Introduction

Sea perch, Lateolabrax japonicus, is one of the most economically important marine fish species in China. Similar with many other highly valued fish species, sea perch culture was affected by several infectious viral diseases that had caused huge economic losses to the aquaculture industry elsewhere in the world. It has been demonstrated that sea perch can be infected by nervous necrosis virus (NNV) and lymphocystis disease virus (Dong et al. 2014; Ciulli et al. 2015). NNV is a pathogen that can cause almost 100% mortality of larvae and juvenile of many fish species (Nishizawa et al. 1997). Over 50 fish species had been reported to be susceptible to NNV (Jia et al. 2015a, b). However, the exact mechanism of NNV infection remains unclear because NNV has different infection mechanisms in distinctive fish species.

Fish cell lines are important materials to study fish virology, immunology, developmental biology, toxicology, physiology, and molecular genetics (Hightower and Renfro 1988; Alvarez et al. 1991; Ferrero et al. 1998; Ma et al. 2001; Chen et al. 2003). Although many fish cell lines from marine and freshwater teleosts had been reported to be susceptible to NNV and were widely used to develop the anti-NNV strategies and to understand the interaction between NNV and host cells (Lakra et al. 2011), few cell lines from sea perch had been developed and none of them had been reported to be susceptible to NNV (Tong et al. 1998; Chen et al. 2003). Given that NNV might have different infection mechanisms in distinctive fish species, the establishment of species-specific cell line from sea perch is important to understand the infection mechanism of NNV in sea perch.

Fish brain is a major target organ for NNV infection as the virus has a tissue tropism for nervous tissue (Munday and Nakai 1997); it is possible to establish a NNV-sensitive cell line from the brain tissue of sea perch. Here, a novel cell line from the brain of sea perch was established and characterized. Furthermore, the transfection efficiency of LJB cells and its susceptibility to red-spotted grouper nervous necrosis virus (RGNNV) were investigated.

Materials and Methods

Primary cell culture and subculture

All procedures carried out with sea perch were approved by the Ethics Committee of Sun Yat-Sen University. Three healthy sea perches (approximately 50 g in weight) were collected from a fish farm (Zhuhai, China) and maintained in an aquarium equipped with seawater recirculation system. Sea perches were anesthetized with MS222 (Sigma, St. Louis, MO) and then wiped with 70% ethanol. The fish were decapitated, and the brains were subsequently removed into phosphate-buffered saline (PBS) with antibiotics (penicillin, 100 U mL−1; streptomycin, 100 μg mL−1) for washing. After rinsing once with 70% ethanol for 10 s and then PBS several times, the brain mass was minced into small pieces (approximately 1 mm3) using surgical scissors. Tissue pieces were seeded into 25-cm2 flasks and cultured in 1 mL of DMEM (supplemented with 20% FBS, 4.76 g L−1 HEPES, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin, pH 7.4) in an incubator at 28°C in air. Three milliliters of DMEM was added to the culture flasks after 24 h. Afterwards, the medium was replaced with fresh medium by half every 2–3 d. Subculture was carried out at 1:2 split subsequently by trypsinization when primary cell cultures grew to 90–100% of confluence. After passage 15, the medium was changed to DMEM with 15% FBS. The cell cultures were designated LJB cells.

DNA extraction and PCR analysis

Total genomic DNA was extracted from LJB cells (at passage 30) and brain tissue of L. japonicus using Tissue DNA Kit (Omega, Norcross, GA). Fragments of L. japonicus 18S ribosomal RNA (rRNA) (556 bp) were amplified using primers listed in Table 1. PCR products were sequenced, and the obtained sequences were aligned against known L. japonicus 18S rRNA sequences from the National Centre for Biotechnology Information (NCBI) database (GenBank Accession No. AB089346.1).

Table 1. Primers used for cloning and expression analysis
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Growth curves

The effects of FBS concentration and culture temperature on cell growth were investigated as previously described (Tong et al. 1997) with some modification. LJB cells at a density of 2.8 × 104 cells per well were seeded into 12-well plates and incubated at different temperatures (20, 24, 28, 32, and 37°C) in DMEM containing 15% FBS or cultured with DMEM containing different concentrations of FBS (10, 15, and 20%) at 28°C. After cultured for 24 h, non-adherent cells were removed by PBS wash and medium change. LJB cells were cultured over 7 d, and the medium was changed every 2 d. Every other day, cells were trypsinized and collected for hemocytometric determination of cell number. All experiments were undertaken in triplicate.

Cytogenetic analysis

Cytogenetic analysis of LJB cells was carried out at passage 45 as described previously (Dong et al. 2014) with some modification. Cells at 60–80% confluence were incubated with colchicine (Sigma) at the concentration of 10 μg mL−1 for 4–6 h and were subsequently harvested by trypsinization. After centrifuged at 700×g for 10 min, cells were suspended in 1 mL of 0.075 M KCl by pipetting. Following a 40-min incubation at room temperature, 0.4 mL of fleshly mixed Carnoy’s fixative (methanol:acetic acid = 3:1) was added for pre-fixation. After three times of fixation, cell pellet was resuspended in 0.2 mL of Carnoy’s fixative, dropped onto cold wet glass slides, air-dried, and stained with 10% Giemsa (Sigma) for 15 min. Chromosome numbers of metaphases were counted using a light microscope (Olympus, Tokyo, Japan).

Cell cryopreservation and recovery

LJB cells at various passages (10, 30, and 40) were used for cryopreservation. Cells at 80–90% confluence were trypsinized and harvested by centrifugation and resuspended in cryopreservation medium (DMEM containing 20% FBS and 10% dimethyl sulfoxide [Sigma]). After stored at −80°C for 24 h in a cryogenic rate-controlled freezing container (Nalgene Nunc, Penfield, NY), cells were transferred into liquid nitrogen for long-term cryopreservation. For cell recovery, cells kept in liquid nitrogen were taken out, thawed in 28°C water bath, and centrifuged at 500×g for 5 min to remove cryopreservation medium. Cell pellet was resuspended in DMEM by pipetting for washing, and then, cells were collected by centrifugation and seeded into a 6-well plate containing growth medium for culture. Cell viability of thawed cells was determined by trypan blue staining assay.

Transfection

LJB cells (at passage 50) were passaged into a 24-well cell culture plate at a density of 2 × 104 cells per well. After being cultured for 24 h at 28°C, LJB cells were transfected with plasmid pEGFP-N3 by Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA). Briefly, 1 μL of Lipofectamine™ 3000 reagent and 1 μg plasmid pEGFP-N3 were diluted by 25 μL of Opti-MEM™ medium in 1.5-mL microfuge tubes, respectively. Then, diluted plasmid pEGFP-N3 was added to the tube containing diluted Lipofectamine™ 3000 reagent and mixed well. After incubation for 10–15 min at room temperature, the mixture was added to each well. Green fluorescence signals were detected using a Zeiss microscope (Zeiss Axio Observer Z1), and the transfection efficiency was determined by counting green fluorescent protein-positive and total cells from 20 random fields at 48 h post transfection.

Viral susceptibility to RGNNV

LJB cells were seeded in a 6-well cell culture plate at a density of 1 × 105 cells per well and incubated for 24 h at 28°C. RGNNV was inoculated into the 6-well plate at a multiplicity of infection (MOI) of 5. After LJB cells were infected for 4 h, the medium containing RGNNV was discarded and 2 mL of growth medium with 15% FBS was added. Virus-specific cytopathic effect (CPE) was observed at 12, 24, and 48 h post infection (hpi), respectively. Meanwhile, infected cells were collected for RT-PCR assay.

RT-PCR

Total RNA was extracted from RGNNV-infected LJB cells using Trizol reagent (Invitrogen) and transcribed into complementary DNA (cDNA) by PrimeScript™ First-Strand cDNA Synthesis Kit (Takara, Dalian, China). A segment of the RNA-dependent RNA polymerase (RDRP) gene was amplified using primers NNV-RDRP-1F and NNV-RDRP-1R (Table 1). The PCR mixture (50 μL) contained 5 μL 10× Ex Taq buffer (Mg2+ Plus), 0.25 μL Ex Taq (5 U μL−1), 4.0 μL dNTPs (5 mM), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1.0 μL DNA template (0.5 μg), and 35.75 μL nucleic acid-free water. All the PCR reagents were obtained from Takara. PCR reactions were carried out as the following: 94°C for 5 min; 94°C for 30 s, 54°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 10 min. The sequences of PCR products were verified by DNA sequencing.

Virus infection and replication

LJB cells in 12-well plates at 70–80% confluence were challenged with RGNNV at an MOI of 5 for 4 h at 28°C. After the medium containing viruses was discarded, LJB cells were washed three times in PBS and then incubated with 1 mL of DMEM for 12, 48, and 72 h at 28°C, respectively. RGNNV-infected cells were harvested for quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the medium was collected for determination of virus titer at 12, 48, and 72 hpi.

Virus titer assay

Virus titer assay was performed as described previously (Reed and Muench 1938). One day before the assay, LJB cells were seeded into a 96-well plate at a density of 1 × 104 cell per well and cultured over night at 28°C. RGNNV was tenfold serially diluted, and 0.1 mL of each dilution was added to LJB cells in the 96-well plate. After the LJB cells were incubated for 5 d, virus titer was determined by 50% tissue culture infective dose (TCID50) assay.

qRT-PCR

To detect RGNNV replication in LJB cells, qRT-PCR was performed as described previously (Jia et al. 2015b). Primers for qRT-PCR are listed in Table 1. The qRT-PCR was conducted in a CFX96 Real-Time System (Bio-Rad, Hercules, CA). Sea perch β-actin was selected as the reference gene. Data represented the average value of three replicates and were expressed as mean ± SD. Statistics were carried out using SPSS version 20. One-way ANOVA was used to determine statistic differences between different groups. p < 0.05 was considered statistically significant.

Transmission electron microscopy

RGNNV-infected LJB cell samples and ultrathin sections were prepared as described previously (Dong et al. 2014). RGNNV-infected LJB cells were harvested by centrifugation, and cell pellets were fixed with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) at 4°C for 24 h. After that, cells were post-fixed with 2.0% osmium tetroxide in 0.1 M PBS (pH 7.4). Ultrathin sections were stained with uranyl acetate/lead citrate and documented using a Philips CM10 electron microscope.

Results

Primary cell culture and subculture of LJB cells

LJB cells were derived from the brain tissue of healthy sea perch. Primary cultures grew from the brain tissue blocks and reached full confluence within 10 d in DMEM (containing 20% FBS, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin) at 28°C. Then, primary LJB cells were subcultured at a split ratio of 1:2 for every 3–4 d. Fibroblast-like cells and epithelial-like cells were present in the first few passages of LJB cells (Fig. 1 A). Fibroblast-like cells became predominant from 18 passages (Fig. 1 B). To date, LJB cells have been subcultured more than 60 times since the initial culture.

Figure 1.
figure 1

Morphology and origination of cell cultures derived from the brain of sea perch. (A) Primary cultures on day 10. (B) LJB cells at passage 18 in 110 d after primary culture. Bar = 20 μm. (C) PCR amplification of partial 18S rRNA gene sequences of LJB cells and sea perch. DNA marker (2000 bp; M), LJB cells (lane 1), brain tissue (lane 2), negative control (lane 3)

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Identification of LJB cell line origination

To identify the origination of LJB cells, a 556-bp PCR product was amplified from LJB cells and brain tissue of L. japonicus, respectively (Fig. 1 C). DNA sequencing and comparative analysis showed that the partial 18S rRNA gene sequences amplified from LJB cells and brain tissue of L. japonicus were 98.8% identity to known sea perch 18S rRNA sequence from the NCBI database (GenBank Accession No. AB089346.1), confirming that the LJB cell line originated from L. japonicus.

Cell growth characteristics

LJB cells at passage 40 were analyzed for cell growth kinetics. The results showed that LJB cells could grow in the range of temperature from 20 to 37°C and exhibited maximum growth rate at 28°C (Fig. 2 A). The growth rate of LJB cells was increased with increase of concentration of FBS (10 to 20%) in DMEM at 28°C and showed maximum growth rate at 20% FBS (Fig. 2 B).

Figure 2.
figure 2

Growth curves of LJB cells at different temperatures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS) (A) and in DMEM supplemented with different concentrations of FBS at passage 40 at 28°C (B)

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Cytogenetic analysis

The chromosome numbers of LJB cells at passage 45 were determined by observing 100 metaphase plates, and the results showed that the chromosome numbers ranged from 20 to 96, with a modal number of 48 (Fig. 3).

Figure 3.
figure 3

Distribution of chromosome numbers of LJB cells at passage 45

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Cryopreservation

LJB cells were cryopreserved at different passages. Our results showed that 80–90% of LJB cells were viable after 1- to 3-mon storage at −196°C. After LJB cells were frozen and thawed, no apparent morphological alterations were observed.

Transfection efficiency

Green fluorescence signals could be detected at 48 h post transfection (Fig. 4), and the transfection efficiency was approximately 40% estimated by cell counting, suggesting that the LJB cell line could serve as an ideal tool for exogenous gene manipulation.

Figure 4.
figure 4

Expression of EGFP in LJB cells. Phase-contrast micrograph (left) and fluorescent micrograph (right) of pEGFP-N3-transfected LJB cells. Bar = 20 μm

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Virus susceptibility

LJB cells were tested for their susceptibility to RGNNV. The typical CPE for RGNNV was observed in LJB cells post RGNNV infection (Fig. 5). Initially, a few round, granular cells and cytoplasmic vacuole cells appeared at 12 hpi (Fig. 5 B); then, the portion of infected cells increased with increasing infection time. Finally, the monolayer was partially or completely disrupted at 24 and 48 hpi (Fig. 5 C, D). Meanwhile, no significant change was observed in the control cells without RGNNV infection (Fig. 5 A). A 129-bp RDRP gene fragment was detected in RGNNV-infected LJB cells (Fig. 5 E, lane 1), and DNA sequencing and comparative analysis showed that the partial RDRP gene fragment was completely identical with the known RDRP sequence (GenBank Accession No. KP455643) (data not shown), which further confirmed the RGNNV infection in LJB cells.

Figure 5.
figure 5

Virus susceptibility of LJB cells to RGNNV. Cytopathic effect of LJB cells infected with RGNNV at 12 (B), 24 (C), and 48 (D) hpi, respectively. A Mock-infected LJB cells. Bar = 20 μm. (E) Agarose gel electrophoresis of PCR products from RGNNV-infected LJB cells using specific primer for RNA-dependent RNA polymerase (RDRP). DNA marker (2000 bp; M), RGNNV-infected LJB cells (lane 1), uninfected LJB cells (lane 2), blank control (lane 3)

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Virus replication in LJB cells

As shown in Fig. 6 A, the transcript of RDRP messenger RNA (mRNA) was also significantly increased in RGNNV-infected LJB cells from 12 to 72 hpi. RGNNV virus titer increased significantly in RGNNV-infected LJB cells from 12 to 72 hpi (Fig. 6 B). Meanwhile, the result of TEM assay indicated that many RGNNV particles existed in the cytoplasm of RGNNV-infected LJB cells. Multiple vacuolation was also observed in RGNNV-infected cells (Fig. 7). All these results confirmed that RGNNV could replicate in LJB cells.

Figure 6.
figure 6

RGNNV replication in LJB cells. (A) Expression analysis of RDRP mRNA in LJB cells at 12, 48, and 72 h post RGNNV infection. (B) Detection of RGNNV production in LJB cells by TCID50 assay. The results were expressed as mean ± SD from three independent experiments performed in triplicates. Asterisks indicate significant differences between 12 and 48 or 72 h post RGNNV infection (p < 0.05)

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Figure 7.
figure 7

Transmission electron micrograph of RGNNV-infected LJB cells under different magnifications. (A) RGNNV-infected LJB cells under low magnification (×27,000). (B) Large numbers of random arrays of virus particles in RGNNV-infected LJB cells (×67,000). Arrows indicate virus particles

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Discussion

In the present study, a cell line derived from the brain tissue of sea perch, a commercially important fish in China, has been established and characterized. The LJB cell line grew well and has been subcultured for more than 60 passages so far. PCR analysis indicated that the LJB cells were originated from L. japonicus.

LJB cells showed the maximum growth rate in the medium containing 20% FBS, which was consistent with other sea perch cell lines (Ye et al. 2006). However, LJB cells could also maintain an ideal growth rate in DMEM with 15% FBS. Taking the cost effect into account, LJB cells were cultured in DMEM with 15% concentration of FBS. LJB cells could grow in the range of temperature from 20 to 37°C and exhibited maximum growth at 28°C, which was also identical with other established sea perch cell lines (Nicholson et al. 1987; Tong et al. 1998). Sea perch was a fish species living in warm water; the range of temperature for its survival was between 20 and 30°C, which encompassed the optimal temperature range for the LJB cell line. Differently, a sea perch heart cell line had an optimal temperature at 30°C (Nicholson et al. 1987). This might be due to the fact that these cell lines were derived from different organs of sea perch. The typical chromosomal number of LJB cell line was 48, which was identical with ES cells derived from sea perch (Chen et al. 2003) and sea perch embryonic cells (unpublished data). Similar with other fish cell lines, aneuploidy and heteroploidy were also found in chromosome preparations of LJB cells. The abnormal chromosomal number together with morphological change might be indications of cell transformation (Frerichs et al. 1996).

It is well established that low transfection efficiency limited the applications of fish cell lines. The transfection efficiency in LJB cells was about 40%, which was higher than that in L. japonicus embryonic cell line (Chen et al. 2003), indicating that the LJB cell line could be employed as an ideal in vitro system to manipulate exogenous genes.

Previous studies indicated that the main target organ where NNV exerted its effects was the central nervous system and NNV infection led to the vacuolation of the brain and retina of many infected fishes (Munday et al. 2002; Shetty et al. 2012). It was speculated that a continuous cell line originated from the brain of NNV-sensitive fish might be a useful tool for NNV isolation, viral pathogenesis studies, and vaccine development against NNV. To date, a few cell lines from different fishes, such as the brain tissue of Dicentrarchus labrax (Frerichs et al. 1996) and Lates calcarifer (Chi et al. 2005) and the spleen of Epinephelus coioides (Qin et al. 2006), have been used for NNV pathogenesis studies. The present study demonstrated that typical CPE was observed in RGNNV-infected LJB cells within 48 hpi, such as cell detachment and heavy cytoplasmic vacuoles, which was consistent with a previous report (Yoshikoshi and Inoue 1990). Furthermore, RGNNV replication was confirmed by qRT-PCR, virus titer, and TEM assay in RGNNV-infected LJB cells. All these results indicated that the LJB cell line could be used as a helpful tool for studying host and NNV interactions.

Conclusion

A brain cell line from sea perch, designated LJB, was established and characterized. The cells exhibited high transfection efficiency and were susceptible to RGNNV. Therefore, the LJB cell line can be used as an ideal in vitro tool for the study of NNV infection mechanism and exogenous gene manipulation.