Elsevier

Aquaculture

Volume 562, 15 January 2023, 738825
Aquaculture

Establishment and characterization of a cell line from the brain of the Japanese flounder (Paralichthys olivaceus) and its application in the study of viral infection

https://doi.org/10.1016/j.aquaculture.2022.738825Get rights and content

Highlights

  • A new cell line JFB from the Japanese flounder brain was established and characterized.

  • The JFB cells could grow at a temperature range of 17 to 29 °C, and 23 °C was the optimum temperature.

  • JFB cells showed susceptibility to HIRRV, LCDV, BIV and GSIV.

  • Viral infection or Poly (I:C) stimulation upregulated the expression of immune-related genes in JFB cells.

Abstract

Fish cell lines are important tools in many biological studies. Here, we established a new Japanese flounder brain (JFB) cell line from the brain tissue of the Japanese flounder (Paralichthys olivaceus), which was successfully sub-cultured for over 120 passages, and consisted predominantly of fibroblast-like cells. Sequencing of the actin gene confirmed that JFB cells were derived from the Japanese flounder, and JFB was also identified as a neural stem cell line on the basis of the mRNA expression of nestin gene. The JFB cells could grow at a temperature ranging from 17 to 29 °C, and 23 °C was the optimum temperature. Karyotype analysis showed that the chromosome mode number was 48, demonstrating a normal diploid chromosome number. The transfection efficiency of pEGFP-N1 in JFB cells was up to 30%. The JFB cells showed susceptibility to Hirame rhabdovirus (HIRRV), Lymphocystis disease virus (LCDV), Bohle virus (BIV) and Chinese giant salamander iridovirus (GSIV), which was demonstrated by varying degrees of cytopathic effects. Viral infection or Poly (I:C) stimulation upregulated the expression of multiple immune-related genes, including IL-1β, IL8, Mx and TNF. These results showed that the newly established JFB cell line is an ideal tool for studying gene manipulation, host-virus interaction and potential vaccines development.

Introduction

In vitro culture of fish cells started in the early 1960s (Wolf and Quimby, 1962). Fish cell line culture is one of the most important research tools in life sciences with many advantages such as low-cost test conditions, accurate control of test results and convenience of operation. They have been widely used in studies in virology, environmental toxicology, fish resource conservation, genetics and breeding, endocrinology, developmental biology and physiology (Buonocore et al., 2006; Gomez-Lechon et al., 2008; Ledermann, 2000; Petersen et al., 2003). Up to date (as of September 2020), over 780 different fish cell lines have been established around the world (Thangaraj et al., 2021). In the past, most of the fish cell lines that have been established are from freshwater fish (Chen et al., 2004; Fernández-Puentes et al., 1993). However, the research on marine fish cells line has developed rapidly in the recent decade due to the continually increasing demand for related studies (Thangaraj et al., 2021).

The Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in China, Japan and Korea. With the development of the Japanese flounder aquaculture, diseases, especially those caused by viral infections, have caused heavy losses to the fish farming industry. The viruses affecting the Japanese flounder include Lymphocystis disease virus (LCDV) (Guo et al., 2019), Hirame rhabdovirus (HIRRV) (Sun et al., 2010; Zhang et al., 2017), Viral hemorrhagic septicemia virus (VHSV) (Isshiki et al., 2001; Kim et al., 2009; Zhang et al., 2019), and Marine birnavirus (MABV) (Inaba et al., 2007; Jung et al., 2008). Appropriate cell lines from the Japanese flounder are required to study these viruses in detail. To this day, there are about 17 cell lines, including brain (Zheng et al., 2015., Gao et al., 2020), fin (Choi et al., 2020), embryo (Chen et al., 2004; Nie et al., 2021), gill (Tong et al., 1997), and skin (Wang et al., 2017), that have been established.

This study established a novel cell line named JFB derived from the Japanese flounder brain. We evaluated the optimal growth condition, modal chromosome number, and transfection efficiency of JFB. Distinguished from all other Japanese flounder brain cell lines (Zheng et al., 2015., Gao et al., 2020), we assessed the susceptibility of JFB cells to different viruses from fish and amphibians for the first time. In addition, we also investigated the immune-related gene expression patterns in JFB cells when exposed to immunostimulant agent Poly (I:C) or viral infection. The current results illustrate the potential application of the JFB cell line in genetic manipulation and viral immunology.

Section snippets

Ethics

This study was performed following the Guide for Care and Use of Laboratory Animals provided by the Chinese Association for Laboratory Animal Sciences (No. 2011–2). All experiment protocols were approved by the animal care and use committee of Beidaihe Central Experiment Station, Chinese Academy of Fishery Sciences.

Primary cell culture and routine maintenance

The Japanese flounder (six months old, weight: 150 g) was obtained from the Beidaihe Central Experimental Station of the Chinese Academy of Fishery Sciences. The fish was

Primary cell culture and routine maintenance

Primary cell cultures were obtained from the brain tissue of the Japanese flounder. After 3 d of culture in a 25 cm2 flask, primary cells migrated from the tissue explant gradually and continued to extend outwards. From the perspective of morphology, the cells were both epithelial-like and fibroblast-like cells in the initial subcultures. The cells grew and formed a 90% monolayer on 28 d, and after that, the cells were sub-cultured every 3–6 d. Up to date, the cells were sub-cultured for >120

Discussion

In the current study, a permanent cell line named JFB, derived from the Japanese flounder brain tissue, was established. The sequence analysis of the actin gene from JFB cells confirmed its origin from Japanese flounder. In addition, the neural stem cells (NSCs)-specific gene nestin, which exists almost exclusively in the brain and plays a fundamental role in neurodevelopment (Gao et al., 2020), was amplified from JFB cell line, indicating that the cell line was derived from Japanese flounder

Declaration of Competing Interest

The authors have declared that no competing financial interests exist.

Acknowledgements

We thank Dr. Yanmiao Tan, Ocean College, Agricultural University of Hebei, for her kindly providing of HIRRV, BIV and GSIV. This work was supported by the Central Public-Interest Scientific Institution Basal Research Fund, CAFS (2022TD38, 2021YJ04), Nature Science Foundation of Hebei Province, China (C2021107002), the Key R&D Program of Hebei Province, China (21326307D), The National Marine Genetic Resource Center, and China Agriculture Research System (CARS-47).

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