| lymphocystis disease (LCD) is one of the most devastating fish diseases in aquaculture, has infected more than 140 species of marine, brackish water and freshwater fish belonging to 42 families. Its causative agent is lymphocystis disease virus (LCDV), which heavily hinders the development of flounder(Paralichthys olivaceus), turbot (Scophthalmus maximus) and sea bass (Lateolabrax japonicus) culture industry in our country. It was reported that the entry and infection of LCDV were mediated by functional receptor. Our previous researches have demonstrated that a 27.8 kDa membrane protein, which is isolated from flounder gill (FG) cells, is a receptor mediating LCDV entry and infection, and monoclonal antibodies against 27.8kDa receptor (27.8R) were produced. In this study, we made an investigation on relationship of 27.8R expression and LCDV infection in FG and hirame natural embryo (HINAE) cells; then tissue distribution of 27.8R in flounder, turbot and sea bass, together with its expression dynamics in response to LCDV infection were investigated; meanwhile, the role of 27.8R in LCDV infection of flounder peripheral leukocyte was analyzed, the resultant data would contribute to the understanding of virus invasion mechanism and transmission route; finally, transcripteomics of flounder gill post LCDV infection were determined by RNA sequencing (RNA-seq), the results would provided important data for the pathogenesis of LCDV. The detailed research contents and results are showed as follows:(1) Relationship between expression of cellular 27.8R and LCDV infection in FG and HINAE cells. Western blotting showed that anti-27.8R MAbs reacted with only one band at a molecular weight of 27.8kDa from both FG and HINAE cell membrane proteins. Co-immunofluorescence staining showed the 27.8R was expressed and co-localized with LCDV in both FG and HINAE cell surface. After LCDV infection, the expression of 27.8R was detected by sandwich ELISA, which exhibited a dose-dependent up-regulation with the increasing of LCDV titers, and demonstrated a tendency to increase firstly and then decrease during a time course up to 9 days; LCDV copies was investigated by real time quantitative PCR, the results showed a similar variation trend to the 27.8R expression, however, it reached the highest level later than did the 27.8R expression. Additionally, the 27.8R expression and LCDV copies in FG cells were higher than those in HINAE cells. In the presence of increasing concentration of the anti-27.8R MAbs, the up-regulation of 27.8R expression and the copy numbers of LCDV significantly declined post LCDV infection, and the cytopathic effect induced by LCDV in the two cells was accordingly reduced, indicating anti-27.8R MAbs pre-incubation could inhibit the up-regulation of 27.8R expression and LCDV infection. These results suggested that LCDV infection could induce up-regulation of 27.8R expression, which in turn increased susceptibility and availability of FG and HINAE cells for LCDV entry.(2) Tissue localization of 27.8R and its expression kinetics induced by LCDV infection in flounder, turbot and sea bass. In this study, the immunofluorescence assay and immunohistochemistry were carried out to locate the distribution of 27.8R by using anti-27.8R monoclonal antibodies in healthy flounder, turbot and sea bass tissues, including gill, stomach, intestine, skin, brain, heart, liver, spleen, head-kidney, kidney and gonad, positive signals were observed in all the detected tissues. Indirect enzyme-linked immunosorbent assay showed 27.8R expression was highest in gill and stomach and lowest in kidney in healthy flounder; in healthy turbot, the expression of 27.8R was relatively higher in stomach, gill, heart and intestine, and lower in ovary and brain; in healthy sea bass,27.8R expression was relatively higher in gill and skin, while lower in kidney and brain. Upon LCDV infection,27.8R expression was generally up-regulated in tissues of fishes. The dynamic changes of LCDV copies were determined by real-time quantitative PCR, the results showed that LCDV copies increased in a time-dependent manner, and were higher in tissues with a higher expression of 27.8R. Moreover, the 27.8R expression and LCDV were detected in partial leukocytes but negative in red blood cells via immunofluorescence assay. These results suggested that 27.8R from flounder also served as a receptor in turbot and sea bass, the leukocytes might play important roles in a systemic infection of LCDV, and a vertical transmission of LCDV might exist in flounder, turbot and sea bass. This study would provide new important insights into pathogenesis and transmission of LCDV in fish.(3) 27.8R was responsible for lymphocystis disease virus infection of peripheral blood leukocytes of flounder. In this study, the blood transmission of LCDV was investigated from the point of 27.8R and LCDV localization in peripheral blood cells of flounder. At 3 h post LCDV infection, the detections of both 27.8R and LCDV by immuno fluorescence assay were negative in red blood cells (RBCs) and positive in some peripheral blood leukocytes (PBLs). Localization of 27.8R in PBLs was confirmed by immunogold electron microscopy, and FACS analysis showed that the percentage of 27.8R+cells in PBLs were 14.28%. By light and transmission electron micrographs, time course of LCDV infection in PBLs in vitro was observed, which showed an initial binding at 1 h and an efficient replication at 36 h post LCDV infection. Finally, a multicolor fluorescent microscopy in PBLs was performed, the results showed that:1) LCDV and 27.8R were well overlapped on the surface of partial PBLs; 2) CD3+ cells were not coincidence with LCDV sensitive cells; 3) Both IgM+and IgD+ cells were sensitive to LCDV infection. Our results showed that 27.8R was expressed in part of PBLs, possibly leading to the susceptibility to the LCDV, and IgM+ and IgD+ cells might be involved in blood transmission of LCDV.(4) Transcriptome analysis of flounder gill in response to LCDV infection. In this study, RNA sequencing (RNA-seq) was employed to investigate differentially expressed genes (DEGs) in the gill of the flounder at one week post LCDV infection. Transcriptome sequencing of gill with and without LCDV infection was performed using the Illumina HiSeq 2500 platform. In total, RNA-seq analysis generated 193,225,170 clean reads aligned with 106,293 unigenes, and 36,537 (34.37%) of them were annotated in public databases. Compared with the unigene expression profile of the control group (without LCDV infection),1812 genes were up-regulated and 1626 genes were down-regulated in the gill with LCDV infection, respectively. These DEGs were significantly enriched in 14 Gene Ontology terms and 22 pathways. A number of genes related to inflammation, ubiquitin-proteasome pathway, cell proliferation, apoptosis, tumor formation and anti-viral defense showed a different expression patterns as a result of LCDV infection. In addition, changes in the pattern of gene expression were validated by quantitative real-time PCR. In conclusion, our comprehensive gene expression study not only provided new information for identification of novel genes in flounder, but also described for the first time the entire transcriptome responses to LCDV infection in the early phase of infection. |
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