A New Cell Line Derived from the Spleen of the Japanese Flounder (Paralichthys olivaceus) and Its Application in Viral Study

SIMPLE SUMMARY: In recent years, with the continuous development of the mariculture industry, the scale of mariculture has gradually expanded, and the problem of mariculture diseases is prominent. Among them, viral diseases and bacterial diseases are the main concerns. Japanese flounder (Paralichthys olivaceus), as the main marine aquaculture fish, has high economic value, mainly distributed in China, Korea, and Japan. At present, the viruses that are extremely harmful to Japanese flounder are Lymphocytes disease virus (LCDV), Hirame rhabdovirus (HIRRV), Viral hemorrhagic septicemia virus (VHSV), et al., were difficult to control in the event of a large-scale epidemic, causing significant economic losses to the Japanese flounder culture industry. Fish cell lines are an important research tool with the advantages of low assay conditions, accurate control of assay results, and ease of operation, and thus have been widely used in virology research. We established a new cell line Japanese flounder spleen (JFSP) from the spleen tissue of the Japanese flounder (P. olivaceus). We explored the optimal growth conditions, chromosome morphology, and number and transfection experiment of JFSP cells in vitro. The susceptibility of JFSP cells to different viruses, including Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV) and the expression changes of immune-related genes after virus infection were detected. The established JFSP cell line has enriched the fish cell resource bank and can be used as an excellent tool for genetic manipulation, studying host-virus interactions, and the development of potential vaccines. ABSTRACT: A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases.