Distribution Of Lymphocystis Disease Virus Receptor In Flounder (Paralichthys Olivaceus) And Identification Of Virion Attachment Proteins

Lymphocystis disease (LCD), is an important chronic disease characterized byformation papilloma-like lesions typically on fish skin and superficial tissue,sometimes in the internal tissues of fish. Lymphocystis disease virus (LCDV), thecausative agent of lymphocystis disease, has a worldwide geographical distributionand has infected more than140species of marine and freshwater fish belonging to42families, including wild, cultured and ornamental fish. The diseased fish lose theirornamental and commercial value, resulting in a great economic loss in marine cultureindustry. Determination of the receptor molecules on the surface of susceptible cellsand virion attachment protein of LCDV will contribute to the understanding of viralinfection, pathogenesis and tissue tropism in hosts, which also will benefit thedevelopment of anti-viral therapeutic agents and control of LCD.In this paper, monoclonal antibodies (Mabs,2G11and3D9) against27.8kDareceptor protein of lymphocystis disease virus (LCDV) produced in our Lab wereapplied to detect LCDV receptor protein in the tissues of flounder (Paralichthys olivaceus),and viral attachment proteins of LCDV were identified. In addition, the existence of27.8kDa receptor in turbot (Scophthalmus maximus) was detected, which provide thetheory basis for the research of extensive infection of LCDV. The results were asfollows.By indirect immunofluorescence assay and immunohistochemical experiment,the peripheral blood, leukocyte, gill, stomach, intestine, epidermis, liver, kidneys,spleen, gonad, brain and heart tissue of flounder (P. olivaceus) were detected. Resultsshowed that LCDV receptor-positive signals were found in leukocyte, gill, stomach,intestine, epidermis, liver, spleen, and head-kidney, which indicated that there isLCDV27.8kDa receptor protein in these tissues, which are the molecular basis that mediate LCDV adsorption and infection. We deduced that LCDV takes gill cells,epidermis and gastrointestinal epithelium as main infection target organs, propagatingin these target cells, and infects the leucocyte in peripheral blood after the cyst-celllysis. Virus achieves its transmission in vivo though blood circulation, and infects theliver, spleen, head-kidney and other organs.By immunoblotting experiment，gill, stomach, intestine, liver and body kidneystissue of turbot (S. maximus) were detected. Results showed that LCDVreceptor-positive signals were found in the major tissue, which indicated that there isLCDV27.8kDa receptor protein in these tissues. Flounder and turbot own the samereceptor， and the distribution of the receptor in the body organs have greatconsistency.Reversal virus overlay protein binding assays (RVOPBA) were carried out toidentify the virion attachment proteins of the LCDV. The protein of LCDV weretransferred to PVDF membrane after SDS-PAGE, and then incubated with27.8kDareceptor protein. Finally one specific LCDV protein band with a molecular mass of32kDa, which could bind with27.8kDa receptor protein, was obtained by chemicalcoloration. The specific protein band was cut from the gel and purified, and thensequenced by mass spectrometry. The results showed that this protein was encoded byOpen reading frame038（ORF038）of LCDV by contrasting the data with genebank.