Berberine hydrochloride (BBH) and EGCG standards were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma (Shanghai, China). DCFDA/H2DCFDA cellular ROS assay kit was purchased from Abcam (Shanghai, China). M199 medium, FBS serum, penicillin/streptomycin, second antibodies, Pierce BCA Protein Assay Kit, and TRIzol reagent were purchased from Thermo Fisher Scientific (Shanghai, China). PrimeSTAR^® Max DNA Polymerase, PrimeScript™ II 1st Strand cDNA Synthesis Kit, TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0, and One-Step SYBR RT-PCR kit were purchased from Takara Bio (Beijing, China). All other reagents were purchased from Sangon Biotech (Shanghai, China). CyHV-2 strain ST-J1 (NCBI: NC_019495) and RyuF-2 cell line were provided by Dr. Motohiko Sano, University of Marine Science and Technology, Tokyo.

Our cell lines are from the generous sharing of Dr. Motohiko Sano from Tokyo Ocean University. Cryopreserved samples of RyuF-2 cell stock at −150°C were thawed in a 37°C water bath before cell passage and virus propagation. Frozen cells were quickly thawed in a water bath, and the cryopreserved liquid was removed by centrifugation at 850 rpm for 3 min. M199 medium was used to resuspend RyuF-2 cells and supplemented with 10% heat-inactivated FBS and penicillin/streptomycin (50 U/ml/50 mg/ml). The suspension cells are adjusted to a density of 5 by the culture medium and then seeded on a 75-cm² culture flask/cm² coated with collagen. The cultured flask was placed in a humidified incubator at 27°C for culture. Before subculturing, the cells were grown to 80% confluence.

The virus was saved in normal M199 medium with 2% FBS (2% medium). After CyHV-2 was taken out from −150°C, the titer was quantified by gradient dilution counting. The 2% medium dilution was used to adjust the virus concentration until the multiplicity of infection (MOI) = 1. To infect the RyuF-2 cells with CyHV-2, the supernatant of the cells was removed, and viral inoculum was added immediately for an incubation time of 2 h. After that, the inoculum was replaced with 2% medium. Infected cells were cultured until CPE was developed in 80% of the cells. The remaining cells were scraped off with a spatula, the suspension was collected, and the cells and cell debris were removed by centrifugation at 12,000 g at 4°C for 20 min. The supernatant was filtered with a 0.22-μm filter to collect the virus. The harvested virus was equally divided and placed at −80°C for subsequent experiments. Before the experiment, the gradient dilution method was used to detect the virus titer again, and the virus concentration was adjusted according to the experimental cell density so that MOI = 1. CyHV-2 virus with a known titer was used to infect RyuF-2, which has a density of about 80%. After incubating for 2 h, the virus solution was removed, and the time point was counted as the start of infection 0 h post-infection (p.i.). The cultures were maintained for up to 96 h and were used between 0 and 96 h in culture.

Berberine hydrochloride and EGCG were separately dissolved in DMSO to prepare a 1-mg/ml preservation solution. RyuF-2 cells were used for experiments when their cell density was close to 80%. To adjust the concentration of BBH storage solution to 15, 20, and 25 μg/ml, 2% M199 medium was used. EGCG storage solution was diluted to 10, 20, and 30 μg/ml in the same way. The pretreatment was performed on RyuF-2 cells with different concentrations of drugs for 30 min. The drug treatment group kept the drug concentration unchanged and continued to incubate. In the virus infection group, the supernatant was removed and replaced with a virus solution, keeping the drug concentration unchanged. After incubating for 2 h, the virus solution was removed and 2% M199 medium was added with the same drug concentration. The cultures were maintained for up to 72 h and were used between 0 and 72 h in culture.

The intracellular ROS levels were estimated using a DCFDA/H2DCFDA cellular ROS assay kit according to a classic method (Heo et al., 2018). Briefly, the RyuF-2 cells were seeded into 96-well microtiter plates at 2.5 × 10⁴ cells/well and were allowed to attach for 24 h at 27°C, and 100 μl of 1 × PBS buffer was added. The cells were then washed with 1 × PBS buffer. The 1 × PBS buffer was removed, and the cells were stained by adding 50 μl of the diluted DCFDA solution. Then, the cells were incubated for 45 min at 37°C in the dark. The fluorescence intensity was measured using a fluorescence microtiter ELISA plate reader at excitation and emission wavelengths of 485 and 535 nm, respectively. For cells infected with CyHV-2, cells were collected at 6, 12, 24, 36, 48, 60, 72, and 96 h after infection, and the intracellular ROS levels were quantified using the DCFDA/H2DCFDA cell ROS detection kit. For CyHV-2-infected cells in the presence of 15, 20, and 25 μg/ml BBR or 10, 20, and 30 μg/ml EGCG, the cells were harvested at 24 and 48 h p.i., and the intracellular ROS levels were quantitated using a DCFDA/H2DCFDA cellular ROS assay kit.

The supernatant of infected cells was obtained at different time points after infection for total DNA extraction. Viral DNA was extracted from the supernatant according to the instructions of the Viral Genomic DNA Extraction Kit (Qiagen). The total RNA of the RyuF-2 sample was extracted with 1 ml TRIzol reagent and purified using phenol/chloroform and ethanol precipitation. The NanoDrop Technologies instrument is used to quantify RNA. Each group of samples used 5 μg of RNA as the sample to generate cDNA for real-time quantitative qPCR, which was achieved by the corresponding reverse transcription reaction of PrimeScript RT Master Mix reagent. TB Green Premix Ex Taq II (2x) was used to amplify 1 μl of the resulting cDNA. According to the gene sequence obtained by the analysis of the transcriptome results in the laboratory, Primer Premier 5.1 (Table 1) was used to design a specific primer set for each open reading frame (ORF). The reaction contained 12.5 μl TB Green Premix Ex Taq II (2x) (Takara, Japan), 0.4 μM forward/reverse primers, and 200 ng cDNA. The thermal cycle program included the following: 95°C for 30 s, then 95°C for 5 s and 59°C for 30 min for 39 cycles. The qPCR reaction was repeated three times. In order to construct a standard curve, the DNA extracted from RyuF-2 cells was used for continuous decimal dilution. Primers from β-actin were successfully used in qPCR, using cDNA prepared from infected and uninfected cells as a template. A one-way analysis of variance (ANOVA) was used to calculate statistical significance. Origin 9.0 was used for data analysis and graphing. Values were considered as significant (*) if they had a p-value of 0.01 to 0.05, very significant (^**) if they had a p-value of 0.001 to 0.01, and extremely significant (^***) if they had a p-value of 0.0001 to 0.001.

Cell samples were collected to extract total protein by boiling with 2X SDS-PAGE protein loading buffer. Thermo Scientific Pierce BCA protein detection kit was used to quantify protein concentration. The protein (20 g) was separated on 10–12% SDS-PAGE and then transferred to PVDF membrane. The membrane was blocked in 1% TBS-T buffer containing 5% skim milk for 1 h at room temperature and then incubated overnight with homemade anti-ORF121 polyclonal antibody (Yu et al., 2019) (1:200 dilution). After washing the membrane with TBS-T three times, horseradish peroxidase-conjugated secondary antibody (diluted 1:1,000) was added and incubated for 2 h at room temperature and then washed with TBS-T three times for 15 min. A chemiluminescence imaging system (Bio-Rad, China) was used to detect the signal with the ECL + western blot kit.

In a recent study to probe host response to viral infection, we identified the differential expression genes in crucian carp cells underlying CyHV-2 infection by transcriptomics analysis (Fei et al., 2020). Primary data analysis of genes involved in the Keap1-Nrf2 pathway indicated that the TPM (transcripts per kilobase million) values of Nrf2, CAT, and SOD2 genes in CyHV-2-infected cells at 48 h post-infection were dramatically higher than those in the negative control (NC), and the values of Keap 1 and GSS were also shown to be slightly higher than those of the control (Figure 1). In order to verify the effect of CyHV-2 infection on the expression of related genes in the Keap1-Nrf2 pathway, we used quantitative real-time RT-PCR analysis to compare the transcriptional expression levels of Keap1, Nrf2, CAT, GSS, and SOD2 in normal cells and CyHV-2-infected cells (Figure 2). At 24, 48, and 72 h after infection with CyHV-2, RyuF-2 cells were collected for total RNA extraction. All qRT-PCR assays were performed in triplicate, with β-actin used as an endogenous control. As shown in Figure 2, CyHV-2 significantly up-regulates all tested genes to higher levels compared to the corresponding low steady-state levels in the control cells. Specifically, the highest induction rate was recorded for Nrf2 and GSS genes at 48 h p.i. (Figures 2B,E). In general, 48 h p.i. seemed to be a time point for all the genes to be upregulated to a peak level.

The Keap1-Nrf2 pathway regulates both mitochondrial and cytosolic ROS production (Kovac et al., 2015); thus, the above data predicted that CyHV-2 infection might interfere with ROS synthesis and activate host oxidative stress response. To clarify whether CyHV-2 infection disturbed the host cellular redox balance, we monitored the ROS level following CyHV-2 infection using DCFDA/H2DCFDA cellular ROS assay. Following CyHV-2 infection at a MOI = 1, the ROS level in RyuF-2 cells was dramatically induced to a higher level with maximum ROS accumulation at 24 h p.i. and then gradually reversed to a constant and lower level (Figure 3A). Successful replication of CyHV-2 in RyuF-2 cells was reflected by the titration of progeny virus from the virus supernatant after infection by quantitating the genome copy number using a real-time polymerase chain reaction (PCR) analysis. As shown in Figure 3B, the produced CyHV-2 in supernatant significantly rose to a higher level of more than 10⁷ genome copy number/ml since 24 h p.i. and then gradually increased until late infection of 96 h p.i. The experiments indicated that higher ROS accumulation was only induced at early phase of CyHV-2 infection, and the high level of ROS was reversed during late infection.

Epigallocatechin-3-gallate, the most abundant polyphenolic substance in green tea, typically plays a critical role in free radical scavenging owing to its antioxidant nature and subsequently exerts a large number of medical activities in cancer control, viral diseases, bacterial diseases, neurodegenerative diseases, etc. (Xu et al., 2017). Thus, we were interested to test the effect of EGCG on the CyHV-2-mediated upregulation of Keap1-Nrf2 pathway. For this purpose, RyuF-2 cells were infected with CyHV-2 at a MOI = 1 in the presence of 10 μg/ml EGCG. At 24, 48, and 72 h post-infection, the infected cells were harvested for total RNA extraction and subjected to gene expression assay by quantitative real-time RT-PCR analysis. MOCK-infected cells treated with 10 μg/ml EGCG served as negative control, and CyHV-2-infected cells without EGCG treatment served as positive control. As shown in Figure 4, the treatment of MOCK-infected cells with EGCG could result in an increased expression of Keap1, Nrf2, GSS, and CAT gene; in comparison to CyHV-2-infected cell controls, the treatment of CyHV-2-infected cells with EGCG could result in a significant increase of Nrf2 at 48 h p.i., Keap 1 at 72 h p.i., GSS at all three time points, and CAT at 72 h p.i. Thus, EGCG could enhance the expression of key genes in the Keap1-Nrf2 pathway except for SOD gene, which was independent of CyHV-2 infection.

Affecting various signaling pathways, berberine has been shown to decrease the activity of xanthine oxidase, COX2, and superoxide dismutase and subsequently reduce ROS levels (Kaboli et al., 2014). As an alternative antioxidant, BBR was subjected for analysis on its effect on the CyHV-2-mediated upregulation of the Keap1-Nrf2 pathway. For this purpose, RyuF-2 cells were infected with CyHV-2 at a MOI = 1 in the presence of 20 μg/ml BBH. At 24, 48, and 72 h post-infection, the infected cells were harvested for total RNA extraction and subjected to gene expression assay by quantitative real-time RT-PCR analysis. MOCK-infected cells treated with 20 μg/ml BBH served as negative control, and CyHV-2 infected cells without BBH treatment served as positive control. As shown in Figure 5, in contrast to MOCK-infected cells treated with BBH, CyHV-2 infection resulted in an increased expression of all tested genes; compared with CyHV-2-infected control cells, the treatment of CyHV-2-infected cells with BBH could result in a significant increase of Nrf2, Keap 1, and GSS at 48 h p.i. The gene expression level was not affected at 48 h p.i. for CAT and at 72 h p.i. for SOD. The results even suggested that these two genes might be downregulated at other time points (Figures 5D,E). Overall, the above experiments suggested that antioxidants, such as EGCG and BBH, could efficiently activate the Keap1-Nrf2 pathway that was induced to a higher level by CyHV-2 challenge in RyuF-2 cells.

The above experiments indicated that CyHV-2 modulated ROS level in infected cells and could induce dramatic ROS accumulation during early infection. We are interested to know what will happen if the ROS accumulation is inhibited by antioxidants such as BBR and EGCG during CyHV-2 infection. RyuF-2 cells were treated with various doses of BBH (0, 15, 20, and 25 μg/ml BBH) or EGCG (0, 10, 20, and 30 μg/ml EGCG) and subjected to CyHV-2 infection at a MOI = 1. At 24 and 48 h post-infection, the cells were harvested for DCFDA/H2DCFDA cellular ROS assay, in which the ROS level was reflected by the fluorescence strength detected from the cellular extract. The NC group referred to normal cells without antioxidant treatment and viral infection. Cells infected with CyHV-2 and treated with 0 μg/ml antioxidant served as positive control (CyHV-2 group). As shown in Figure 6, both BBH and EGCG effectively counteracted the accumulation of ROS induced by CyHV-2 infection at 24 and 48 h post-infection. In contrast to the dramatic increase of ROS level at indicated time points post-infection, treatment with BBH (Figure 6A) or EGCG (Figure 6B) resulted in low ROS accumulation in CyHV-2-infected cells, which was close to the level of the NC group.

The above experiments indicated that ROS accumulation was efficiently inhibited by BBH or EGCG during CyHV-2 infection. Then, it will be interesting to know the effect of these antioxidants on CyHV-2 replication. For this purpose, we determined the progeny virus in the supernatants of infected cells by quantifying the viral genome copy number through real-time PCR analysis. The antiviral activity of antioxidants in vitro was evaluated according to the reduced level of progeny virus produced from CyHV-2-infected RyuF-2 cells in the presence of different concentrations of EGCG or BBH. The supernatants of infected RyuF-2 cells, in the presence of BBH at 0, 15, 20, and 25 μg/ml or EGCG at 0, 10, 20, and 30 μg/ml, were collected at 8, 24, and 48 h post-infection for total viral genome extraction and quantification. As shown in Figure 7, both BBH and EGCG dose-dependently suppressed the production of progeny virus from infected cells, and the overall efficiency of these two antioxidants was similar at 48 h post-infection. At 8 h post-infection, the treatment of EGCG (Figure 7B) displayed a lower level of CyHV-2 (Figure 7A) in supernatants than that of BBH, suggesting a more robust effect of EGCG in antagonizing viral replication at this time point.

To confirm the antiviral effects of BBH and EGCG, we continued to investigate the transcriptional and translational expression levels of ORF121, an immediately early gene encoded by CyHV-2 (Tang et al., 2020). The infected RyuF-2 cells in the presence of 20 μg/ml BBH or 10 μg/ml EGCG were harvested at 24, 48, and 72 h post-infection, and total protein samples were extracted from the infected cells for ORF121 detection by western blot analysis; total RNA samples were extracted from the infected cells for ORF121 mRNA detection by real-time RT-PCR analysis. As shown in Figure 8A, the RyuF-2 cells infected with CyHV-2 resulted in robust transcriptional expression of ORF121, which lasted until 72 h post-infection; treatment with either BBH or EGCG significantly suppressed the transcriptional expression of ORF121 throughout the infection course. As shown in Figures 8B,C, in contrast to the strong signal of ORF121 protein from CyHV-2-infected RyuF-2 cells in the immunoblot analysis, the treatment of BBH or EGCG at indicated concentrations demonstrated a dramatic decrease of protein signal, which indicated that either BBH or EGCG was effective in inhibiting viral protein synthesis.

Thus, the above experiments suggested that BBH and EGCG could significantly block CyHV-2-induced ROS accumulation, inhibit viral gene transcription and protein synthesis, and reduce the production of progeny virus.