to maintain the cells, when 20% serum medium to quick up the cell growth and 10% serum MEM as normal culture medium.In the second chapter, monoclonal antibodies against LCDV were raised using purified virus as antigen. By indirect immunofluorescence assay test (IFAT), a lot of positive hybridomas were found and 2 of them (1A6, 1H9) were cloned because of secreting high titer antibodies.The study in the third chapter is about the replication of LCDV in FG cells. FG cells exhibit cytopathic effect (CPE) 1-2 days after infected by LCDV obtained from lymphocystis cells. The virus reaches a tilter of 22 57 tissue culture infectious does with 50% endpoint (TCIDso) per 40u.l. CPE occurs also when FG cells infected by cell-culture-replicated virus. The results of detecting LCDV in FG cells using immunocytochemistry (ICC) and IFAT by monoclonal antibodies against LCDV show that virus begins to replicate within 24 hours, and reaches the top in 48-72 hours. The result of detection LCDV in culture medium by dot blotting is negative.