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ABSTRACT:In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDVbinding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and β-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells. KEY WORDS: Lymphocystis disease virus · Receptor · Virus entry · Flounder gill cells · Co-immunoprecipitation Resale or republication not permitted without written consent of the publisherDis Aquat Org 94: [9][10][11][12][13][14][15][16] 2011 tions showed that the virus could attach to the cell surface and then penetrate the cells by membrane fusion or by endocytosis, suggesting that there were virusspecific receptors located on the FG cell surface (Lv et al. 2003), and that the FG cell line is a good candidate for studying LCDV-cell receptor interaction.Several protein-protein interaction methods can be used to study the virus receptor, such as the the yeast 2-hybrid system, glutathione S-transferase (GST) pull down, virus overlay protein binding assay (VOPBA), and co-immunoprecipitation (Melegari et al. 1998, Ryu et al. 2000, Imajoh et al. 2003. Coimmunoprecipitation has become a widely accepted method for identification of receptors for viruses such as mouse mammary tumor virus, fish rhabdovirus, SARS coronavirus and dengue 2 virus (Golovkina et al. 1998, Bearzotti et al. 1999, Liu et al. 2004.To date, the cellular receptor involved in the attachment and entry of LCDV to target cells is unknown. Thus, the present study attempted to isolate and characterize the putative receptor molecule that is responsible for binding LCDV. The identification and molecular characterization of the cellular receptor molecules is of importance in understanding viral replication, pathogenesis and tissue tropism of LCDV in the host. MATERIALS AND METHODSCell culture and virus purification. The flounder gill cell line FG-9307 was obtained from the Marine Science College of Ocean University of China (Tong et al. 1997). Monolayer cultures of FG cells were grown at 22°C in Eagle's MEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone), 100 IU ml -1 penicillin and 100 µg ml -1 streptomycin. The fetal bovine serum was reduced to 2% in maintenance medium following inoculation of the cultures with virus.Japanese flounder Paralichthys olivaceus with lymphocystis nodules on the body surface were obtained from a farm in Qingdao, ...