key: cord-0918042-6xogcdp1
authors: Zhang, Zhe-Rui; Zhang, Hong-Qing; Li, Xiao-Dan; Deng, Cheng-Lin; Wang, Zhen; Li, Jia-Qi; Li, Na; Zhang, Qiu-Yan; Zhang, Hong-Lei; Zhang, Bo; Ye, Han-Qing
title: Generation and characterization of Japanese encephalitis virus expressing GFP reporter gene for high throughput drug screening
date: 2020-08-01
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104884
sha: f07f24eb0dab3b60423198c9ce86e90cd9538760
doc_id: 918042
cord_uid: 6xogcdp1

Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1,443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection.

Japanese encephalitis virus (JEV) is a mosquito-borne virus that belongs to the genus Flavivirus in the family Flaviviridae, which includes many other important human pathogens such as yellow fever virus (YFV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and dengue virus (DENV) (Buescher et al., 1959; Ellis et al., 2000) . JEV is mainly epidemic in the Asia Pacific region, putting more than 3 billion people at the risk of JEV infection (Turtle and Driver, 2018) . It is estimated that more than 67,900 Japanese encephalitis cases caused by JEV occur worldwide each year and the mortality rate is as high as about 20%-30% (Campbell et al., 2011; Griffiths et al., 2014; Hills et al., 2010) . Although the use of live-attenuated vaccine has greatly reduced the incidence of JEV infection, there is theoretically a risk of possible reversion toinfectivity. So far, no clinically approved antiviral drugs or therapies are available for treatment of JEV infection. Therefore, it is important to develop antiviral agents to control the virus infection.

JEV genome is a single-stranded plus-sense RNA that is approximately 11-kb in length, containing a single open reading frame (ORF) flanked by untranslated regions (UTRs) at the 5' and 3' ends. The ORF encodes a polyprotein which is cleaved by viral and host proteases into three structural proteins (capsid [C] , precursor membrane or membrane [preM/M] and envelope [E] ) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Sampath and Padmanabhan, 2009 ). The 5' and 3' UTRs form various highly conserved secondary structures involved in the replication, translation and packaging of viral genome. The structural proteins assemble into viral particles (Li et al., 2008; Roby et al., 2015) , while the non-structural proteins play essential roles in genome replication, viral particle assemble and immune escape (Brinton, 2013; Morrison et al., 2012; Murray et al., 2008; Nikonov et al., 2013; Shi, 2014) .

There are usually two strategies for high-throughput screening (HTS) of antiviral drugs. The first one is to screen active molecules targeting specific viral proteins through in vitro functional assays. For flaviviruses, the non-structural proteins NS3, NS5 and the structural protein E are the main targets. Some candidate agents targeting the flavivirus E-domain III (Zu et al., 2014) , NS3-helicase (Fang et al., 2016) , NS2B-NS3 interaction (Li et al., 2017b) and NS5-methyltransferase (Brecher et al., 2015; Han and Lee, 2017) have been successfully identified. The second strategy is based on in vivo cellular antiviral assays. In this case, the replicon-containing cell line (Li et al., 2018; Zhang et al., 2017) , virus-like particle (VLP) (Wang et al., 2017) , and the reporter virus (Li et al., 2017a) are the main screening tools. In contrast to VLP and replicon-containing cell line, the propagation of reporter virus contains the entire viral life cycle as wild type (WT) virus, it can be used to screen antiviral drugs targeting the complete infection steps. Currently, several reporter flaviviruses have been developed for different research purposes, including Rluc-JEV (Li et al., 2017a) , Rluc-DENV (Zou et al., 2011) and eGFP-DENV (Schoggins et al., 2012) .

In this study, we constructed and characterized a JEV reporter virus with an eGFP gene (eGFP-JEV). An eGFP-JEV-based HTS assay was established in a 96-well format and used for screening of 1,443 compounds from an FDA-approved drug library. Using this system, 16 hit drugs inhibiting JEV infection were identified, and five of them were firstly reported to have inhibitory effect on flavivirus replication, offering potential new therapies for the treatment of JEV infection.

Aedes albopictus mosquito C6/36 cells were cultured in RPMI-1640 medium (Invitrogen, Darmstadt, Germany) with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin (PS) at 28 °C. All other cells were grown at 37 °C with 5% CO 2 . Baby hamster kidney fibroblast (BHK-21) cells, and human hepatoma (HuH-7) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Darmstadt, Germany) with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. JEV virus was derived from the infectious cDNA clone of pACYC-JEV-SA14 (Li et al., 2014b) . The 4G2 antibody against the E protein of Texas Red-conjugated goat anti-mouse IgG was purchased from Protein Tech Group.

Nucleoside analogue inhibitor NITD008 was synthesized as reported previously (Yin et al., 2009) . A library of FDA-approved drugs was purchased from Selleck Chemicals.

The reporter JEV genome carrying eGFP (green fluorescent protein) was constructed with pACYC-JEV-SA14 (Li et al., 2014a ) as a backbone. The "KpnI-T7 promoter-5' UTR-Capsid-38 amino acids-AscI" and "PacI-C-prM-E" fragments were amplified using pACYC-JEV-SA14 as a template. The "AscI-eGFP-2A-PacI" fragment was amplified using EV71-eGFP-2A as a template (Shang et al., 2013) . The three fragments were fused together by overlapping PCR to obtain a fragment "KpnI-T7 promoter-5' UTR-Capsid-38 amino acids-AscI-eGFP-2A-PacI-C-prM-E" as shown in Fig. 1 . This fragment was engineered into pACYC-JEV-SA14 by KpnI and BsrGI sites to generate an eGFP-JEV cDNA clone. The complete sequence of the cDNA clone of eGFP-JEV was validated by DNA sequencing analysis before the subsequent experiments.

The JEV infectious clone and the reporter cDNA plasmids were linearized with XhoI and purified by extraction with phenol/chloroform. The linearized cDNA was transcribed using mMESSENGER mMACHINE T7 Kit (Ambion, Austin, TX, USA).

All procedures were performed according to the manufacturer's protocols. RNA was dissolved in RNase-free water and stored at -80 ℃. The RNA was transfected into cells with DMRIE-C reagent (Invitrogen) following the protocol described previously (Deng et al., 2016) .

The eGFP-JEV genomic RNA was transfected into BHK-21 cells. At 24, 48, and 72 hour post-transfection (hpt), the cells on the coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature. The fixed cells were washed three times with PBS and incubated with 4G2 antibody (1:500 dilution in PBS) for 1 h at room temperature. After washing three times with PBS, the cells were incubated with Texas Red-conjugated goat anti-mouse IgG antibody for 40 min in the dark.

After washing three times with PBS, the cells were mounted on a glass slide with 95% glycerol and cell images were captured under a fluorescence microscope.

To examine the genetic stability of the eGFP gene of eGFP-JEV, total RNAs were extracted from cells transfected with eGFP-JEV or cells infected with each passaged viruses using Trizol reagent (Takara). The fragment from 5'UTR to prM which covers the eGFP gene was amplified by one-step RT-PCR using a PrimeScript 

To clarify whether the compounds specifically inhibit viral replication or generally suppress cellular RNA transcription, the viral RNA and β-actin RNA were 

The viral titer and morphology were determined by single-layer plaque assay.

The virus was serially diluted 10-fold, and 100 μL of each dilution was added into a single well of a 24-well plate containing BHK-21 cells (1 × 10 5 cells per well). The plates were incubated at 37 °C with 5% CO 2 for 1 h before adding DMEM-2% FBS containing 1% methylcellulose. After 3 days of incubation at 37 °C with 5% CO 2 , these cells were fixed and stained with 3.7% formaldehyde and 1% crystal violet in water. The morphology and number of plaques were recorded after washing with water.

BHK-21 cells was seeded in 12-well plates (1 × 10 5 cells per well) were infected with WT-JEV or eGFP-JEV at an MOI of 0.1. Huh7 was seeded on 12-well plates (2 × 10 5 cells per well) were infected with WT-JEV at an MOI of 0.5. Thereafter, the infected cells were incubated with various concentrations of NITD008, lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene respectively. The supernatants were collected at 48 hpi or 72 hpi, and viral titers were determined by the single-layer plaque assay. For eGFP-JEV based antiviral assay, the expression of eGFP gene was recorded at 72 hpi. The antiviral activity of the compounds was expressed as 50% effective concentration (EC 50 ) which meant the drug concentration required to achieve 50% of viral titer reduction and was calculated by nonlinear regression using GraphPad Prism 5.0 software.

The eGFP-JEV based HTS assay was developed in a 96-well plate format using NITD008 as a positive control and 0.5% DMSO as a negative control. The compounds of FDA-approved drug library were all dissolved in DMSO at a stock concentration of 1 mM. We diluted each of the compound to 5 µM with DMEM-2% FBS as the working concentration. Huh7 cells were seeded into 96-well plates at the cell density of 1.0 × 10 4 per well and cultured for 24 h at 37 °C. Then the cells were infected with eGFP-JEV (MOI= 0.5). At the same time, the compound or DMSO was added to the culture medium. At 48 hpi, the number of the eGFP-positive cells was read by a PerkinElmer high content screening system. To evaluate the performance of the HTS assay, the Z' factor values were calculated. The Z' factor between 0.5 and 1

indicates an excellent assay with good separation between controls (Zhang et al., 1999) .

To determine the step inhibited by lonafarnib during viral life cycle, a time-of-addition assay was performed. Huh7 cells were infected with WT-JEV at an MOI of 10 for 1 h at 37°C, and lonafarnib (5 μM) was added to the infected cells at Supernatants were harvested at 12 hpi, and viral titers were determined by plaque assay. Inhibition rates are calculated as the percentage of of viral titer realtive to control.

Huh7 cells were seeded in 96-well plates (1 × 10 4 cells per well) and allowed to grow for 24 h before treatments. Afterward, compounds at concentrations in a 2-fold dilution series were added to the cell. 

Using the infectious clone pACYC-JEV-SA14 as a backbone (Li et al., 2014b) , we constructed a cDNA clone of JEV encoding an eGFP reporter gene. As shown in gradually increased from day 1 to day 3, but were lower than that of WT-JEV at each time point (Fig. 2D) . These results showed that the growth trend of eGFP-JEV was similar with WT-JEV and the replication of eGFP-JEV reporter virus could be easily tracked by eGFP signals.

To test the stability of eGFP-JEV, we serially passaged the virus in C6/36 cells. (Fig. 3B ). In addition, the P1-P4 viruses exhibited homogeneously small plaque morphology as P0 virus, but the plaque size of P5 virus turned as large as WT-JEV (Fig. 3C) . These results demonstrated that the eGFP gene of the eGFP-JEV reporter virus could be stably maintained within four rounds of passage and was significantly lost at P5. Therefore, we chose the P1 virus for the following antiviral study.

NITD008, a well-known anti-flavivirus compound, has shown to be able to inhibit JEV replication (Li et al., 2017a) . To determine the availability of the reporter virus for antiviral drug screening, we evaluated the antiviral activity of NITD008 using the eGFP-JEV reporter virus. Two-fold serial dilutions of NITD008 were used in this study with a maximum concentration of 3 µM. NITD008 showed consistently dose-dependent inhibitory effects on both eGFP expression (Fig. 4A ) and viral titers ( Fig. 4B ) of eGFP-JEV. In addition, the EC 50 of NITD008 for eGFP-JEV was 0.22 µM, which was similar to that of WT-JEV (EC 50 = 0.27 µM) (Fig. 4C) . The results not only confirmed that the expression level of eGFP can represent the replication of WT-JEV, but also indicated that the eGFP-JEV reporter virus system is suitable for screening potential anti-JEV compounds.

We replication in a 96-well plate to determine the optimal solvent concentration for the screen assay. As shown in Fig. 5D , DMSO had little effect on the expression of eGFP at ≤ 0.5% (v/v) concentration, but 1% DMSO significantly reduced the percentage of eGFP-positive cells to 30%. Therefore, we chose 0.5% DMSO as the work concentration to dissolve the compounds. We used the known inhibitor NITD008 to verify the feasibility of these identified conditions. Under the above experimental conditions, the calculated Z' value was 0.83 at 0.3 µM of NITD008 (HTS assays with Z' ≥ 0.5 are considered robust) ( Fig. 5E and F) . These results demonstrate that eGFP-JEV reporter virus could be used in a high throughput assay for screening inhibitors of JEV.

We screened 1443 compounds in an FDA-approved drug library using the eGFP-JEV based HTS to select the potential antiviral drugs. A schematic diagram of eGFP-JEV-based HTS method was shown in Fig. 6A . All drugs were used at a concentration of 5 µM, the drugs with an inhibition rate greater than 90% were selected as candidates. The Z' values of each screening were between 0.5 and 1, with an average value of 0.7 (data not shown), indicating that our results were reliable.

Twenty-six hits were identified though the HTS assay ( Interestingly, 5 drugs (listed in Group III), which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV in our study (Fig. 6C ).

We next confirmed the anti-JEV effects of these newly discovered compounds (Fig. 7C) . None of the drugs had any effect on the mRNA level of cellular actin (Fig. 7D) , suggesting that the inhibitory effect of these newly discovered compounds on virus replication was due to their antiviral activity rather than generally inhibiting cellular RNA transcription.

The selective index (SI) of each newly discovered compound was calculated as the ratio of CC 50 to EC 50 (Fig. 7E) . Lonafarnib, hexachlorophene and cetylpyridinium chlorid possessed good selective index (>10). Since lonafarnib is an orally administered drug and showed lower cytotoxicity, we chose lonafarnib to perform a time-of-addition assay to explore at which stage the drug blocked during viral life cycle. Huh7 cells were infected with WT-JEV at an MOI of 10 for 1h, and washed three times with PBS subsequently to remove the unattached viruses. Lonafarnib was added to the cells either pre-, during, or post infection at a concentration of 5 μM (Fig.   8A ). The supernatants were collected at 12 hpi and subjected to plaque assay. As shown in Fig. 8B , viral titers were significantly reduced when the compound was added at different time points post infection, suggesting the inhibition of virus replication. In addition, treatment with the compound during viral infection (0-1) resulted 60% reduction in viral titer, implying that lonafarnib also affected virus entry stage. However, there was no effect on virus propagation when lonafarnib was added pre-infection, indicating that a cellular response may be not required for lonafarnib to exert its antiviral activity. These results primarily demonstrated that lonafarnib might block viral propagation mainly through significant suppression of virus replication and modest inhibition of virus entry process during viral life cycle.

Currently, vaccine is the only approach to prevent JEV infection, and no antiviral therapeutics are available. Thus, development of HTS assay is important for JEV drug discovery. In this study, we constructed an eGFP-JEV reporter virus containing eGFP gene, and developed the eGFP-JEV-based HTS assay for antiviral screening. For the construction of eGFP-JEV reporter virus, the eGFP-2A fragment was inserted between the 5'UTR and C protein of JEV genome (Fig. 1) . Upon the cleavage by the FMDV 2A peptide, the eGFP protein was released and visible under a fluorescence microscope. As shown in Fig. 2A , C, and D, the expression level of eGFP correlated well with the level of JEV replication. In addition, a dose-dependent reduction in eGFP fluorescence signals was observed in eGFP-JEV-infected cells with NITD008 (a well-known anti-flavivirus inhibitor) treatment, implying that this reporter virus could be used as a convenient and effective HTS system for screening anti-JEV compounds (Fig. 4) . As the stability of the reporter virus is crucial for the reproducibility and reliability of HTS assay, eGFP-JEV was serially passaged in C6/36 cells to investigate its genetic stability. It was found that the eGFP reporter gene was stably retained in P4

virus, indicating that the reporter virus was stable at least within four serial passages.

It can meet the measurement requirements of the eGFP-JEV based HTS system.

Under the optimized HTS condition (Fig. 5) , 26 hit drugs were firstly identified out of the screened 1,443 FDA approved drugs. Considering the possible interference of the unspecific fluorescence and drug cytotoxicity, the first set of compounds were subjected to the dose-dependent inhibition assay using eGFP-JEV. At this stage, 16 drugs with significant inhibitory activity on eGFP expression and low cytotoxicity were screened out (Fig. 6 ). Among them, 5 drugs (mycophenolic acid, cyclosporin A, manidipine, pimecrolimus and lacidipine) had been reported to be effective in inhibiting JEV infection (Sebastian et al., 2011; Tu et al., 2012; Wang et al., 2017) .

Having these five drugs independently identified as hits in our screening further confirmed the reliability of the eGFP-JEV based HTS system in identifying potential anti-JEV inhibitors. In addition, it was found that some drugs that had been reported to inhibit other flaviviruses (Balasubramanian et al., 2017; Bullard-Feibelman et al., 2017; de Freitas et al., 2019; Dong et al., 2019; Fryk et al., 2016; Fryk et al., 2017; Gan et al., 2018; Kuivanen et al., 2017; Papin et al., 2005; Shahen et al., 2018; Simanjuntak et al., 2015) could also inhibit JEV. For instance, sofosbuvir, an initially approved nucleotide polymerase inhibitor for the distantly related hepatitis C virus Notably, 5 new hit drugs were identified for the first time to possess anti-JEV activity using this HTS system. It included three approved antiseptics:

cetylpyridinium chloride, cetrimonium bromide and hexachlorophene; and two orally administered drugs: lonafarnib and nitroxoline. Nitroxoline is a quinoline antibiotic for the treatment or prophylaxis of acute urinary tract infections with potential antitumor activity (Veschi et al., 2018) . To our knowledge, this was also the first time for nitroxoline associated with the interference with viral infection. Lonafarnib is an orally active inhibitor of farnesyltransferase that has entered clinical trials for treatment hepatitis delta virus (HDV) infection (Koh et al., 2015) . Lonafarnib abrogates HDV production by inhibiting prenylation of the large delta hepatitis antigen to prevent its interaction with hepatitis B surface antigen to form secreted particles (Koh et al., 2015) . Here, using a time-of-additon assay, we primarily demonstrated that lonafarnib dramatically inhibit virus replication, and also partially affected virus entry process during the viral life cycle of JEV. A recent study had identified that lonafarnib can combine with the RNA-dependent RNA polymerase of SARS-CoV and SARS-CoV-2 by virtual screening (Ruan et al., 2020) . Therefore, the possible anti-JEV mechanisms of lonafarnib might be the inhibition of prenylation modification of the viral proteins, or the binding with the RdRp to inhibit viral replication, or other unknown factors, which needs further investigation. In the future studies, we are going to investigate the anti-JEV mechanisms of these newly discovered hit drugs, especially those two oral drugs.

In summary, we have developed a novel eGFP-JEV based HTS system. The use of eGFP-JEV for HTS assay provided direct visibility of the suppression of viral infection by JEV inhibitors, expediting greatly the process of antiviral development.

Using this system, sixteen drugs that effectively inhibited JEV were identified from the FDA-approved drug library, and five of them are newly discovered compounds which have antiviral activity against JEV and flavivirus, providing potential new therapies for the treatment of JEV infection. respectively, and viral titers were quantified using the plaque assay on BHK-21 cells.

Error bars indicate the standard deviations from three independent experiments. 

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The eGFP-JEV reporter virus was established.The eGFP-JEV -based high throughput screening (HTS) assay was developed with a calculated Z′ factor of >0.5.A library of 1,443 FDA-approved drugs was screened to determine their ability to inhibit JEV infection using this HTS assay.