Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Nov 24;11(12):1697.
doi: 10.3390/biology11121697.

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

Yucong Yang  1   2   3 , Yuqin Ren  3 , Yitong Zhang  3 , Guixing Wang  3 , Zhongwei He  3 , Yufeng Liu  3 , Wei Cao  3 , Yufen Wang  3 , Songlin Chen  4 , Yuanshuai Fu  1   2 , Jilun Hou  3
Affiliations

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

Yucong Yang et al. Biology (Basel). .

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.

Keywords: Bohle virus; Lymphocystis disease virus; Paralichthys olivaceus; cell line; cell transfection; virus susceptibility.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphology of the Japanese flounder spleen (JFSP) cells. (A–D) Morphology of JFSP cells at Primary passage (A), passage 40 (B), passage 60 (C), and passage 80 (D). Scale bar = 50 μm.
Figure 2
Figure 2
Agarose gel electrophoresis of PCR products from JFSP cells using specific primers for 18s rRNA. M, 2000 bp DNA maker; lane 1, 18s rRNA; lane 2, Blank.
Figure 3
Figure 3
Growth kinetics of JFSP cell line at passage 55. (A) Effect of temperature on cell growth in L-15 medium containing 15% FBS. (B) Effect of L-15 medium with different concentrations of FBS on cell growth at 23 °C. (C) Effect of different media added with 15% FBS concentration on cell growth at 23 °C. Data are means ± SD of three measurements.
Figure 4
Figure 4
Chromosome morphology and frequency distribution of the number of entries in JFSP cells. (A) Chromosome morphology and (B) chromosome number statistics of JFSP cells treated with colchicine at 65 passage. Scale bar = 100 μm.
Figure 5
Figure 5
The expression of the GFP gene in JFSP cells 24 h after pEGFP-N1 transfection at passages 40 (A,B) and 80 (C,D). (A,C) Bright-field. (B,D) The expression of pEGFP-N1 in JFSP cells. Scale bar = 50 μm.
Figure 6
Figure 6
Observed CPE of JFSP cells infected by BIV, VHSV, HIRRV, IHNV, or LCDV through an inverted microscope. (A) Control group. CPE of JFSP cells infected with (B) BIV, (C) VHSV, and (D) HIRRV at 36 h p.i. (E) CPE of IHNV-infected JFSP cells at 48 h p.i. (F) CPE of LCDV-infected JFSP cells at 3 d p.i. Scale bar = 50 μm.
Figure 7
Figure 7
Replication characteristics of BIV, VHSV, HIRRV, IHNV, and LCDV in JFB cells. (A) BIV, (B) VHSV, (C) HIRRV, (D) IHNV, and (E) LCDV. Different letters denote significant differences among groups (p < 0.05).
Figure 8
Figure 8
Ultrastructural observations of BIV, VHSV, HIRRV, IHNV, or LCDV-infected JFSP cells. Abundant virus particles were observed in BIV, VHSV, HIRRV, IHNV, or LCDV-infected cells. (A,B) Virus particles of BIV-infected cells. Scale bar = 200 μm. (C,D) Virus particles of VHSV-infected cells. (C) Scale bar = 500 μm. (D) Scale bar = 200 μm. (E,F) Virus particles of HIRRV-infected cells. Scale bar = 500 μm. (G,H) Virus particles of IHNV-infected cells. Scale bar = 200 μm. (I,J) Virus particles of LCDV-infected cells. Scale bar = 200 μm. ★: BIV, ❋: VHSV, ▶: HIRRV, ➞: IHNV, ➤: LCDV.
Figure 9
Figure 9
Expression of immune-related genes during infection of JFSP cells by BIV, VHSV, HIRRV, IHNV, or LCDV. (A–D) Relative expression levels of immune-related genes (TRAF3, IL-1β, TNF-α, and TLR2) in JFSP cells infected with the five viruses for 36 h p.i. Uninfected JFSP cells were used as a control group. All data are expressed as mean ± SEM (n = 3). * indicates a significant level of difference at p < 0.05, ** indicates a very significant level of difference at p < 0.01, and *** indicates an extremely significant level of difference at p < 0.001.
See this image and copyright information in PMC

References

    1. Guo Y.N., Nan X.Y., Zhang X.Y., Wang G.X., Ren Y.Q., Wang Y.F., Fu Y.S., Hou J.L. Molecular characterization and functional analysis of Japanese flounder (Paralichthys olivaceus) thbs2 in response to lymphocystis disease virus. Fish Shellfish Immunol. 2019;93:183–190. doi: 10.1016/j.fsi.2019.07.055. - DOI - PubMed
    1. Zhang J.L., Tang X.Q., Sheng X.Z., Xing J., Zhan W.B. Isolation and identification of a new strain of hirame rhabdovirus (HIRRV) from Japanese flounder Paralichthys olivaceus in China. Virol. J. 2017;14:73. doi: 10.1186/s12985-017-0742-4. - DOI - PMC - PubMed
    1. Zhang W.W., Li Z.L., Xiang Y., Jia P., Liu W., Yi M., Jia K. Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China. J. Fish Dis. 2019;42:1563–1572. doi: 10.1111/jfd.13078. - DOI - PubMed
    1. Kim W.S., Kim S.R., Kim D., Kim J.O., Park M.A., Kitamura S.I., Kim H.Y., Kim D.H., Han H.J., Jung S.J., et al. An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea. Aquaculture. 2009;296:165–168. doi: 10.1016/j.aquaculture.2009.07.019. - DOI
    1. Gong C.G., Zhang Y.T., Wang G.X., Liu Y.F., He Z.W., Ren Y.Q., Cao W., Zhao H.T., Xu Y.H., Wang Y.F., et al. The isolation and full-length transcriptome sequencing of a novel nidovirus and response of its infection in Japanese flounder (Paralichthys olivaceus) Viruses. 2022;14:1216. doi: 10.3390/v14061216. - DOI - PMC - PubMed