Novel antiviral discoveries for Japanese encephalitis virus infections through reporter virus‐based high‐throughput screening

Japanese encephalitis (JE) caused by JE virus (JEV), remains a global public health concern. Currently, there is no specific antiviral drug approved for the treatment of JE. While vaccines are available for prevention, they may not cover all at‐risk populations. This underscores the urgent need for prophylaxis and potent anti‐JEV drugs. In this context, a high‐content JEV reporter system expressing Nanoluciferase (Nluc) was developed and utilized for a high‐throughput screening (HTS) of a commercial antiviral library to identify potential JEV drug candidates. Remarkably, this screening process led to the discovery of five drugs with outstanding antiviral activity. Further mechanism of action analysis revealed that cepharanthine, an old clinically approved drug, directly inhibited virus replication by blocking GTP binding to the JEV RNA‐dependent RNA polymerase. Additionally, treatment with cepharanthine in mice models alleviated JEV infection. These findings warrant further investigation into the potential anti‐JEV activity of cepharanthine as a new therapeutic approach for the treatment of JEV infection. The HTS method employed here proves to be an accurate and convenient approach that facilitates the rapid development of antiviral drugs.

[13] The development of robust antiviral assays has significantly aided the drug discovery process.High-throughput screening (HTS) enables the rapid evaluation of numerous compounds for their antiviral activities at the cellular level.Phenotypic HTS, which combines cell-based imaging with the quantification of viral RNA replication, has been widely used. 14,15However, it typically requires long and tedious staining protocols with fluorescently labeled antibodies that target a specific viral protein or complicated nucleotide extraction and amplification procedures.Recently, JEV viruses with reporter proteins such as luciferase or fluorescent proteins have been developed for HTS, including Rluc-JEV and green fluorescent protein (GFP)-JEV. 16,17Nevertheless, maintaining their genetic stability and reporter expression during long-term passage has remained a challenge. 16,17In this study, a novel, genetically stable JEV reporter virus system for HTS was constructed and evaluated.In comparison to the standard cytopathic effect (CPE)-based antiviral screening method, the reporter protein-based HTS offers a faster and more convenient way to authentically screen for the antiviral activity of compounds.Furthermore, the reporter system serves as a valuable tool to screen, test, and optimize antiviral compounds and elucidate their mechanism of action.Using this reporter system, an antiviral library of 227 compounds was tentatively screened.Notably, a novel FDA-approved drug, cepharanthine, demonstrated excellent antiviral activity and competed with GTP in binding to JEV RNA-dependent RNA polymerase (RdRp) during RNA replication.The top drugs identified in this study may provide new and additional therapeutic options for the treatment of JEV infection and the associated disease.
The fused fragment was then engineered into pCC1-JEV through NotI and BstBI sites to generate pCC1-JEV-Nluc clone.The recombinant pCC1 vectored plasmids were transformed into EPI300 competent E. coli cells, and colonies were selected on LB plate containing 25 μg/mL chloramphenicol.Colonies were picked and cultured in LB broth containing 25 μg/mL chloramphenicol at 37°C for 16 h, before 1:10 dilution into fresh LB broth containing 25 μg/mL chloramphenicol and 1 mM arabinose at 37°C for 5 h for plasmid induction.Following verification through Sanger sequencing, plasmid DNA with the correct sequence was employed for linearization and subsequent in vitro transcription below.

| RNA in vitro transcription and electroporation
The reporter virus construct (pCC1-JEV-NLuc) was first linearized by ClaI (NEB), and then purified by phenol/chloroform extraction and ethanol precipitation.The purified linear DNA was dissolved in nuclease-free water.Subsequently, the mMESSAGE mMACHINE T7 transcription kit (Invitrogen) was employed to synthesize the genomic RNAs from the linearized DNA according to manufacturer's instruction.The yielded RNA was then subjected to electroporation.In brief, BHK-21 cells were washed in PBS and counted before electroporating 8 × 10 6 cells in ice-cold Electroporation Solution (Ingenio) with 10 μg RNA at 850 V, 25 µF, pulse three times with 3 s interval in the chilled 4 mm electroporation cuvette (BioRad).Cells were resuspended in an appropriate volume of complete medium and then seeded into 96-well plates and 24-well plates at the required density for luciferase or Immunofluorescence assays (IFA), respectively.Supernatant was harvested when CPE occurred obviously (referred to P0), and clarified after centrifugation for storage at −80°C for subsequent antiviral analysis.
Additionally, the harvested viruses were serially passaged in BHK-21 cells for up to ten passages (P1−P10).

| IFA
Viral RNA electroporated cells were seeded into 24-well plates, the cells were fixed in 4% paraformaldehyde (PFA) every 24 h and cellular membranes were permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 7 min.To detect E protein of JEV, the cells were stained using a mouse monoclonal antibody 4G2 (GTX57154, dilution 1:500), followed by staining of the Alexa Fluor-488 conjugated secondary anti-mouse IgG (Thermo Fisher Scientific) diluted 1:500.
The images were acquired with EVOS microscope (Invitrogen).

| Titration of virus by plaque assay
One day before the assay, BHK-21 cells (2 × 10 5 per well) were seeded into 24-well plate with 0.5 mL of medium and then incubated at 37°C with 5% CO 2 .On the day of the assay, the virus was serially diluted in 10-fold.Subsequently, 100 μL of correspondingly diluted viruses were added into the 24-well plate and allowed to incubate for 1 h.After incubation, the inoculum was removed and replaced with 0.5 mL of methyl cellulose.The plates were fixed with 4% PFA for 30 min after 3 days of incubation when CEP occurred.The plates were then stained with crystal violate and counted to determine the virus titer.
F I G U R E 1 Construction and characterization of the JEV-Nluc reporter virus.(A) The strategy for constructing the full-length cDNA clone of JEV-WT (pCC1-JEV) and JEV-Nluc reporter virus (pCC1-JEV-Nluc).The reporter gene, NanoLuc (Nluc), was flanked by the first 34 amino acid of the capsid protein encoding a sequence at the 5′-end and a 2A self-cleaving peptide derived from Thosea asigna virus (T2A) at the 3′-end.To ensure the full-length expression of the capsid, a duplicate capsid 1−34 coding sequence was placed downstream of the T2A site, utilizing a distinct codon to prevent the risk of RNA recombination.At the 3′ end of the viral genome, an HDV ribozyme (HDVr) sequence was incorporated to generate an authentic 3′ end of viral RNA sequence.(B) Kinetics of Nluc expression post-electroporation determined by measuring the nanoluciferase activity of (RLU) and plaque assay.BHK-21 cells were electroporated with in vitro-transcribed reporter virus RNA.Cell cultures were harvested at the indicated time points for luciferase activity measurement.Supernatants harvested post-electroporation at the indicated time points were titrated by plaque assay to determine viral titers.(C) Immunofluorescence analysis was performed to evaluate the expression of JEV E protein in cells electroporated with reporter virus RNA at the indicated time points using 4G2 antibody immunostaining (E in green, nuclei in blue).(D) Comparison of plaque phenotype between JEV-WT and JEV-Nluc.(E, F) Comparison of the viral growth kinetics between JEV-Nluc reporter virus and JEV-WT using plaque assays (E) or Nluc activity (F).BHK-21 cells were infected with JEV-Nluc or JEV-WT at an MOI of 0.1.Supernatants of the virus-infected cells were harvested every 12 h for up to 96 h.Viral titers were determined by plaque assays in BHK-21 cells (E).The JEV-Nluc-infected cells were lysed in Glo lysis buffer (GLB, Promega) for measurement of Nluc activity (F).(G) Stability of the inserted Nluc reporter gene during virus propagation.The rescued virus was serially passaged in BHK-21 cells from P1 to P10 after genomic RNA electroporation (P0).Viral RNA from virus stocks of each passage was extracted and subjected to RT-PCR using specific primers targeting the JEV 5′ UTR and prM genes.Viral RNA from the rescued JEV-WT (P0) was used as a control, lacking the Nluc reporter gene.(H) Stability of Nluc activity of reporter virus post propagation from P1 to P10.BHK-21 cells were infected with viruses of JEV-Nluc (P0−P10) at 0.01 MOI, and cells were harvested 48 h post-infection for measurement of Nluc activity.The results represent at least two independent experiments and are expressed as the mean ± SEM.JEV, Japanese encephalitis virus; MOI, multiplicity of infection; Nluc, nanoluciferase; RT-PCR, reverse transcription and PCR; SEM, standard error of the mean.

| High-throughput screening of chemical compounds
Antiviral assays were conducted in the 96-well plates by combination of JEV-Nluc virus and individual compounds.The drug library, purchased from Selleck, comprised of 227 compounds, which were initially dissolved in DMSO to create at a stock concentration of 1 mM.In the primary screening, Vero cells (1.5 × 10 4 per well) were seeded in 96-well plates and incubated for approximately 20 h before being infected with JEV-Nluc at a multiplicity of infection (MOI) of 0.01.Simultaneously, each compound was individually added to the virus-infected cells at a working concentration of 10 μM, with DMSO serving as the control for virus-only conditions.The luciferase activities of the compound-treated cells were quantified at 48 h post-infection, by measuring luciferase activity with Nano-Glo Luciferase assay system (Promega) with the Synergy H1 plate reader (BioTek).In the second round of screening, the compounds that showed promise were subjected to a 12-point serial threefold dilution, starting from the initial concentration of 100 μM.When combined with JEV-Nluc virus (MOI = 0.01) infection, the luciferase activity of treated cells was also measured to calculate the EC 50 , which represents the concentration at which the drug exerts 50% of its maximal effect.

| Cytotoxicity assay
The toxicity of compound to Vero cells were determined via the CellTiter-Glo (CTG) luminescent assay (Promega), which quantifies the number of viable cells based on ATP levels.Briefly, Vero cells seeded in 96-well plates were treated with threefold diluted compounds as used in antiviral assay.After 48 h of treatment, the CTG assay was carried out following the manufacturer's instructions.
Luminescent signals were recorded using the Synergy H1 plate reader (BioTek).Cell viability was expressed as a percentage of the compound treated cells to the control cells treated with the same concentration of DMSO.The CC 50 value, representing the cytotoxic concentration at which 50% of the cells remain viable, was calculated.

| Plaque reduction assay (PRA)
A viral titer reduction assay was performed to assess the antiviral efficacy of the compound in cell culture.Briefly, Vero cells were seeded in a 24-well plate (2 × 10 5 cells per well).Approximately 20 h after seeding, the cells were infected with indicated viruses at a MOI of 0.01.Simultaneously, the cells were treated with threefold serially diluted compound.Following a 1 h incubation period to permit virus entry, the mixtures of the compound and virus were carefully removed and replaced with the corresponding dilution of the compound.The culture supernatant was collected at 48 h postinfection.Viral titers of all samples were determined by plaque assays as described previously.

| In silico docking simulation of compound binding to target proteins
The crystal structures of the key JEV proteins including E (PDB ID: 3P54), NS1(PDB ID: 5O19), NS3 protease (PDB ID: 4R8T), and RdRp (PDB ID: 4MTP) proteins, were obtained from RCSB Protein Data Bank (RCSB PDB).The 3D conformations of ligands were downloaded from the PubChem database (GTP CID: 135398633; Cepharanthine CID: 10206).These structures were further refined for docking simulations using the Protein Preparation Wizard Script (Schrödinger; LLC).In silico molecular docking was performed using molecular docking program AutoDock Vina (v1.1.2). 19,20 BIOVIA Discovery Studio Visualizer and Pymol was used to visualize interacting residues between molecules and protein for structural analysis.The binding free energy (ΔG) for each protein−ligand complex was computed using the Molecular Mechanics/Generalized Born Surface Area method within the Schrödinger suite (Schrödinger Release 2018.3:Prime; Schrödinger; LLC; 2018).The net free binding energy (ΔG) was calculated using the following equation: ΔG bind = Δ G complex (minimized) −(ΔG receptor (minimized) + ΔG ligand (minimized) ), where ΔG bind denotes the binding free energy, ΔG complex signifies the free energy of the complex, ΔG receptor and ΔG ligand represents the energy associated with receptor and the ligand, respectively.Additionally, 4-week-old C57BL/6J mice (Male; Vital River Laboratories) were infected with 10 6 PFU of JEV-SA-14 and were also treated with cepharanthine or PBS following the same regimen as described above (n = 6 for each group).These mice were monitored for their survival rate for a period of 21 days.The in vivo mice assays above were performed once, respectively.

| Statistical analysis
The values are presented as mean plus standard error of the mean.The statistical analysis was performed with GraphPad Prism 9.0 software.A Student's two-tailed unpaired t-test was performed to analyze the statistical differences between two experimental groups.One-way ANOVA with a Dunn's multiple comparisons were applied to compare more than two experimental groups.p Values < 0.05 were significant and n.s.indicated as not significant.***p < 0.001, **p < 0.01, *p < 0.05.

| Construction and confirmation of JEV-NLuc reporter virus
A JEV reporter virus expressing NLuc was engineered using a previously reported strategy. 21In this strategy, the Nluc gene followed by the ribosome-skipping 2A sequence of Thosea asigna virus (T2A) was positioned within the ORF of the JEV polyprotein (Figure 1A).The T2A autoproteolytic cleavage sequence allows for the cleavage of Nluc protein from the nascent JEV polyprotein during translation.Since the 5′ cyclization sequence (CS) is located within the coding sequence of the capsid protein 136−146 nucleotides from the 5′ end of the JEV genome, the first 34 codons of the capsid protein (C1-34) upstream of the Nluc gene were preserved 22 (Figure 1A).To restore full-length JEV capsid expression, the fulllength capsid coding sequence was duplicated downstream of the T2A, but with the use of another codon to preserve its amino acid identity while avoiding the risk of RNA recombination from tandem repeat nucleotide sequences.A T7 promoter was linked to the 5′ UTR of JEV to facilitate the initiation of JEV genomic RNA transcription in vitro.The hepatitis D virus ribozyme (Rbz), a selfcleaving catalytic RNA, was adjacent to the 3′ end of the JEV 3′ UTR to yield an authentic viral genome end (Figure 1A).1B,C).The supernatant was collected at around 4 days post-electroporation when cells showed obvious CPE.Subsequently, a plaque assay was conducted to compare the plaque morphology of JEV-Nluc with that of its parental JEV strain (JEV-WT).The plaque size of JEV-Nluc retained unaltered in comparison with JEV-WT (Figure 1D).This observation suggests that the insertion of Nluc reporter gene had no discernible impact on the infectivity of JEV.To further substantiate whether JEV-Nluc exhibited similar antiviral activity to JEV-WT, the viral production fitness of JEV-Nluc was compared with that of JEV-WT.To this end, BHK-21 cells were infected with JEV-Nluc or JEV-WT at a MOI of 0.1, and the viruses produced in the supernatant were harvested for growth kinetics analysis.Up to 96 h post-infection (hpi), no significant differences in virus yields were observed between JEV-Nluc and JEV-WT (Figure 1E).Moreover, the observed increase in Nluc activity detected in JEV-Nluc-infected cells was correlated with the virus growth curve (Figure 1F).These results strongly supported the notion that the replication phenotype of JEV-Nluc closely resembles that of JEV-WT, thereby validating the use of Nluc activity as a robust surrogate marker for JEV infection.F I G U R E 2 Schematic diagram of standard and high-throughput screening (HTS) procedures and comparison of the JEV-Nluc reporter system and JEV-WT in antiviral activity analysis.(A) Standard antiviral screening (left panel) is a two-step procedure consisting of viruscompound treatment and viral titration steps.This approach This approach typically takes almost 6 days and necessitates the use of a substantial number of consumables.To elaborate, Vero cells were seeded in 24-well plates at a density of 2 × 10 5 cells per well 1 day before virus infection.These cells were subsequently infected with the virus at a MOI of 0.01 and treated with varying compound concentrations for 1 h at 37°C.After this initial incubation, the virus-compound mixture was removed, and only the corresponding concentration of compound was added for an additional 48 h incubation at 37°C.Supernatants were collected, titrated by plaque assay, and quantified at 72 h post-infection (hpi).In contrast, the one-step reporter virus antiviral analysis for HTS (right panel) significantly streamlines the process and can be completed in just 3 days.Vero cells were plated at a density of 1.5 × 10 4 cells per well in a 96-well plate.The compounds were diluted as needed and coincubated with the reporter virus for 48 h at 37°C.The plates were then directly read using a plate reader after the addition of the luciferase substrate.To assess the suitability of the reporter virus for antiviral drug screening, the system was initially validated for drug discovery employing a known inhibitor of flaviviruses, NITD008. 23JEV-Nlucinfected cells were treated with NITD008, which was serially diluted in threefold increments starting from a concentration of 100 μM, following the protocols of both traditional and HTS.In parallel, JEV-WT was treated in the same way as NITD008 to allow a comparison of the efficacy of the JEV reporter virus for high-throughput antiviral screening.Samples at various concentrations of NITD008 were collected for titration using a plaque assay.As demonstrated in These results firmly confirmed the sensitivity of JEV-Nluc for antiviral screening.In summary, this study establishes that the JEV-Nluc reporter virus is a dependable and convenient tool for screening antiviral drugs targeting the entire viral life cycle.

| HTS of a drug library for potential antivirals against JEV infection
After confirming that the assay conditions were suitable for a largescale screening, we applied this experimental design to screen a chemical library consisting of 227 compounds.These compounds were selected because they were previously documented to possess antiviral activities against RNA viruses or were known to target cell signaling pathways potentially associated with virus production.In the primary screening, we evaluated the potential antiviral activity of these compounds against JEV-Nluc in Vero cells (Figure 3A).The assay employed a final compound concentration of 10 μM, and Vero cells were infected with JEV-Nluc at a MOI of 0.01.Compounds that demonstrated a signal reduction exceeding 90% and exhibited less than 10% cell cytotoxicity were selected for further confirmation (Figure 3B and Supporting Information S3: Table 1).Subsequently, 25 compounds that exhibited both suitable antiviral activity and mild cytotoxicity through the HTS assay (Figure 3B and Supporting Information S3: Table 1).These compounds were then subjected to a threefold serial dilution to treat JEV-Nluc virus.The EC 50 and CC 50 were calculated under conditions of a dose-dependent inhibitory effect (Supporting Information S3: Table 1).Ultimately, we singled out five compounds with a selection index (SI) value (CC 50 /EC 50 ) greater than 10 (Figure 3C−H).These five compounds demonstrated a promising balance between their antiviral efficacy and low cytotoxicity, making them potential candidates for further evaluation as antivirals against JEV infection.
Out of the five compounds listed in Figure 3H, remdesivir (GS-5734) and Cepharanthine have been clinically approved.On the other hand, ADH-503 (GB1275), JG98, and SEA0400 are still in the preclinical research phase.Given their FDA-approved status, the approved drugs, remdesivir and cepharanthine, were prioritized for further investigation to explore their potential for repurposing.
Notably, remdesivir is a well-established antiviral drug with documented efficacy against a range of RNA viruses, spanning multiple viral families such as Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae.This effectiveness is attributed to its inhibition of RdRp activity.Cepharanthine, an old drug, is an alkaloid compound derived from the plant Stephania cepharantha (Figure 4A), and it has gained attention in the context of potential COVID-19 treatment.While it is not a widely recognized or FDA-approved treatment for COVID-19, research has suggested that cepharanthine may have antiviral properties and could be a promising candidate for further study in the fight against the disease.In our study, it was identified as a novel inhibitor against JEV.

| Confirmation of antiviral activity of cepharanthine against JEV and other flaviviruses
Following the HTS using the JEV-Nluc reporter virus system, we further validate the antiviral activity of cepharanthine against both JEV-Nluc and wild-type JEV (JEV-WT) using the traditional plaque reduction method.Vero cells were infected with JEV-Nluc or JEV-WT at a MOI of 0.01.Cepharanthine was introduced in threefold serially diluted concentrations.After 48 h post-infection (hpi), viral fractions were collected and subjected to plaque assays to determine the EC 50 values.Consistent with the inhibitory outcomes observed in the HTS screening, the EC 50 values for cepharanthine against JEV-Nluc and JEV-WT were found to be 1.08 and 1.53 μM, respectively (Figure 4B,C).
To evaluate the potential broad-spectrum inhibitory effects of cepharanthine against other flaviviruses, comprehensive antiviral analyses were conducted using reporter viruses for ZIKV, YFV, and DENV2.Intriguingly, cepharanthine demonstrated varying degrees of inhibition against these flaviviruses, with EC 50 values spanning from 2.52 to 15.82 μM (Figure 4D−F).These findings indicate the presence of a broad-spectrum antiviral activity against flaviviruses.Moreover, we extended our investigations to assess the protective effect of cepharanthine against JEV-induced lethality in mouse models in vivo.
The administration of cepharanthine at a dosage of 25 mg/kg effectively curtailed virus replication in various tissues, including the blood, brain, spinal marrow, and spleen, predominantly during the early stages of infection (Figure S1A,B).In the A129 mouse model, within 14 days post-infection, all mice in the JEV-infected group succumbed to the infection, while cepharanthine treatment following JEV infection reduced the mortality rate to 80% (one out of five animals survived) (Figure S1C).In the case of C57BL/6J mice, which exhibited lower susceptibility to JEV infection, cepharanthine treatment provided enhanced protection.The mortality rate decreased from 83.3% to 66.7%, with a notable extension in survival time (Figure S1D).These results collectively suggest that cepharanthine offers a degree of protection against JEV-induced mortality in mice, highlighting its potential as a valuable candidate for further exploration in the context of antiviral therapeutics.F I G U R E 3 Discovery of novel and potent JEV antiviral drugs based on high-throughput screening (HTS) of a drug library purchased from Selleck.(A) The workflow for HTS utilizing the JEV-Nluc reporter virus is illustrated, outlining the process employed to identify inhibitors of JEV replication.(B) A scatter plot representing the primary screening data of 277 compounds from the Selleck purchased drug library is displayed.Compounds demonstrating over 90% antiviral activity and 90% cell viability (samples within the square box) were selected for a secondary round of screening.The screening was performed in duplicate.(C−G) Dose-dependent inhibitory effects (circle dot) and cell viability analysis (square dot) of the five chosen compounds were assessed in Vero cells.Vero cell monolayers were treated with each drug at the indicated concentrations and inoculated with JEV-Nluc at a MOI of 0.01.At 48 hpi, cells were harvested, and Nluc substrate was added for luciferase activity analysis and CellTiter-Glo luminescent cell viability assay.The results represented at two independent experiments and are expressed as the mean ± SEM. (H) Five selected compounds exhibiting dose-dependent inhibitory effects and a SI greater than 10 from the reconfirmation screen are listed out.CC 50 , 50% cytotoxic concentration; EC 50 , 50% inhibitory concentration; JEV, Japanese encephalitis virus; MOI, multiplicity of infection; Nluc, nanoluciferase; SEM, standard error of the mean; SI, selection index, indicating the selectivity of a particular compound in inhibiting JEV replication while maintaining cell viability.

F I G U R E 5 (See caption on next page).
YIN ET AL.
| 11 of 15 substantial inhibition, signifying that cepharanthine predominantly hampers intracellular viral replication (Figure 5B).Viral attachment and entry were also affected to a certain extent, with reductions of approximately 40% and 50%, respectively, in comparison to control virus levels.This suggests that cepharanthine exerts a relatively mild impact on the early stages of the viral infection process (Figure 5B).
Analysis of the interaction between cepharanthine and JEV viral proteins (E, NS3 protease, and NS1) was performed by in silico docking simulation (Figure S2 and Supporting Information S3: Table 2).Our docking model unveiled that cepharanthine interacts specifically with the active site pocket of the JEV NS5 RdRp, the site where GTP is recruited for initiating RNA synthesis (Figure 5C−H).
According to the docking results, cepharanthine forms two hydrogen bonds with Arg 474 and Trp 477, as well as four hydrophobic interactions with Arg 460, Asp 669, and Trp 800 within JEV RDRP (Figure 5E).These interactions are in close proximity to residues that form the binding interface with GTP (Figure 5H).Notably, the binding free energy of cepharanthine molecules was estimated at −11.3 kcal/ mol.In contrast, the binding free energy of GTP with RdRp was found to be −9.5 kcal/mol, which was higher than that of cepharanthine (Figure 5I).This result underscores the competitive inhibition exerted by cepharanthine during viral replication, positioning it as a potential antagonist in the process.

| DISCUSSIONS
In recent decades, significant advancements in reverse genetics have facilitated the creation of replicon-based or reporter virus systems for a wide range of RNA viruses, including HCV, SARS-CoV-2, DENV, WNV, ZIKV, EV71, HEV, CHIKV, and many others. 21,24,25These reporter viruses have become invaluable tools in antiviral screening assays.They play a pivotal role in identifying potential antiviral compounds, evaluating their effectiveness, and gaining insights into their mechanisms of action.Viruses within the flavivirus family, including ZIKV, DENV, JEV, YFV, WNV, TBEV, and Langat virus (LGTV), have been genetically modified for the construction of reporter viruses. 21The most robust scheme for reporter flavivirus construction to date is the capsid duplication strategy developed for the YFV-eGFP reporter virus, in which the reporter gene eGFP was placed between the 5′ UTR and the capsid gene. 26Notably, a portion of the capsid gene is duplicated upstream of the reporter gene to preserve the essential capsid-coding region hairpin element (cHP), which is crucial for circularizing the viral RNA genome and, consequently, for RNA replication and transcription. 27The codon sequence of the complete capsid gene is often optimized to minimize homology and usage of the downstream 5′ CS 26 In previous work, two infectious JEV reporters were developed, one linked with GFP and the other linked with Renilla luciferase (RLuc). 16,17However, due to homology issues, the reporter gene typically becomes unstable within five passages in cell culture.In the present study, the JEV-Nluc reporter virus with C34 duplicates exhibited exceptional genetic stability, with the Nluc gene remaining intact for up to 10 passages.This genetic stability of the JEV-Nluc reporter virus enabled us to conduct a HTS of potential antiviral compounds against JEV infection.
Before HTS, we initially evaluated the feasibility and effectiveness of the reporter virus by comparing it to wild-type JEV (SA14-14-2) through the gold standard PRA methods.PRAs are a well-established and sensitive method for assessing antiviral activity, but they are lower in throughput, relatively expensive, and time-consuming.In contrast, reporter virus-based HTS is more efficient and versatile, especially when dealing with diverse viruses, making it ideal for large-scale compound screening endeavors. 21In this study, NITD008, a widely recognized broad-spectrum flavivirus inhibitor, was employed to evaluate JEV-Nluc in an antiviral activity analysis.As shown in Figure 2, the results obtained from the JEV-Nluc reporter system closely align with those from the conventional plaque assay method, indicating that the JEV-Nluc reporter virus holds significant promise for widespread utilization in HTS assays.
First, we compiled a library of antiviral compounds consisting of 227 substances with documented inhibitory effects against flaviviruses or known to target cellular proteins involved in flavivirus replication, based on an extensive review of the existing literature.From this library, we identified five promising drugs with a SI exceeding 10 (Table 1).One of them is Remdesivir, a well-established antiviral agent, F I G U R E 5 Antiviral mechanism of action of cepharanthine.(A) Schematic of the time-of-addition analysis to examine steps in JEV life cycle.Cepharanthine was introduced at different time points during virus infection (i, whole; ii, attachment; iii, entry; or iv, post-entry): (i) administered throughout the entire life cycle, including during the 1 h virus inoculation at 4°C followed by a 23 h infection period; (ii) added during the 1 h virus inoculation at 4°C and removed thereafter; (iii) introduced after the virus inoculation, with a 2 h treatment at 37°C to target entry and then removed; (iv) added postinoculation and entry, remaining present for the remaining 21 h of infection.Solid and dashed boxes delineate periods with and without treatment, respectively.which has been demonstrated to have broad-spectrum antiviral activity against multiple RNA viruses. 28,291][32] CD11b, acting as a transmembrane transporter, plays a modulatory role during ZIKV infection, mediating cell-to-cell transmission. 33JG98 is an analog of Hsp70 inhibitors (JG40) known for their broad-spectrum inhibition of the replication of various including KUNV, YFV, and LGTV. 345][36][37] The impressive anti-JEV activity exhibited by JG98 suggests the need for further preclinical and clinical studies on its potential for treating JEV and other flaviviruses.SEA0400 is a specific Na + /Ca 2+ exchanger (NCX) inhibitor that restricts viral replication, likely by blocking the activation of the p38 MAPK pathway. 38,39Among the top five drugs, cepharanthine stands out as one of the two clinically approved drugs, indicating its potential for future applications in the treatment of JEV.This selection of compounds holds promise for the development of antiviral strategies against JEV and warrants further investigation and potential clinical application.
Cepharanthine is a natural alkaloid compound derived from the plant S. cepharantha, a member of the bis-benzylisoquinoline family, commonly used in traditional Chinese medicine. 40Dating back to the 1950s, cepharanthine found clinical applications in the treatment of various acute and chronic ailments, including alopecia, leukopenia, xerostomia, and snake bites. 41This versatile compound boasts an array of medicinal properties, encompassing inhibitory effects on signaling pathways, immunomodulation, and antiviral capabilities. 42In clinical settings, the combination of cepharanthine with antitumor medications has demonstrated the reversal of immunosuppression and treatment of chemotherapy-and radiation-induced thrombocytopenia, all while exhibiting minimal side effects.4][45][46][47][48][49] More recently, researchers have explored its potential in combatting COVID-19 by disrupting various stages of the SARS-CoV-2 life cycle, particularly viral entry and the release of viral particles. 50This study revealed that cepharanthine exerts its influence at different stages of viral infection, primarily post-entry stage.A subsequent structure-based analysis unveiled that cepharanthine could bind within the active RdRp pocket of JEV, where GTP is recruited to initiate RNA synthesis. 51It is thereby inferred that Cepharanthine competes with GTP for RdRp binding, thus impeding JEV replication.Other studies have suggested that Cepharanthine promotes autophagy, potentially involved in the inhibition of JEV replication. 52,53Moreover, the efficacy of cepharanthine was monitored in vivo but the results showed it did not provide complete protection against JEV.This limitation is believed to stem from its poor water solubility, resulting in low oral bioavailability in vivo. 40To address this issue and enhance solubility and bioavailability, novel forms such as dry powder inhalers and delivery systems like liposomes and nanoparticles can be developed and utilized in future research to establish more effective dosing regimens for viral infections via pulmonary, oral, and intravenous administration (Table 1).

| CONCLUSIONS
In conclusion, the JEV-Nluc reporter virus system proves to be a dependable, precise, user-friendly, and efficient tool for conducting antiviral screening.Leveraging this system, we conducted a HTS of a drug library, focusing on compounds with potential anti-flavivirus properties.Among the compounds examined, cepharanthine, an already clinically approved drug, stood out with its remarkable antiviral activity.This discovery opens up new avenues for potential therapies in the treatment of JEV infections.

AUTHOR CONTRIBUTIONS
Chunhong Yin designed the experiment and wrote the manuscript.| 13 of 15

F
I G U R E 1 (See caption on next page).
In the time of addition assay, we introduced compounds at four distinct time points, which corresponded to different stages of the viral life cycle, as illustrated in Figure 5A: (i) Attachment phase: In this stage, the compound (5 μM) was present during the 1 h virus attachment process at 4°C.Subsequently, both the compound and virus were removed, and the cells were subjected to an additional 23 h of infection.(ii) Entry phase: The compound was added after the initial 1 h virus attachment, allowed to for 2 h at 37°C, and then removed; (iii) Post-entry phase: The compound was present during the remaining 21 h of infection after viral entry.(iv) Whole life cycle: Vero cells were treated with the compound from the moment of virus inoculation.As a reference, cells treated with 0.5% DMSO, either with or without virus, was included as virus or cell controls.The inhibition ratio of the drug in each stage was calculated as the percentage of Nluc activity relative to the virus control.

2. 13 |
In vivo efficacy of cepharanthine in JEV mouse models A129 mice aged 6−8 weeks were housed at the Laboratory Animal Center of Wuhan Institute of Virology, CAS.The mice were intraperitoneally infected with 10 PFU of JEV per mouse and were randomly divided into three groups: JEV-infected group (n = 8), cepharanthine-treated group (n = 7), and a vehicle-treated group (n = 5).For the infection, mice were inoculated intraperitoneally with 10 PFU of JEV-SA-14 strain in a 100 μL volume.The cepharanthinetreated group received 25 mg/kg/day of the drug.Cepharanthine and vehicle treatments were administered intragastrically, with 25 mg/kg of cepharanthine or PBS with 5% DMSO, respectively.These treatments were initially administered twice a day for the first 2 days and then consecutively administered once per day.The animals were closely observed for clinical signs, morbidity and mortality for 10 days.Blood samples were collected daily via retro-orbital blooding method for viremia detection.On Day 3 post-infection, three mice from each group were killed, and their tissues (including the brain, spinal marrow, spleen) were harvested and homogenized for viral burden analysis (n = 3).All animal experiment protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS (Wuhan, China).
Following linearization of JEV-Nluc plasmid by ClaI, genomic RNA of JEV-Nluc was transcribed in vitro under the control of the T7 promoter and then electroporated into BHK-21 cells.The expression of Nluc was detected as early as 4 h post-electroporation, with the luciferase activity signal showing a progressive increase over time (Figure 1B).Consistent with the results of the viral titration analysis (plaque assay) and IFA, Nluc activity, viral tires, and JEV E protein expression peaked at approximately 72 h post-electroporation (Figure To assess the stability of the Nluc transgene during consecutive passaging, the rescued virus (P0) underwent blind passage in BHK-21 cells from the first generation (P1) to the 10th generation (P10) (Figure1G).Viruses from each passage were harvested for extraction of genomic RNA.The presence of the Nluc gene was determined via RT-PCR using primers designed to target regions in the JEV 5′ UTR and prM genes situated up-and downstream of the Nluc insertion sites.This amplification yielded amplicons of 1200 bp in case of viral RNA carrying the Nluc insert or 490 bp in the absence of the insert (Figure1G).It is noteworthy that the P0−P10 viruses consistently exhibited 1200 bp fragments upon amplification.This observation demonstrates the remarkable stability of the Nluc gene within the JEV reporter virus, as it remained intact throughout at least 10 consecutive rounds of passage.Luciferase activity of the viruses post passages retain the comparable level with the freshly rescue virus (P0) (Figure1H).Consequently, virus stocks up to P10 can be confidently employed in antiviral assays without any concerns about a decrease in signal intensity.It is important to note that the virus used throughout this research was derived from the P0 generation, collected approximately 4 days post-electroporation.

3. 2 |
Establishment and evaluation of high-throughput antiviral screening using JEV reporter virusThe standard antiviral screening of JEV involves a series of laboratory assays and tests aimed at assessing the effectiveness of compounds or drugs in inhibiting JEV replication.Among these methods, the PRA is a commonly employed technique to gauges the antiviral activity of compounds against JEV and other viruses.This assay measures the ability of compounds to inhibit viral replication by quantifying the reduction in the number of viral plaques that form in a cell monolayer when treated with varying concentrations of compounds.As illustrated in Figure2A, this process encompasses multiple steps, including the coincubation of virus and compound with cells, compound treatment and titration of compound-treated virus fractions.In contrast, the reporter virus-based screening method primarily involves the first step and subsequently quantifies antiviral activities using an automated high-throughput approach.
(B−D) Comparison of the inhibitory effect of NITD008 on JEV-Nluc reporter virus and JEV-WT determined by luciferase activity analysis (B) and plaque assay titration (C, D). (B) As described above, Vero cells seeded in 96-well plates were treated with NITD008 at the indicated concentrations and infected with JEV-Nluc at a MOI of 0.01.The antiviral activity of NITD008 (EC 50 ) was determined by comparing the luciferase activity of the NITD008-treated cells to that of DMSO-treated cells.(C, D) Vero cells, seeded in 24-well plates, were treated with indicated concentrations of NITD008 and then infected with JEV-Nluc (C) or JEV-WT (D) at an MOI of 0.01.The viral titers in harvested supernatants were determined by plaque assay at 48 hpi.EC 50 values were calculated based on the reduction in luciferase activity (Luminescence inhibition, %) and viral titers (plaque reduction, %) when exposed to varying concentrations of NITD008.The results represented three independent experiments and are expressed as the mean ± SEM.JEV, Japanese encephalitis virus; MOI, multiplicity of infection; Nluc, nanoluciferase; SEM, standard error of the mean.

Figure
Figure 2B−D, the EC 50 values, calculated based on the inhibition of Nluc signal of JEV-Nluc and the reduction in virus titers for both JEV-NLuc and JEV-WT were 0.44, 0.37, and 0.28 µM (Figure 2B−D).

5 |
Action mechanism of cepharanthine against JEV Within cell-based systems, the viral life cycle unfolds through distinct stages of infection, including viral entry events (such as attachment, fusion, and uncoating), the replication phase (encompassing viral genome replication and protein translation), and virion egress (assembly, maturation, and release).Tailored assays can be employed to scrutinize each stage of the viral life cycle, utilizing an array of tools and methods.This allows for the assessment of candidate antivirals with specific mechanisms of action at particular stages.In our quest to uncover how cepharanthine impacts the viral replicative life cycle, we conducted a time-of-addition assay (Figure 5A).This assay involved measuring the antiviral activity of cepharanthine (at a concentration of 5 μM) when introduced at different time points: (i) "Whole life cycle": Cepharanthine was added during the initial 1 h virus inoculation stage at 4°C, facilitating viral attachment, and it was maintained throughout the subsequent 23 h infection period.(ii) "Attachment": Cepharanthine was introduced during the 1 h virus inoculation step at 4°C and then removed.(iii) "Entry": Cepharanthine was added after viral inoculation, with a 1 h treatment at 37°C, and then removed.(iv) "Post-entry": Cepharanthine was added after the viral attachment and entry phases and remained present for the remaining 21 h of infection.Luciferase activity was measured to gauge the inhibitory impact of cepharanthine.Results revealed that the most pronounced inhibition occurred when cepharanthine was present throughout the entire viral life cycle.Following this, the post-entry stage exhibited the next most F I G U R E 4 Inhibitory activities of cepharanthine against other flaviviruses.(A) Structure of cepharanthine.(B, C) The antiviral activity of cepharanthine against both JEV-Nluc (B) and JEV-WT (C) is validated using the plaque assay method.Vero cells were infected with JEV-Nluc or JEV-WT at an MOI of 0.01 and subjected to different concentrations of cepharanthine.Viral titers in the supernatants were determined by plaque assay at 48 hpi.(D−F) Antiviral activities of cepharanthine against ZIKV (D), YFV (E), and DENV2 (F) reporter viruses.The results represented at least two independent experiments and are expressed as the mean ± SEM.DENV, dengue virus; JEV, Japanese encephalitis virus; MOI, multiplicity of infection; Nluc, nanoluciferase; SEM, standard error of the mean; YFV, yellow fever virus; ZIKV, Zika virus.
(B) Inhibitory effects of cepharanthine under different conditions were estimated by measuring the luciferase activity 24 hpi.The results represented four independent experiments and are expressed as the mean ± SEM.The significance of the difference between mean values was determined by Student's t-test.**p < 0.01, ****p < 0.0001.(C−H) Interactions of cepharanthine with JEV RdRp (PDB: 4MTP).(C and F) RdRp colored in palecyan was shown in cartoon and cepharanthine molecule (C) or GTP (F) colored in lighpink or green was shown as sticks.(D and G) Zoom-in view of the RdRp with cepharanthine (D) or GTP (G) in 3D structures.Cepharanthine/GTP and the interacting amino acid residue are shown in sticks.Hydrogen bonds are shown as yellow dashed lines and hydrophobic bonds are shown as red dashed lines.(E and H) A 2D schematic representation for the interaction profile of the RdRp protein docked with cepharanthine (E) or GTP (H).(I) Binding free energy (ΔG) calculation performed for the RdRp protein docked with cepharanthine (CEP) and GTP.JEV, Japanese encephalitis virus; RdRp, RNA-dependent RNA polymerase; SEM, standard error of the mean.
Chunhong Yin, Peipei Yang, and Qingcui Xiao conducted the experiments and analyzed the data.Peipei Yang and Xuekai Zhang assisted with HTS and mice assays.Qingcui Xiao performed molecular docking analysis.Jiaxuan Zhao provided the commercial drug library.Xue Hu assisted with assay implementation.Peng Sun contributed critical ideas and edited the manuscript.Chunhong Yin and Chao Shan conceived and supervised the experiments.Chao Shan revised the manuscript.

T A B L E 1
Lists of five hit compounds showed dose-dependent inhibition and SI > 10.RNA-dependent RNA-polymerases (RdRp)inhibitor, broad-spectrum antiviral activity YIN ET AL.