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
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).
References (51)
- et al.
Propagation and isolation of ranaviruses in cell culture
Aquaculture.
(2009) - et al.
Establishment and characterization of a new cell line derived from the fin of olive flounder (Paralichthys olivaceus)
Aquaculture.
(2020) - et al.
Molecular characterization and functional analysis of Japanese flounder (Paralichthys olivaceus) thbs2 in response to lymphocystis disease virus
Fish Shellfish Immun.
(2019) - et al.
Expression of MHC class I pathway genes in response to infectious salmon anaemia virus in Atlantic salmon (Salmo salar L.) cells
Fish Shellfish Immun.
(2006) - et al.
An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed olive flounder Paralichthys olivaceus in Korea
Aquaculture.
(2009) - et al.
Virion-associated viral proteins of a Chinese giant salamander (Andrias davidianus) iridovirus (genus Ranavirus) and functional study of the major capsid protein (MCP)
Vet. Microbiol.
(2014) - et al.
Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line
Dev. Comp. Immunol.
(2021) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method
Methods.
(2001) - et al.
Ultrastructural morphogenesis of an amphibian iridovirus isolated from Chinese giant salamander (Andrias davidianus)
J. Comp. Pathol.
(2014) - et al.
An assessment of immunostimulants as mx inducers in Atlantic salmon (Salmo salar L.) parr and the effect of temperature on the kinetics of mx responses
Fish Shellfish Immun.
(2004)
Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR
J. Virol. Methods
In vitro and in vivo differential expression of rainbow trout (Oncorhynchus mykiss) Mx isoforms in response to viral haemorrhagic septicaemia virus (VHSV) G gene, poly I:C and VHSV
Fish Shellfish Immun.
The establishment and partial characterization of a continuous fish cell line FG-9307 from the gill of flounder Paralichthys olivaceus
Aquaculture.
Isolation and identification of a new strain of hirame rhabdovirus (HIRRV) from Japanese flounder Paralichthys olivaceus in China
Virol. J.
Production and characterization of a continuous embryonic cell line from sea bass (Dicentrarchus labrax L.)
Mar. Biotechnol.
Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation
Dis. Aquat. Org.
Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus
Arch. Virol.
Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans
Dis. Aquat. Org.
Initiation of a cell line from turbot (Scophthalmus maximus L.).
In Vitro Cell. Dev. Biol.
Establishment and characterization of four long-term cultures of neural stem/progenitor cell lines from the Japanese flounder Paralichthys olivaceus
J. Ocean Univ. China.
Cell lines: a tool for in vitro drug metabolism studies
Curr. Drug Metab.
Functions of cytokines in immune response to influenza a virus infection
Prog. Vet. Med.
Early interactions of marine birnavirus infection in several fish cell lines
J. Gen. Virol.
Annual dynamics of marine birnavirus (MABV) in cultured Japanese flounder Paralichthys olivaceus and sea water
Fish. Sci.
An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan
Dis. Aquat. Org.
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