Study On Virion Attachment Proteins And Cellular Receptors Of Lymphocystis Disease Virus Of Japanese Flounder

Lymphocystis disease (LCD) is a chronic worldwide fish disease, characterized by papilloma-like lesions typically on the skin, fins and tail. It has more than 100 hosts including marine and freshwater species. Virus attachment to a cellular receptor through virion attachment proteins is the first step in viral infection. Understanding of how viral proteins and host cell receptors mediate this initial interaction will contribute to understanding the virus infection mechanism and provide a new method for virus disease prevention. In this paper, flounder gill (FG) cell line was used for study of virion attachment proteins and cellular receptors of lymphocystis disease virus (LCDV) isolate from Japanese flounder. An efficient method to detect neutralizing antibodies against LCDV was established using FG cells, through which we screened and obtained two strains of neutralizing monoclonal antibodies against LCDV; Virus overlay protein binding assays (VOPBA) were carried out to find out the virus binding proteins of the LCDV target organs and FG cells. The characteristics and function of the virus binding protein of FG cells were also studied.FG cell culture was the foundation of this study. FG cells were derived from flounder gill tissue and they grew fast and well at 20℃in Eagle's MEM supplemented with 10% of fetal calf serum. According to the growth curve, the density of cells could reach the peak 3-4 days after inoculation.Obvious cytopathic effect (CPE) could be found on FG cells 1-2 days after inoculation with LCDV purified from lymphocystis cells of Japanese flounder, and the half tissue culture infection dosage (TCID50) was 22.88 per 40μL. Two kinds of CPE were observed including plaques and syncytia. The results of detecting LCDV in FG cells using immunofluoresence assay test (IFAT) and immunocytochemistry (ICC) by monoclonal antibodies against LCDV show that virus could propagated in FG cells. The location of the virions was detected in virus-adsorbed cells by IFAT, suggesting the specific LCDV receptors on the surface of FG cells.Micro-cytopathic effect neutralization test (MCPENT) demonstrated that infectivity to FG cells was reduced by pretreatment of virus with serum obtained from LCD-diseased Japanese flounder and rabbit anti-LCDV serum, the neutralization titers were respectively 1:64 and 1:160. As a sensitive cell line for LCDV, FG cells could be used in the detection of neutralizing antibodies to LCDV.Monoclonal antibodies against LCDV were raised using virus purified by differential centrifugation and sucrose density gradient centrifugation as antigen. By IFAT and dot blotting assay, 9 positive hybridomas were found and then cloned.MCPENT demonstrated that Mab 3G3 and 1B2 were able to neutralize LCDV infection to FG cells. Western blotting analysis showed that Mab 3G3 reacted with a polypeptide with molecular weight of of 38.2 kDa, while Mab 1B2 reacted with a polypeptide with molecular weight of 32.9 kDa.Tissues of LCDV-diseased Japanense flounder were detected by IFAT using Mabs against LCDV. The result showed the gill, stomach, intestine, kidney and epidermis were the target organs of LCDV. Proteins of target organs were extracted and then subjected to VOPBA for LCDV binding protein identification. A gill tissue protein of 37.6 kDa could bind LCDV. A kidney tissue protein of 56 kDa could bind LCDV. The stomach, intestine and epidermis tissue shared the same binding protein of 27.8 kDa. FG cell membrane proteins was extracted by NP-40 lysis buffer and separatedfrom other cellular components by differential centrifugation. The cell membrane proteins were subjected to VOPBA. A protein of 37.6 kDa located on the surface of FG cells bound LCDV. This virus binding protein was purified by electroelution and then analysed by 2D gel electrophoresis. The profile of 2D gel electrophoresis showed it was a single polypeptide with pI of 6.0. After treated by Trypsin and sodium periodate, the ability of virus binding with 37.6 kDa protein was weakened, which suggested this protein was a glycosylated binding protein. Balb\c mice were immunized by 37.6 kDa protein to product polyclonal antibodies. IFAT found the polyclonal antibodies could react with the surface of the FG cells. Virus binding and infection inhibition test demonstrated that the polyclonal antibodies against the 37.6 kDa binding protein could block the virus binding and infection to FG cells, so that the 37.6 kDa binding protein might be a putative receptor of LCDV to FG cells.