Human lung adenocarcinoma A549 cells (cat. no. CCL-185), human neuroblastoma SH-SY5Y cells (cat. no. CRL-2266), African green monkey kidney Vero cells (cat. no. CCL-81) and porcine kidney PK-15 cells (cat. no. CCL-33) were kindly provided by Professor Rong Ye (School of Basic Medical Sciences, Fudan University, Shanghai, China) and were originally sourced from ATCC. All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc.), 1% L-glutamine and 1% non-essential amino acid solution, 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C in a humidified atmosphere of 5% CO₂. Unless otherwise stated, the products for cell culture were from Invitrogen (Thermo Fisher Scientific, Inc.).

TBEV (provided by Professor Rui-Wen Ren; Center for Disease Control and Prevention of Southern Theater Command, Guangzhou, China) propagation in Vero cells was previously described (26). Vero cells seeded in a T-75 culture flask were cultured and reached 90% confluency. The medium was then replaced with a mixture of 1 ml TBEV stock and 5 ml culture medium and allowed to proceed for 2-h adsorption at 37°C. After the adsorption, fresh culture medium was added to the cells, followed by an additional 72-h incubation at 37°C. The culture supernatants were collected at 72 h post incubation and centrifuged at 1,000 × g for 10 min to remove cell debris at room temperature. TBEV stock obtained was frozen in aliquots at -80°C until use. Titers of TBEV were determined by plaque assay with PK-15 cells before use. Infection experiments with TBEV were conducted in the Biological Safety Level 3 Laboratory in accordance with the guidelines by the Committee on Safety of Biomedicine at Naval Medical University (Shanghai, China).

Plaque assay was performed to determine TBEV titers in the culture supernatants collected from TBEV-infected cells with and without cepharanthine treatment. PK-15 cells were cultured overnight in 12-well culture plates to form a confluent monolayer. Serial ten-fold dilutions of the culture supernatants were prepared and added to the confluent monolayer of PK-15 cells (three wells per dilution). After 3 h of adsorption at 37°C, the viral inoculum was removed. The cells were washed once with phosphate-buffered saline (PBS) and incubated with 2% carboxymethylcellulose sodium (cat. no. SLBN8656V; Sigma-Aldrich; Merck KGaA) overlay medium. Following an incubation period of 6 days, the cells were washed once with PBS and fixed for 15 min with 4% paraformaldehyde at room temperature. The cells were then stained with 1% crystal violet for 15 min at room temperature. Plaques formed were counted and the plaque-forming unit (PFU) was calculated. The virus titers are expressed as PFU/ml.

Cepharanthine (cat. no. S4238; Selleck Chemicals) was dissolved in dimethyl sulfoxide (DMSO; cat. no. D2650; Sigma-Aldrich; Merck KGaA) to yield a stock solution of 10 mM. Cytotoxicity of cepharanthine in A549, SH-SY5Y and Vero cells was assessed. Cells seeded into 96-well culture plates were cultured overnight to form a confluent monolayer. After removal of the culture medium, the cells were treated for 48 h at 37°C with fresh culture medium containing cepharanthine at increasing concentrations ranging from 10 to 60 μM (experiment group). The cells maintained in fresh culture medium were used as the control group. DMSO was added to the cell monolayer at a final concentration of 0.1% as the dissolvent group. The culture medium was directly added to cell-free wells as a blank group. All the groups were examined in three separate wells. Cell viability was determined using CellTiter 96^® AQueous One Solution Cell Proliferation Assay Kit containing MTS (cat. no. G3582; Promega Corporation) according to the manufacturer's protocol. The optical density (OD) was measured at 490 nm on a Synergy 2 Multi-Mode Microplate Reader (BioTek; Agilent Technologies, Inc.). Cell viability was determined using the following formula: (OD_{experiment group/dissolvent group}-OD_{blank group}/OD_{control group}-OD_{blank group}) ×100%.

Cells were seeded into 12-well culture plates and cultured overnight to form a confluent monolayer. The cells were then infected with TBEV at a multiplicity of infection (MOI) of 0.1 and allowed to proceed for 2 h adsorption at 37°C. Following the adsorption, unbound virus was removed by washing with PBS. The cells were subsequently maintained in fresh culture medium for 48 h. The time point for cepharanthine addition experiments was measured from the end of the 2 h adsorption. Cells were infected with TBEV at an MOI of 0.1 throughout the present study.

In total, three cepharanthine addition experiments were adopted to treat A549, SH-SY5Y and Vero cells. Confluent monolayer cells in 12-well culture plates were treated with cepharanthine at increasing concentrations (5, 10 and 20 μM; three wells per concentration). For co-treatment of cepharanthine, the cells were maintained in fresh culture medium containing TBEV and cepharanthine for 48 h. For pre-treatment of cepharanthine, the cells were pre-treated with cepharanthine in fresh culture medium for 12 h, washed once with PBS and incubated for 2 h with TBEV. Following adsorption for 2 h, the cells were washed once with PBS and cultured with fresh culture medium for another 48 h. For post-treatment with cepharanthine, the cells were incubated for 2 h with TBEV. After removal of the viral inoculum, the cells were washed once with PBS and cultured in fresh culture medium containing cepharanthine for 48 h. Cells without TBEV infection served as a mock control (Mock; three wells). Cells infected with TBEV served as an untreated control (0 μM; three wells). TBEV-infected cells were treated with DMSO at a final concentration of 0.1% as a dissolvent control (DMSO; three wells).

A549 cells cultured in 96-well culture plates were treated with cepharanthine at increasing concentrations (10 and 20 μM; three wells per concentration) and TBEV. Cells just infected with TBEV served as an untreated control (0 μM cepharanthine; three wells). TBEV-infected A549 cells treated with 0.1% DMSO served as the dissolvent control (DMSO; three wells). Following a 48 h incubation, the cells were subjected to immunofluorescence staining for TBEV-positive cells as described previously (26). Briefly, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature, washed with PBS and permeabilized with methanol for 20 min at -20°C. After washing with PBS, blocking was achieved with 3% bovine serum albumin (BSA; lot. no. 4255715; Affymetrix Inc.) for 2 h at room temperature. The cells were incubated with formaldehyde-inactivated TBEV immunized mouse ascites recognizing the viral surface antigen (1:500; provided by Professor Rui-Wen Ren) overnight at 4°C, washed with PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse antibody (1:1,000; cat. no. ab150113; Abcam) for 1 h at room temperature. The cells were washed with PBS and subsequently treated with mounting medium with 4',6'-diamidino-2-phenylindole (cat. no. ab104139; Abcam) for 5 min at room temperature to visualize cell nuclei. The cells were counted and analyzed with Gen5 3.10 software (BioTek; Agilent Technologies, Inc.) by a Cytation 5 imaging reader (BioTek; Agilent Technologies, Inc.). Finally, the cells were imaged under a fluorescence microscope (Olympus Corporation).

Culture supernatants from TBEV-infected A549 and SH-SY5Y cells with and without cepharanthine treatment were collected and tested for cytokine production. ELISA was conducted with Quantikine ELISA kits (R&D Systems China Co., Ltd.) for human tumor necrosis factor-α (TNF-α; cat. no. VAL105), human IL-1β (cat. no. VAL101), human C reactive protein (CRP; cat. no. VAL120) or human IL-11 (cat. no. D1100) following the manufacturer's protocol. The OD was measured at 450 nm on the Synergy 2 Multi-Mode Microplate Reader. Cytokine concentrations were calculated based on standard curves generated for each assay with Sigma Plot version 10.0 software (Systat; Grafiti LLC) and are shown as pg/ml.

For extraction of total RNA, cells were washed once with ice-cold PBS and RNA was extracted using TRIzol^® Reagent (cat. no. 15596026; Thermo Fisher Scientific, Inc.). The concentration and purity of the extracted RNA were examined using the Synergy 2 Multi-Mode Microplate Reader. Subsequently, cDNA was synthesized from the RNA with moloney murine leukemia virus reverse transcriptase (cat. no. M1705; Promega Corporation) and dNTP mix (cat. no. C1141S; Promega Corporation) following the manufacturer's protocol. qPCR was carried out to detect gene expression of TBEV and C/EBP homologous protein (CHOP). The reaction was performed in a Rotor-Gene 3000 Thermal Cycler (Corbett Life Science) in volumes of 20 μl containing 10 μl SYBR Green 2X PCR Master Mix (cat. no. LS2062; Promega Corporation), 2 μl cDNA template, 0.4 μl of each primer (10 μM) and 7.2 μl nuclease-free water. The thermocycler conditions for qPCR were as follows: An initial denaturation at 2 min at 95°C and 40 cycles of 10 sec at 95°C, 10 sec at 55°C and 25 sec at 72°C. Expression of genes was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of TBEV RNA and CHOP mRNA were quantified compared with the relative control by the threshold cycle (2^−ΔΔCq) method (27). The primer sequences for CHOP were as follows: 5'-AGCTGGAACCTGAGGAGAGA-3' (forward) and 5'-TGGATCAGTCTGGAAAAGCA-3' (reverse). The primer sequences for TBEV and GAPDH were described previously (26).

For total protein extracts, cells were washed once with ice-cold PBS and lysed in blue loading buffer containing dithiothreitol (cat. no. 7722; Cell Signaling Technology, Inc.) according to the manufacturer's protocol. Protein samples were prepared and total protein concentration was determined by BCA protein assay. Equal amounts of protein samples (30 μg/lane) were separated by SDS-PAGE on 12% gels and transferred onto a polyvinylidene difluoride membrane (cat. no. 1620177; Bio-Rad Laboratories, Inc.). The membranes were blocked for 2 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) with 5% non-fat milk powder. Mouse monoclonal antibody for CHOP (cat. no. 2895) and rabbit antibodies specific for eukaryotic initiation factor 2α (eIF2α; cat. no. 5324), phospho-eIF2α (cat. no. 3398), β-actin (cat. no. 4967) or GAPDH (cat. no. 5174) were used for immunoblotting overnight at 4°C. All primary antibodies (1:1,000; Cell Signaling Technology, Inc.) were diluted with 5% BSA in TBST. After three washes with TBST, the membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse (1:2,000; cat. no. 1706516; Bio-Rad Laboratories, Inc.) or goat anti-rabbit secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) at room temperature. The membranes were washed three times with TBST and the bound antibodies were detected with Clarity Western ECL Substrate (cat. no. 1705060; Bio-Rad Laboratories, Inc.) on a GeneGnome HR image capture. The bands were quantified by densitometric analysis with GeneTools version 4.01.02 software (SynGene; Synoptics Ltd.).

All data are presented as the mean ± standard deviation of three independent experiments. Group comparisons were conducted using one-way ANOVA followed by Tukey's post hoc test with GraphPad Prism software (version 8.0; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

Human lung adenocarcinoma A549 cells are highly susceptible to TBEV and are therefore used to characterize strains of TBEV (28). Some compounds have been evaluated for their antiviral activities against TBEV in A549 cells (29). Human neuroblastoma SH-SY5Y cells have been used to explore the replication and pathogenesis of the Zika virus, which is a major pathogen of human neurological disorders (30). Food Drug Administration-approved bioactive small molecules have been screened for their abilities to inhibit the death of SH-SY5Y cells infected with La Crosse virus, which is a mosquito-borne Orthobunyavirus that leads to viral encephalitis (31). In the present study, the effects of cepharanthine on TBEV propagation and pathogenicity were assessed using both A549 and SH-SY5Y cells.

Time-of-drug addition experiments were adopted to explore which step(s) of the viral life cycle may be disturbed by cepharanthine (32). Anti-TBEV activity of polyphenol complex isolated from Zosteraceae seagrass (main components containing rosmarinic acid, luteolin disulfate and bioflavonoids) has been characterized using several versions of the addition experiments (33). In the present study, three schemes including co-treatment, pre-treatment and post-treatment with cepharanthine were adopted for evaluating anti-TBEV efficacy. A schematic presentation of the co-treatment and pre-treatment cepharanthine experiments is shown in Fig. 1A. First, the co-treatment and pre-treatment experiments were carried out to investigate the anti-TBEV efficacy of cepharanthine. TBEV RNA replication, viral protein expression and virus propagation were synchronously detected in cells following cepharanthine treatment.

It was necessary to analyze the cytotoxicity of cepharanthine prior to determining its antiviral efficacy. For this, A549 and SH-SY5Y cells were treated with increasing concentrations of cepharanthine. MTS assay showed that cepharanthine was not cytotoxic to A549 cells at concentrations of up to 30 μM (Fig. 1B). A significant reduction in A549 cell viability was observed upon treatment with cepharanthine at concentrations of 40, 50 and 60 μM (P<0.05 or P<0.001). As for SH-SY5Y cells, cepharanthine did not exert a cytotoxic effect at concentrations of up to 20 μM. Cepharanthine at concentrations ranging from 30 to 60 μM significantly decreased SH-SY5Y cell viability (P<0.01 or P<0.001). There was no significant difference in the cell viability between the DMSO dissolvent and the untreated control groups. The maximal concentration of 20 μM of cepharanthine was thereby selected for evaluation of its antiviral efficacy.

Whether co-treatment or pre-treatment with cepharanthine displays antiviral efficacy against TBEV was next assessed. The cellular RNA obtained from the infected cells treated with cepharanthine at the increasing concentrations was analyzed for TBEV RNA expression. RT-qPCR analysis showed that TBEV RNA replication was inhibited by cepharanthine in a concentration-dependent manner (Fig. 1C). The TBEV RNA levels were significantly reduced in the infected A549 cells with co-treatment or pre-treatment with 20 μM cepharanthine (P<0.05 or P<0.01). There was no notable reduction in TBEV RNA levels in the infected A549 cells treated with the DMSO dissolvent. Co-treatment or pre-treatment with cepharanthine also inhibited TBEV RNA replication in SH-SY5Y cells (Fig. 1D). For co-treatment, the TBEV RNA levels were significantly reduced in SH-SY5Y cells treated with cepharanthine at 5, 10 or 20 μM (P<0.05 or P<0.001). For pre-treatment, a significant reduction in TBEV RNA levels was observable in SH-SY5Y cells treated with the cepharanthine at 5, 10 and 20 μM (P<0.001 or P<0.0001, respectively). While the TBEV RNA levels were significantly decreased in SH-SY5Y cells treated with the DMSO dissolvent (P<0.05; Fig. 1D, pre-treatment) compared with the untreated group, the cepharanthine at 5, 10 and 20 μM significantly inhibited the viral RNA replication compared with the DMSO dissolvent (P<0.01 or P<0.001). In summary, co-treatment or pre-treatment with cepharanthine displayed a potent inhibitory effect on TBEV RNA replication.

Next, immunofluorescence staining was performed to detect TBEV protein expression in infected A549 cells with cepharanthine co-treatment. As shown in Fig. 2, expression of TBEV protein was detectable in TBEV-infected A549 cells and that treatment with cepharanthine at 20 μM effectively inhibited such expression. TBEV protein expression was undetectable in the mock group. The DMSO group showed a minor influence on viral protein expression. In parallel with the marked inhibition of TBEV RNA replication by cepharanthine at 20 μM (Fig. 1C, co-treatment), the percentage of TBEV-positive cells was significantly decreased upon cepharanthine treatment (P<0.001). These results demonstrated that TBEV RNA replication and protein expression were inhibited by cepharanthine.

The antiviral efficacy of cepharanthine was further confirmed by plaque assay to detect TBEV titers in the culture supernatants obtained from cells treated with cepharanthine via different schemes. The virus titers were significantly decreased in infected A549 cells co-treated with cepharanthine at 20 μM (P<0.05) or pre-treated with cepharanthine at 10 and 20 μM (P<0.05 or P<0.01; Fig. 3A). In SH-SY5Y cells, the TBEV titers were also significantly decreased upon co-treatment with cepharanthine at 10 and 20 μM (P<0.001) or pre-treatment with cepharanthine at concentrations ranging from 5 to 20 μM (P<0.01) (Fig. 3B). The DMSO group showed no or a minor inhibitory effect on the virus titers, suggesting that the inhibition of TBEV propagation was attributed to the cepharanthine treatment. These results were consistent with the significant inhibition of TBEV RNA replication in A549 and SH-SY5Y cells co-treated or pre-treated with cepharanthine (Fig. 1C and D).

In addition to the co-treatment and pre-treatment schemes, post-treatment of cepharanthine was also adopted for evaluating antiviral efficacy. A schematic presentation of cepharanthine post-treatment is shown in Fig. 4A. TBEV titers were not influenced in the A549 cells with cepharanthine post-treatment (Fig. 4C). Furthermore, whether cepharanthine exhibited antiviral efficacy against TBEV was analyzed using Vero cells. Cepharanthine at concentrations ranging from 20 to 60 μM exerted a marked cytotoxic effect on Vero cells, implying variable cytotoxicity of cepharanthine among different cell types (Fig. 4B). Vero cells were thus treated with cepharanthine at 5 and 10 μM for subsequent assays. Plaque assays showed that TBEV titers were markedly decreased upon co-treatment with cepharanthine at 10 μM (P<0.05) and that a significant reduction in the virus titers was observed in Vero cells with cepharanthine pre-treatment at 5 and 10 μM (P<0.01 or P<0.001) (Fig. 4D). Conversely, post-treatment with cepharanthine did not lead to a notable reduction in TBEV titers.

In A549, SH-SY5Y and Vero cells, TBEV propagation was inhibited by cepharanthine co-treatment and pre-treatment. This characterization allowed for further examination of the mechanisms underlying the effects of cepharanthine on TBEV propagation and pathogenicity based on the co-treatment and pre-treatment schemes.

Inflammatory cytokines are closely linked to severe inflammatory symptoms during TBEV infection. In mouse brain and human neural cells, TBEV is responsible for the elevated production of a variety of cytokines and chemokines that cause pathological neuroinflammation (34). In the present study, the influence of cepharanthine on the TBEV-induced inflammatory response was evaluated. ELISA was carried out to detect the production of inflammatory cytokines in the culture supernatants obtained from infected cells treated with the cepharanthine. The levels of TNF-α, IL-1β and IL-11 were notably increased in TBEV-infected A549 cells compared with the mock group (P<0.01 or P<0.001; Fig. 5A and B). The co-treatment with cepharanthine at 10 and 20 μM led to a significant reduction in the production of TNF-α, IL-1β and IL-11 induced by TBEV (P<0.05, P<0.01 or P<0.001; Fig. 5A). The amounts of TNF-α, IL-1β and IL-11 induced by TBEV were significantly decreased in A549 cells pre-treated with cepharanthine at 10 and 20 μM (P<0.001; Fig. 5B). DMSO dissolvent showed no notable inhibitory effects on the production of these cytokines. The production of TNF-α, IL-1β and IL-11 induced by TBEV was reduced by co-treatment or pre-treatment with cepharanthine.

CRP is an acute inflammatory protein that plays important roles in inflammation processes and the host response to pathogen infection (35). However, little is known about the contribution of TBEV infection to CRP release. Therefore, whether CRP release is associated with TBEV infection was assessed. As shown in Fig. 6A, TBEV infection showed little influence on the levels of CRP in A549 and SH-SY5Y cells and co-treatment with cepharanthine had a minor effect on the CRP levels, indicating that CRP was not likely involved in the inflammatory response to TBEV infection. Similarly, the release of TNF-α and IL-1β was not notably affected in the TBEV-infected SH-SY5Y cells (Fig. 6B), suggesting differential regulation of TNF-α and IL-1β by TBEV in a cell type-dependent manner. There were no notable changes in the production of TNF-α and IL-1β in infected SH-SY5Y cells co-treated with cepharanthine. Based on these data, the influence of cepharanthine pre-treatment on the release of CRP, TNF-α and IL-1β was not further analyzed in TBEV-infected SH-SY5Y cells. However, TBEV infection markedly enhanced IL-11 release from SH-SY5Y cells and a significant reduction in the IL-11 production induced by TBEV was observed upon co-treatment with cepharanthine at 10 and 20 μM (P<0.01 or P<0.001; Fig. 6C). Pre-treatment with cepharanthine at 20 μM significantly reduced IL-11 production in infected SH-SY5Y cells (P<0.05; Fig. 6D). Notably, 10 μM cepharanthine resulted in a significant increase in IL-11 production (P<0.05), implying that IL-11 management may be related to the cell type and cepharanthine concentration. Inhibitory effects of the DMSO dissolvent on IL-11 release were ruled out. These results suggested that co-treatment and pre-treatment with cepharanthine exhibited differential inhibitory effects on the TNF-α, IL-1β and IL-11 production induced by TBEV.

CHOP, a major component of ER stress-induced apoptosis, is implicated in the progression of human diseases as well as in microbial infection processes (36). Host cells maintain ER homeostasis by triggering the defense response to viral infection, which in turn impacts viral survival and replication. Fig. 7A showed that CHOP mRNA expression was significantly upregulated in TBEV-infected A549 cells (P<0.001). Similarly, significant upregulation of CHOP mRNA expression was observed in infected SH-SY5Y cells (P<0.01; Fig. 7B). In infected A549 cells, the CHOP mRNA levels were significantly decreased upon co-treatment (20 μM; P<0.01) or pre-treatment (10 and 20 μM; P<0.05) with cepharanthine (Fig. 7A). In infected SH-SY5Y cells, co-treatment as well as pre-treatment with cepharanthine also led to a significant decrease in the CHOP mRNA levels (P<0.05; Fig. 7B). The DMSO dissolvent showed a minor inhibitory effect on the CHOP mRNA expression.

Moreover, the expression of CHOP was verified at the protein level. Western blot analysis showed that TBEV infection markedly induced CHOP expression in A549 cells and that co-treatment (20 μM) or pre-treatment (10 μM) with cepharanthine significantly reduced such an induction (P<0.05; Fig. 7C). The CHOP protein levels were increased in infected SH-SY5Y cells and these levels were decreased upon treatment with cepharanthine (Fig. 7D), but these results were not statistically significant. Pre-treatment with cepharanthine displayed an inhibitory effect on CHOP expression in A549 and SH-SY5Y cells. Thus, co-treatment and pre-treatment with cepharanthine effectively downregulated TBEV-induced CHOP expression.

eIF2α plays pivotal roles in stress response and eIF2α/ATF4/CHOP signaling is involved in viral infection (37,38). Since CHOP expression was upregulated in TBEV infected cells, a potential association between TBEV infection and eIF2α phosphorylation was investigated, together with an evaluation of the effects of cepharanthine on eIF2α phosphorylation. Western blotting was performed to measure the total and phosphorylated levels of eIF2α in the cell lysates. In parallel with the upregulation of CHOP expression (Fig. 7), the levels of phosphorylated eIF2α were increased in TBEV-infected A549 and SH-SY5Y cells. Compared with levels of phosphorylated eIF2α in the mock group, TBEV infection resulted in a notable (co-treatment) or minor (pre-treatment) elevation in eIF2α phosphorylation in the A549 cells and a marked elevation in the SH-SY5Y cells (Fig. 8A and B), suggesting the phosphorylation and activation of eIF2α induced by TBEV. The levels of phosphorylated eIF2α were significantly increased in the A549 and SH-SY5Y cells co-treated or pre-treated with cepharanthine at 20 μM (P<0.05 or P<0.01; Fig. 8A and B). DMSO dissolvent did not exert enhancement of eIF2α phosphorylation in the infected cells. Treatment with cepharanthine via the two schemes enhanced eIF2α phosphorylation during TBEV infection.

Taken together, co-treatment or pre-treatment with cepharanthine displayed an anti-TBEV effect in A549 and SH-SY5Y cells. The management of eIF2α phosphorylation by cepharanthine may contribute to the antiviral effect against TBEV.