Identification And Characteristics Of The Lymphocystis Disease Virus Cellular Receptor In The Flounder (Paralichthys Olivaceus)

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystisdisease (LCD), an infectious virosis of fish manifested by hypertrophy ofconnective cells mainly in the integument of the fish body surface and fins. Thedisease has affected over140different wild and cultured fish species worldwide,including species of particular commercial importance, e.g. flounder Paralichthysolivaceus. Electron microscopic observations showed that the virus could attach tothe cell surface and then penetrate the cells by membrane fusion or by endocytosis,suggesting that there were probable virus-specific receptors located on theflounder gill (FG) cell surface. Research on host cell receptors of LCDV willcontribute to the understanding of viral replication and pathogenesis.In this paper, co-immunoprecipitation and virus overlay protein binding assay(VOPBA) were used to isolate and identify the LCDV cellular receptors in FG andflounder tissues; two-dimensional (2D) electrophoresis, mass spectrometry andbiochemical methods were used to analyze the characteristics of receptors; virusinfection inhibition assay verified the function of the receptor; and thedistributions of LCDV cellular receptor in flounder were studied by receptormonoclonal antibodies. The details are as follow:(1) FG cells and LCDV are prepared initially in this study. The LCDV waspurified by differential sucrose density gradient centrifugation, and the virions wereintact, round polygon in shape, with envelope outside. FG cells were derived fromflounder gill tissue and they grew fast and well at22℃,2%CO2in Eagle’s MEMsupplemented with10%fetal calf serum.(2) The FG cell membrane proteins were extracted by NP-40lysis buffer and separated from other cellular components by differential centrifugation. Theco-immunoprecipitation assay with LCDV and anti-LCDV monoclonal antibodiesdetected a27.8kDa molecule protein from FG cell membrane that bound to LCDV.Mass spectrometric analysis established that the27.8kDa protein had a strongassociation with β-actin. Balb/c mice were immunized by27.8kDa protein to productpolyclonal antiserum. In western blotting, the antiserum were able to react stronglywith the27.8kDa protein and weakly cross-react with the42.7kDa protein; and theβ-actin antibody reacted strongly with42.7kDa protein band and weakly with the27.8kDa protein band. Of interest, β-actin protein in different species has highhomology as well as a molecular weight of about42kDa. Thus, the27.8kDa proteinmight be a part of β-actin protein or an unknown protein sharing some epitopes withβ-actin.(3) Monoclonal antibodies (Mabs) very often achieve greater sensitivity thanpolyclonal antibodies. In this study, two MAbs designated as2G11and3D9wereproduced after cells fusion, screened by ELISA and clone by limiting dilution.Analysed by the indirect enzyme-linked immunosorbent assay (ELISA) and westernblotting, the MAbs specifically reacted with the27.8kDa protein of FG cells.Confocal fluorescence microscopy and immunogold electron microscopy (IEM)provided visualized evidence that the epitopes recognized by these MAbs weremainly located on the cell membrane and occasionally in the cytoplasm near the cellmembrane of FG cells. Blocking ELISA results showed that the blocking rate of MAb2G11and3D9was83.6%and79.4%, respectively; for LCDV infection inhibitionassay, the MAbs could inhibit LCDV infection by the MAbs pre-incubation with FGcells. These results strongly supported the possibility that the27.8kDa protein wasthe putative receptor specific for LCDV infection of FG cells.(4) Native-PAGE and virus overlay protein binding assay (VOPBA) were used toidentify the receptor proteins of LCDV on the flounder gill (FG) cells. One135kDaprotein molecule was found to be the LCDV binding protein on the FG cells. SDS-PAGE and two dimensional (2D) gel electrophoresis analysis of this proteinindicated it had three proteins with molecular weight of58.3kDa,44.6kDa and37.6kDa. While VOPBA after SDS-PAGE showed that only37.6kDa polypeptidecould bind to LCDV. The co-immunoprecipitation results were subjected to VOPBAto analyze the relation of27.8kDa and37.6kDa receptor proteins in FG cells. Inco-immunoprecipitation, we observed the27.8kDa protein but no37.6kDa protein.While in VOPBA after co-immunoprecipitation, the37.6kDa protein was presentedbut there was no27.8kDa protein. It suggested that both27.8kDa and37.6kDaproteins were the LCDV receptor proteins in FG cells, and the27.8kDa protein had aLCDV binding activity only in natural state, but the37.6kDa protein could bind theLCDV in non-natural state.(5) Co-immunoprecipitation was used to identify the LCDV receptor proteins ongill, stomach, intestine and skin of flounder (P. olivaceus). The results showed that thegill, stomach and intestine tissues were detected to share a binding protein with thesame molecular weight of27.8kDa, and a skin tissue protein of37.6kDa was foundto specifically bind LCDV. Then Alcian blue staining was used to identify theglycoprotein characteristics on the samples of co-immunoprecipition, and the resultsrevealed that the27.8kDa proteins in the gill, stomach and intestine were defined asnon-glycoprotein while the37.6kDa protein in the skin as a glycoprotein. Westernblotting was used to investigate the distribution of the27.8kDa protein in floundertissues, and the results showed the MAbs could react with the27.8kDa protein in thetissues of gill, stomach, intestine and liver. But to other three tissues, including skin,kidney and spleen, no reactions were observed.