# Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling

Yunlong Li, Pengfei Li, Qiyu He, Ruyi Zhang, Yang Li, Nassim Kamar, Maikel P Peppelenbosch, Robert A de Man, Lin Wang, Qiuwei Pan

► To cite this version:

Yunlong Li, Pengfei Li, Qiyu He, Ruyi Zhang, Yang Li, et al.. Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling. Antiviral Research, 2022, 197, pp.105228. 10.1016/j.antiviral.2021.105228. hal-04243172

HAL Id: hal-04243172

https://hal.science/hal-04243172v1

Submitted on 16 Oct 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
<figure><img src="image_2.png" /></figure>

# Niclosamide inhibits hepatitis E virus through suppression of NF-kappaB signalling

Yunlong Li<sup>a</sup>, Pengfei Li<sup>a</sup>, Qiyu He<sup>b</sup>, Ruyi Zhang<sup>a</sup>, Yang Li<sup>a</sup>, Nassim Kamar<sup>c</sup>, Maikel P. Peppelenbosch<sup>a</sup>, Robert A. de Man<sup>a</sup>, Lin Wang<sup>b</sup>, Qiuwei Pan<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

Check for
updates

## ARTICLE INFO

**Keywords:**

Niclosamide

HEV

Cell culture model

Human liver organoids

Antiviral therapy

NFkB

## ABSTRACT

Hepatitis E virus (HEV) infection can cause severe acute hepatitis in pregnant women and chronic infection in immunocompromised patients, promoting the development of effective antiviral therapies. In this study, we identified niclosamide, a widely used anthelmintic drug, as a potent inhibitor of HEV replication in a range of subgenomic and full-length HEV models, which are based on human cell lines and liver organoids harbouring genotype 1 and 3 HEV strains. Niclosamide is known to have multiple cellular targets including the inhibition of STAT3 and NFkB signaling pathways. Although HEV activates STAT3, we excluded its involvement in the anti-HEV activity of niclosamide. Interestingly, HEV infection activated NFkB and activation of NFkB promoted viral replication. Consistently, stable silencing of NFkB by lentiviral RNAi inhibited HEV replication. By targeting NFkB signaling, we further revealed its role in mediating the anti-HEV action of niclosamide. These results demonstrated that niclosamide potently inhibits HEV replication by inhibiting NFkB signaling but independent of STAT3. Our findings support the potential of repurposing niclosamide for treating HEV infection.

## 1. Introduction

Hepatitis E virus (HEV) infection is a major cause of acute viral hepatitis worldwide and occurs in sporadic and epidemic forms, especially in resource-limited regions (Hakim et al., 2018). Generally, HEV infection is self-limiting with very low mortality. However, acute infection with genotype 1 HEV in pregnant women can develop severe complications such as acute liver failure, miscarriage, preterm delivery, stillbirth, and perinatal mortality, resulting in a high death rate of up to 25% (Hakim et al., 2017). Infection with genotype 3 and occasionally genotype 4 HEV in organ transplantation patients is prone to develop chronic hepatitis E (Kamar et al., 2008; Wang et al., 2018; Zhou et al., 2013). Thus, for treating the infection in these specific vulnerable populations, safe and effective antiviral therapy is urgently required.

Since no FDA-approved medication is available, besides supportive care, (peg)-interferon-alpha (IFNα), ribavirin, or their combination have been explored as an off-label treatment for treating some chronic HEV cases in the clinic (Haagsma et al., 2010; Kamar et al., 2010, 2014; Todt et al., 2016). Ribavirin monotherapy is effective in approximately 80%

of eligibly treated chronic hepatitis E patients (Kamar et al., 2020). However, ribavirin treatment failure has been frequently reported, probably attributed to resistance development related to viral mutations (Debing et al., 2014, 2016; Todt et al., 2018). Furthermore, many patients are not eligible or do not tolerate IFNα or ribavirin treatment. For example, IFNα is generally contraindicated in kidney, pancreas, heart, and lung-transplant recipients because it stimulates the immune system and increases the risk of acute rejection (Rostaing et al., 1995). Ribavirin therapy is contraindicative in pregnancy due to teratogenicity (Wede-meyer et al., 2012). Thus, there is a clinical need for further developing new antiviral therapies against HEV.

Drug repurposing has emerged as an attractive strategy to discover new applications of existing drugs. This approach offers various advantages such as reduced time, cost, and risk over the development of new drugs (Pushpakom et al., 2019). In a previous study, we screened a library of safe-in-human broad-spectrum antiviral agents to identify potential anti-HEV agents. Niclosamide, widely used to treat tapeworm infestations, was among the top hits (Li et al., 2020). Thus, this study aims to concretely evaluate the anti-HEV activity of niclosamide and to

<footnote><sup>a</sup> Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Rotterdam, the Netherlands</footnote>

<footnote><sup>b</sup> Department of Microbiology and Infectious Disease Centre, School of Basic Medical Sciences, Peking University Health Science Centre, Beijing, China</footnote>

<footnote><sup>c</sup> Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, INSERM U1043, IFR-BMT, University Paul Sabatier, Toulouse, France</footnote>

<footnote>* Corresponding author. Department of Gastroenterology and Hepatology, Erasmus MC, room Na-1005, Wytemaweg 80, NL-3015 CN, Rotterdam, the Netherlands. E-mail address: q.pan@erasmusmc.nl (Q. Pan).</footnote>
explore the underlying mechanism-of-action.

## 2. Materials and methods

### 2.1. Reagents and antibodies

Ribavirin and human IFNα were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) and were dissolved in Dimethyl sulfoxide (DMSO). Niclosamide, the potent STAT3 activators Colivelin TFA and recombinant human interleukin-6 (IL6), and recombinant human tumor necrosis Factor-alpha (TNFα) were obtained from MedChemExpress. Antibodies including anti-HEV capsid protein antibody (EMD Millipore, MAB8002), anti-dsRNA antibody (Scicons J2), anti-STAT3 antibody, anti-NFκB p65 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-albumin antibody and anti-β-actin antibody (Santa Cruz Biotech, Santa Cruz, CA) were used. Anti-rabbit and anti-mouse IRDye-conjugated secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA) were also used.

### 2.2. Viruses, cell lines and human liver organoids culture models

Multiple cell lines including human hepatoma Huh7 and PLC/PRF/5 (PLC) cell lines, human embryonic kidney epithelial cell line (293T), and human glioblastoma cell line (U87) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin.

Tissue samples (≤0.5 cm³) of donor liver biopsies (n > 40) used for organoids isolation and culture were collected during liver transplantation at the Erasmus Medical Center Rotterdam. Use of liver tissues for research purposes was approved by the Medical Ethical Council of the Erasmus MC and informed consent was given (MEC-2014-060 for adult liver tissues). Liver organoids were cultured as previously described (Huch et al., 2015). Briefly, human liver organoids were cultured in organoid expansion medium (EM), based on advanced DMEM/F12 (Invitrogen), supplemented with 1% penicillin/streptomycin (Life Technologies), 1 M HEPES (Life Technologies), 200 mM Ultraglutamine (Life Technologies), 1% (vol/vol) of N2 (Gibco), 2% (vol/vol) of B27 (Gibco), 1 mM N-acetylcysteine (Sigma-Aldrich), 10 mM Nicotinamide (Sigma-Aldrich), 5 µM A83.01 (Tocris), 10 µM Forskolin (Tocris), 10 nM Gastrin (Sigma-Aldrich), 50 ng/mL EGF (Peprotech), 10% (vol/vol) of R-spondin-1 (conditioned medium), 100 ng/mL FGF10 (Peprotech), 25 ng/mL HGF (Peprotech) and 10 µM Y27632 (Sigma-Aldrich).

For genotype 3 HEV models, HEV genomic RNA was generated from a plasmid construct containing the full-length HEV genome (Kernow-C1p6 clone, GenBank Accession Number JQ679013) or a construct containing subgenomic HEV sequence in which ORF2 was replaced by a Gaussia luciferase reporter gene (p6-luc), using the Ambion mMESSAGE mMACHINE® in vitro RNA transcription Kit (Life Technologies Corporation). Cells and organoids were electroporated with p6 full-length HEV RNA or p6-Luc subgenomic RNA to generate infectious or luciferase-based replicon models, respectively. Similarly, HEV genotype 1 replication cell model was electroporated with Sar 55/S17/luc (GenBank accession number AF444002) HEV RNA, and viral replication was quantified by Gaussia luciferase activity. Furthermore, infectious HEV particles were produced from Huh7 cells harbouring p6 full-length HEV RNA, and viruses were harvested to inoculate naïve huh7 cells.

NFκB stable firefly luciferase expressing cells were generated by transducing Huh7 cells with a lentiviral vector expressing the firefly luciferase gene under control of the promoters containing the NFκB motifs (NFκB-Luc).

### 2.3. Measurement of luciferase activity

For Gaussia luciferase (HEV-Luc), the activity of secreted luciferase in the cell culture medium was monitored by the activity of secreted

Gaussia luciferase measured by QUANTI-Luc™ Gold (InvivoGen). For firefly luciferase (NFκB-Luc), luciferin potassium salt (100 mM; Sigma) was added to cells and incubated for 30 min at 37 °C. STAT3 promoter elements related luciferase activity (STAT3-luc) was measured using the Luciferase Assay System (Promega, Madison, WI). Data were normalized for the transfection efficiency by using the Dual Luciferase Reporter Assay system (Promega) according to the manufacturer's instructions. All the luciferase activity was quantified with a LumiStar Optima luminescence counter (BMG Lab Tech, Offenburg, Germany).

### 2.4. MTT assay

10 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) was added to the cells seeded in 96-well plate and cells were maintained at 37 °C with 5% CO2 for 3 h. Medium was removed and 100 µL of DMSO was added to each well. The absorbance of each well was read on the microplate absorbance readers (BIO-RAD) at wavelength of 490 nm.

### 2.5. Quantitative real-time polymerase chain reaction

Total RNA was isolated using a Machery-Nucleo Spin RNA II kit (Bioke, Leiden, The Netherlands) and quantified using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA). cDNA was prepared from total RNA using a cDNA Synthesis Kit (Takara Bio Inc, USA). The HEV RNA level was quantified using a SYBR Green-based real-time PCR assay (Applied Biosystems SYBR Green PCR Master Mix, Life Technologies, CA, USA) according to the manufacturer's instructions. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) was used as housekeeping gene to normalize gene expression using the 2^ΔΔCt method. The HEV primer sequences were 5'-GGTGGTTTCTGGGGTGAC-3' (sense) and 5'-AGGGGTTGGTTGGATGAA-3' (antisense), and the primers of GAPDH were 5'-GTCTCCTCTGACTTCAACAGCG-3' (sense) and 5'-ACCACCCTGTTGCTGTAGCCAA-3' (antisense).

### 2.6. Western blot assay

Proteins in cell lysates were heated at 95 °C for 5 min, followed by loading onto a 10% sodium dodecyl sulfate polyacrylamide gel (SDSPAGE), separated at 90 V for 120 min, and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (pore size: 0.45 mm; Thermo Fisher Scientific Life Sciences) for 120 min with an electric current of 250 mA. Subsequently, the membrane was blocked with blocking buffer (Li-Cor Biosciences) for 1 h at room temperature. Membrane was followed by incubation with primary antibodies anti-HEV ORF2, anti-STAT3, anti-NFκB p65 and anti-β-actin (1:1000) overnight at 4 °C. The membrane was washed 3 times followed by incubation for 1 h with anti-mouse or anti-rabbit IRDye-conjugated secondary antibodies (1:5000; Li-Cor Biosciences) at room temperature. After washing 3 times, protein bands were detected with Odyssey 3.0 Infrared Imaging System.

### 2.7. Immunofluorescence staining

Organoids were washed in cold AdMEM/F12 medium for 3 times to remove all basal matrix. Subsequently, organoids were added into the CytoSpin II Cytocentrifuge (Shandon Scientifi Ltd, Runcorn, England) and spun down into slides at 1000 rpm for 5 min. Then organoids or cells grown on µ-Slide 8 Well (ibidi GmbH, 80826) were fixed with 4% (w/v) paraformaldehyde (PFA) for 10 min at room temperature. The slides containing organoids or well plate with cells were then rinsed 3 times with PBS for 5 min each time, followed by permeabilizing with PBS containing 0.2% (vol/vol) tritonX100 for 5 min. Then the slides or plates were twice rinsed with PBS for 5 min, followed by incubation with blocking solution (5% donkey serum, 1% bovine serum albumin, 0.2% tritonX100 in PBS) at room temperature for 1 h. Next, slides or plates
were incubated in a humidity chamber with primary antibody diluted in blocking solution at 4 °C overnight. Primary antibodies used in this study are as follows: Anti-HEV ORF2 antibody (1:250, mouse mAb), anti-dsRNA antibody (1:500, mouse mAb), anti-albumin antibody (1:500, goat mAb). Excess primary antibodies were removed and the slides or plates were washed 3 times for 5 min each in PBS prior to 1 h incubation with 1:1000 dilutions of the anti-mouse IgG (H + L, Alexa Fluor® 594), the anti-rabbit IgG (H + L, Alexa Fluor® 488) and anti-goat IgG (H + L, Alexa Fluor® 594) secondary antibodies. Nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole; Invitrogen).

## 2.8. Statistical analysis

Statistical analysis was performed using the non-paired, non-parametric test (Mann-Whitney test; GraphPad Prism software, GraphPad Software Inc., La Jolla, CA). All results were presented as mean ± standard errors of the means (SEM). P values < 0.05 were considered as statistically significant.

# 3. Results

## 3.1. Niclosamide effectively inhibits HEV replication in multiple cell culture models

In this study, the potential anti-HEV effect of niclosamide was investigated in a variety of human hepatic cell models, based on sub-genomic replicon and full-length genome HEV infectious model with genotype 1 or 3. In the hepatic Huh7 cells harboring subgenomic genotype 3 HEV replicon, niclosamide treatment significantly inhibited viral replication-related luciferase activity in a time and dose-dependent manner. For example, at 72 h post-treatment, 5 or 10 µM niclosamide almost completely inhibited viral replication (Fig. 1A). The 50% inhibition concentration (IC50) of niclosamide against HEV replication was 0.96 µM, and the 50% cytotoxicity (CC50) value in Huh7 cells was 16.17 µM (Fig. 1B). The anti-HEV effect of niclosamide was further confirmed in the Huh7 infectious p6 cell model of HEV genotype 3 shown by both qRT-PCR assay (Fig. 1C) and western blotting assay (Fig. 1D). For instance, treatment with 5 µM of niclosamide for 48 h resulted in a 55.46% ± 3.14 (mean ± SEM, n = 20, p < 0.0001) (Fig. 1C) and 65.83% ± 2.88 (mean ± SEM, n = 4, p < 0.05) (Fig. 1D) inhibition of HEV RNA and the ORF2 capsid protein level, respectively. The inhibitory effect was further confirmed by immunofluorescence staining of ORF2 protein. As shown, niclosamide treatment resulted in a dose-dependent decrease of ORF2 protein expression, and the inhibition is remarkable even at the low concentration (1 µM) and much stronger at higher concentration (10 µM) (Fig. 1E). Furthermore, niclosamide also dose-dependently inhibited viral replication in Sar55 clone-based genotype 1 HEV subgenomic replicon (Fig. 1F).

Since the p6 clone is capable of producing infectious viral particles, the anti-HEV effect of niclosamide was further verified by a re-infection assay. We found the amount of produced HEV with infectivity was profoundly reduced by niclosamide treatment shown at both viral RNA and protein levels upon re-infection in Huh7 cells (Fig. 1G). Notably, HEV is more sensitive to niclosamide than ribavirin. For example, 10 µM niclosamide exhibited almost complete inhibition (Fig. 1G), whereas there was hardly antiviral activity of ribavirin at this concentration as potent anti-HEV activity of ribavirin requires concentrations ranging from 50 to 100 µM (Li et al., 2020; Wang et al., 2014). Furthermore, we confirmed the anti-HEV activity of niclosamide in Huh7 cells inoculated with cell culture derived infectious HEV particles shown at both viral RNA and protein levels (Fig. S1). Drug resistance is one of the main factors that limit the effectiveness of antiviral treatment. We next examined the effect of long-term treatment of niclosamide at a suboptimal concentration, which is intuitively prone to induce drug resistance mutagenesis. However, treatment with 2.5 µM niclosamide retained the inhibition of HEV viral RNA even after 21 days, and this effect was

further confirmed at ORF2 protein level (Fig. 1H).

Although primarily causing hepatitis, HEV infection associates with a broad range of extrahepatic manifestations, particularly kidney injury and neurological diseases (Drave et al., 2016; Huang et al., 2016; Zhou et al., 2017b). We thus further profiled the antiviral activity of niclosamide in a variety of cell models, including hepatic and non-hepatic cell lines harboring the HEV genotype 3 p6-Luc replicon. Similarly, niclosamide significantly inhibited viral replication-related luciferase activity in human kidney 293T (Fig. 2A), hepatic PLC (Fig. 2B), and neuronal U87 (Fig. 2C) cells. The IC50 and CC50 concentrations of niclosamide were 1.09 µM and 10.7 µM in 293T cell model, 0.16 µM and 134.9 µM in PLC cell model, and 0.16 µM and 6.74 µM in U87 cell model, respectively. These results indicate that the anti-HEV effect of niclosamide is universal across different cell types.

## 3.2. Niclosamide inhibits HEV in human liver organoids

We have previously established HEV infection in liver organoids, which allows the study of virus-host interactions and assessment of antiviral agents. Liver organoids, also called mini-liver, are stem cells-derived epithelial cultured in a 3D structure. They are much better at recapitulating the architecture, composition, diversity, organization, and functionality of cell types. In human liver organoids harboring subgenomic genotype 3 HEV replicon, niclosamide treatment significantly inhibited viral replication-related luciferase activity (Fig. 3A). This effect was further confirmed in liver organoids harbouring the genotype 3 p6 infectious HEV strain shown by both qRT-PCR quantification of viral RNA (Fig. 3B) and immunofluorescence staining of ORF2 protein (Fig. 3C). Notably, HEV is more sensitive to niclosamide than ribavirin. For instance, 5 µM niclosamide exhibited 60% inhibition, whereas there was hardly an antiviral activity of ribavirin at 10 µM. To more specifically examine at HEV replication, we performed immunofluorescence staining of viral double-strand RNA (dsRNA), the replicating intermediate. We found treatment with 5 µM niclosamide significantly reduced the level of dsRNA (Fig. 3D). In addition, niclosamide also inhibited viral replication in Sar55 clone-based genotype 1 HEV subgenomic replicon in liver organoids (Fig. 3E).

## 3.3. Niclosamide antagonizes the anti-HEV effects of IFNα and ribavirin

Since monotherapy of IFNα, ribavirin or their combination has been used as an off-label treatment for chronic hepatitis E and combination of multiple antiviral drugs is a common strategy to enhance efficacy and avoid resistance development. Thus, we evaluated their combinatorial effects with niclosamide. Surprisingly, we found that combination with niclosamide exhibited an antagonistic effect with IFNα (Fig. 4A) and ribavirin (Fig. 4B).

## 3.4. The anti-HEV effects of niclosamide is independent of the STAT3 signaling pathway

It has been reported that niclosamide confers multiple therapeutic effects for the treatment of cancer, infection, and metabolic diseases by interfering with activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway (Chen et al., 2018; Ren et al., 2010). Here, we found HEV infection potently activates STAT3 by using a transcriptional reporter (Fig. 5A). Functionally, activation of STAT3 by overexpression or treated with TFA or IL6 significantly promoted HEV replication in Huh7 cells harbouring the p6 infectious clone, shown by qRT-PCR quantification of viral RNA, and western blotting and immunofluorescence staining of ORF2 protein (Fig. 5B). Although niclosamide treatment significantly inhibited STAT3 activation (Fig. 5C), STAT3 overexpression or activation by TFA or IL6 hardly affected the anti-HEV activity of niclosamide (Fig. 5D).

To further confirm, we used lentiviral shRNA vectors to stably knockdown STAT3 gene in Huh7 cells (Fig. 5E). Two clones with
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

(caption on next page)
**Fig. 1.** Niclosamide inhibits HEV replication in Huh7-based cell culture models. (A) The effects of niclosamide treatment for 24, 48 or 72 h on viral replication related luciferase activity in the genotype 3 subgenomic Huh7-p6-Luc cell model. The untreated group serves as control (CTR) (set as 1) (n = 8–10). (B) The 50% inhibitory concentration (IC50) and 50% cytotoxic concentration (CC50) of niclosamide in Huh7-p6-Luc cell model and Huh7 cell line were calculated using GraphPad Prism software (n = 6). (C) The infectious Huh7-p6 cell model was treated with indicated concentrations of niclosamide for 48 h. The effects on viral RNA was quantified by qRT-PCR (n = 8–12). (D) Western blot analysis of HEV capsid protein level in Huh7-p6 cells treated with niclosamide for 48 h. The uninfected group (mock) serves as the negative control, and the infected but untreated group serves as the positive control (set as 1%) (n = 4). (E) Immunofluorescence analysis of viral ORF2-encoded capsid protein (red) in Huh7 cells treated with indicated concentrations of niclosamide for 48 h. Huh7 cells incubated with the anti-HEV capsid protein antibody serves as the negative control. HEV infected Huh7 cells untreated and incubated with the anti-HEV capsid protein antibody serves as the positive control. DAPI (blue) was applied to visualize nuclei. (Scale bar, 100 µm. 40× oil immersion objective). (F) Huh7 cell based genotype 1 HEV replicon (Sar55 clone) was treated with niclosamide for 24, 48 or 72 h and viral replication related luciferase activity was measured (n = 8). (G) Naïve Huh7 cells were inoculated with HEV particles produced from HEV infected Huh7 cells 48 h post-treatment with different concentrations of niclosamide or ribavirin (untreated set as control group). After the inoculation for 48 h, the Huh7 cells were subjected to qRT-PCR analysis of HEV RNA (n = 5–8) and Western blotting and Immunofluorescence analysis of HEV ORF2 protein. (Scale bar, 100 µm. 40× oil immersion objective). (H) The effects of long-term treatment with 2.5 µM niclosamide on HEV RNA in Huh7-p6 cell model were quantified by qRT-PCR. The untreated (CTR) group serve as control (set as 1) (n = 4). Western blot analysis of HEV ORF2-encoded capsid protein was performed on 21 days of treatment. RLU: relative luciferase unit. Data are presented as means ± SEM. (*P < 0.05; **P < 0.01; ***P < 0.001).

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

**Fig. 2.** Niclosamide inhibits HEV replication in different human cell culture models. The effects of niclosamide treatment on viral replication related luciferase activity and the 50% inhibitory concentration (IC50) against HEV replication and 50% cytotoxic concentration (CC50) of niclosamide were calculated using GraphPad Prism software in kidney 293T cells (n = 6–12) (A), PLC/PRF/5 cells (n = 6–12) (B), or neuronal U87 cells (n = 6–12) (C). The untreated (CTR) group serve as control (set as 1). RLU: relative luciferase unit. Data are presented as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).

optimal gene knockdown levels were selected for subsequent functional experimentation. Compared with control, knockdown of STAT3 by the shSTAT3 clones 2 and 4 led to 65.25% ± 5.44 (mean ± SEM, n = 4, p < 0.05) and 63.95% ± 9.12 (mean ± SEM, n = 4) reduction of intracellular

HEV RNA respectively (Fig. 5F), suggesting that STAT3 is essential for efficient HEV replication. However, the anti-HEV activity of niclosamide was not affected in STAT3 knockdown cells (Fig. 5G), demonstrating that the anti-HEV effect of niclosamide is independent of inhibiting the
<figure><img src="image_15.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_19.png" /></figure>

**Fig. 3.** Niclosamide against HEV replication in human liver organoids models. (A) The effects of niclosamide treatment for 24, 48 or 72 h on viral replication related luciferase activity in the genotype 3 subgenomic human liver organoids model. The untreated group serves as control (CTR) (set as 1) (n = 9–10). (B) The infectious p6 human liver organoids model was treated with indicated concentrations of niclosamide and ribavirin for 48 h. The effects on viral RNA was quantified by qRT-PCR (n = 5–8). Immunofluorescence analysis of viral ORF2-encoded capsid protein (C), or viral double-strand RNA (dsRNA) (D) in human liver organoids treated with 5 µM niclosamide for 48 h. Uninfected liver organoids incubated with the anti-HEV capsid protein antibody or anti-dsRNA antibody serves as the negative control. HEV infected liver organoids untreated and incubated with the anti-HEV capsid protein antibody or anti-dsRNA antibody serve as the positive control. DAPI (blue) and Epcam (green) were applied to visualize nuclei and cytomembrane. (Scale bar, 100 µm. 40× oil immersion objective). (E) Human liver organoids based genotype 1 HEV replicon (Sar55 clone) was treated with niclosamide for 24, 48 or 72 h and viral replication related luciferase activity was measured (n = 10). The untreated (CTR) group serve as control (set as 1). RLU: relative luciferase unit. Data are presented as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).

STAT3 signaling pathway.

### 3.5. Niclosamide inhibits HEV through targeting NFκB

Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) plays a central role in promoting the expression of genes involved in inflammation and is activated in various infections (Tai

et al., 2000). It has been reported that HEV infected cells showing activation of NFκB at p65 protein level (Devhare et al., 2013). Moreover, niclosamide has been shown to inhibit NFκB activity (Jin et al., 2010). We thus investigated the potential role of NFκB signaling in niclosamide-mediated anti-HEV effect. A lentiviral vector expressing the firefly luciferase gene under the control of the NFκB promoter was transduced into Huh7 cells. We found that inoculation of HEV particles
<figure><img src="image_20.png" /><figcaption>**Fig. 4.** Niclosamide antagonizes the anti-HEV effects of IFNα and ribavirin. The antiviral effects of niclosamide in combination with IFNα (A) and ribavirin (B) were analyzed by the MacSynergyII model. The three-dimensional surface plot represents the differences (within 95% confidence interval) between actual experimental effects and theoretical additive effects of the combination at various concentrations (n = 4).</figcaption></figure>

significantly activates the NFκB transcription activity (Fig. 6A). By supplementation of the potent NFκB activator TNFα, NFκB transcription activity was significantly activated and correlated with enhanced viral replication at both viral RNA and protein levels (Fig. 6B). In contrast to TNFα, treatment with niclosamide dose-dependent inhibited the NFκB transcription activity and protein expression (Fig. 6C). Interestingly, supplementation with TNFα almost completely reversed the anti-HEV activity of niclosamide (Fig. 6D), suggesting the possible involvement of NFκB in the anti-HEV action of niclosamide.

To further confirm, lentiviral shRNA vectors were used to stably knockdown the NFκB p65 gene in Huh7 cells. The gene silencing efficiency was confirmed at both mRNA and protein levels (Fig. 6E), as compared to control. Two clones with optimal gene knockdown were selected for subsequent functional experimentation. Compared with control, knockdown of NFκB p65 by the shNFκB p65 clone 1 or 2 led to 44.58% ± 4.87 (mean ± SEM, n = 8, p < 0.01) and 61.83% ± 5.46 (mean ± SEM, n = 8, p < 0.001) reduction of intracellular HEV RNA, respectively (Fig. 6F), suggesting that NFκB p65 supports HEV replication. Importantly, the anti-HEV activity of niclosamide was almost completely abolished in the NFκB p65 knockdown clone 1 or 2 cells (Fig. 6G), demonstrating the requirement of NFκB for the anti-HEV action of niclosamide.

## 4. Discussion

To facilitate the discovery of new anti-HEV agents that can be expeditiously developed into clinical application, we have recently screened a library of known safe-in-human broad-spectrum antiviral agents (Andersen et al., 2020; Li et al., 2020). Among the identified several candidates, this study focused on validating the anti-HEV activity of niclosamide and exploring the potential mechanism-of-action.

Niclosamide is an FDA-approved anthelmintic drug to treat worm infections for approximately 50 years and is currently listed on the World Organization's list of essential medicines (Andrews et al., 1982; Organization, 2019). Interestingly, niclosamide has shown antiviral activity against a broad range of RNA and DNA viruses including dengue virus (Kao et al., 2018), Zika virus (Xu et al., 2016), hepatitis C virus (Stachulski et al., 2011), Epstein-Barr virus (Huang et al., 2017), Ebola virus (Hulseberg et al., 2019), human rhinoviruses (Jurgeit et al., 2012), human adenoviruses (Marrugal-Lorenzo et al., 2019), chikungunya virus (Wang et al., 2016), and coronaviruses such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus (Gassen et al., 2019; Wen et al., 2007). In this study, the anti-HEV effects of niclosamide were demonstrated on viral genome, viral protein expression, dsRNA replication, and viral release in multiple models including cell line and human liver organoids based systems with both genotype 1 and 3 strains.

More importantly, niclosamide is cheap and well-tolerated in vivo, for example with an extremely high acute oral LD₅₀ value of >5000 mg/kg in rats (Andrews et al., 1982). In human, single oral doses of 0.5, 1, and 2 g of niclosamide are recommended for children under 2 years, children between 2 and 6 years, and children older than 6 years and adults, respectively. The serum concentration of niclosamide after a single dose of 2 g leads to maximal serum concentrations of 0.25–6.0 µg/mL (corresponding to 0.76–18.35 µM) in rats (Andrews et al., 1982), which is in the range showing anti-HEV activities in our models. Nevertheless, niclosamide has several limitations such as poor aqueous solubility and oral bioavailability (F = 10%), which may hinder its further clinical development for drug repurposing such as treating HEV infection (Chang YE, 2006). In this context, efforts are dedicating to the development of innovative formulations aiming at efficient drug delivery. A series of O-alkylamino-tethered derivatives based on
<figure><img src="image_21.png" /><figcaption>**Fig. 5.** STAT3 facilitates HEV replication and the anti-HEV activity of niclosamide is independent of the STAT3 signaling. (A) Mock or HEV infected Huh7 cells were transiently transfected with STAT3-luc and TK-Renilla (used to normalize the transfection efficiency). The luciferase activity was determined 48 h post-transfection. The uninfected group serves as control (CTR) (set as 1) (n = 8). (B) The infectious Huh7-p6 cell model was transiently transfected with STAT3 or treated with TFA (1 µM)/IL6 (50 ng/mL) for 48 h. The effects were quantified by qRT-PCR on viral RNA (n = 4–6) and Western blotting and immunofluorescence analysis of HEV ORF2 protein. (Scale bar, 100 µm. 40× oil immersion objective). (C) The Huh7 cells based STAT3-luc and TK-Renilla reporter model was treated with 5 µM niclosamide for 48 h before testing the luciferase activity, and the uninfected group serves as control (CTR) (set as 1) (n = 8). Western blot analysis of STAT3 protein level in Huh7 cells treated with indicated concentrations of niclosamide for 48 h. (D) HEV RNA in Huh7-p6 cells was quantified by qRT-PCR after 48 h treatment with 5 µM niclosamide co-incubation of STAT3 overexpression, 1 µM of TFA, or 50 ng/mL of IL6. The untreated group serves as control (CTR) (set as 1) (n = 4). (E) Knockdown of STAT3 by lentiviral shRNA vectors. The effects on STAT3 gene of 5 shSTAT3 clones were quantified by qRT-PCR and Western blot analysis of STAT3 protein level. The expression of control vector transduced cells was set as 1. (F) The effects of selected STAT3 knockdown on cellular HEV RNA levels in HEV infected Huh7 cell model. Viral RNA was quantified by qRT-PCR (n = 4). (G) STAT3 knockdown and control Huh7 cells harboring the infectious HEV model were treated with 5 µM niclosamide for 48 h. The effect on viral RNA was quantified by qRT-PCR (n = 4). Data are presented as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).</figcaption></figure>

niclosamide with improved aqueous solubility and orally bioavailable in *vitro* and *in vivo* have been developed (Chen et al., 2013). The acyl derivative of niclosamide was also reported to increase its systemic drug exposure and extend the duration of exposure when dosed orally (Mook et al., 2015). Nanostructured systems are also explored to improve the pharmacological and pharmacokinetic properties of niclosamide demonstrated in mice and rabbits (Lin et al., 2016; Rehman et al., 2018). Thus, the advanced drug delivery systems would likely facilitate drug repurposing of niclosamide for possibly treating many disease like HEV infection. Genotype 1 HEV exclusively causes acute infection, whereas most of the chronic cases are attributed to genotype 3. Therefore, our *in vitro* models based on genotype 1 and 3 HEV strains can recapitulate acute and chronic HEV infections to some extent, but validation in animal models with acute and chronic infections is highly recommended

for future research.

In addition to its function as an anthelmintic drug, niclosamide has multiple cellular targets, such as blocking the mTOR, STAT3, and NFκB signaling pathways (Chen et al., 2018; Li et al., 2014). These signaling cascades are essentially involved in viral infections and cancer development (Diehl and Schaal, 2013; Li et al., 2014). STAT3 regulates inflammation and immunity exhibiting dual regulatory activities during viral infection. We found HEV infection potently activates STAT3 and overexpression or activation of STAT3 facilitates HEV infection. However, we demonstrated the anti-HEV activity of niclosamide is independent of the STAT3 signaling pathway.

Many viruses such as dengue virus and influenza virus have been reported to activate NFκB to support their replication (Cassens et al., 2003; Nimmerjahn et al., 2004). We (Fig. 6) and other groups (Devhare
<figure><img src="image_22.png" /><figcaption>**Fig. 6.** Activating NFκB facilitates HEV replication and the anti-HEV effect of niclosamide requires its inhibition on NFκB activity. (A) The Huh7 cells based NFκB reporter model was inoculated with culture medium containing HEV particles. After 24 h inoculation, the medium was changed to normal cell culture medium without HEV particles and the NF-κB luciferase was measured at 48 h post-infection. The uninfected group serves as control (CTR) (set as 1) (n = 16). (B) The Huh7 cells based NFκB reporter model or Huh7 based HEV infectious model was treated with 200 ng/mL TNFα for 48 h before testing NFκB luciferase value (n = 12), HEV RNA level by qRT-PCR assay (n = 6), and HEV ORF2 protein by immunofluorescence staining. (Scale bar, 100 µm. 40× oil immersion objective). The untreated group serves as control (CTR) (set as 1). (C) The Huh7 cells based NFκB reporter model or Huh7 cells treated with indicated concentrations of niclosamide for 48 h before testing the luciferase activity (n = 8–12) and protein level. The untreated mock group serves as control (set as 1). (D) HEV RNA in Huh7-p6 cells was quantified by qRT-PCR after 48 h treatment with 5 µM niclosamide by adding 200 ng/mL TNFα. Data in niclosamide-treated group were presented relative to the untreated control group (set as 1). Data in the combination group of niclosamide with TNFα were presented relative to TNFα-solely treated group (set as 1) (n = 6–10). (E) Knockdown of NFκB p65 by lentiviral shRNA vectors. The effects on NFκB p65 gene of 5 shNFκB p65 clones were quantified by qRT-PCR and Western blot analysis of NFκB p65 protein level. The expression of control vector transduced cells was set as 1. (F) The effects of selected NFκB p65 knockdown on cellular HEV RNA levels in HEV infected Huh7 cell model. Viral RNA was quantified by qRT-PCR (n = 8). (G) NFκB p65 knockdown and control Huh7 cells harboring the infectious HEV model were treated with 5 µM niclosamide for 48 h. The effect on viral RNA was quantified by qRT-PCR (n = 7–8). Data are presented as means ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001).</figcaption></figure>
et al., 2013; Qu et al., 2019) have extensively demonstrated that HEV infected cells showed activation of NFkB transcription, and the NFkB activator enhanced HEV replication. Consistent with a study in patients, liver biopsies from pregnant women infected with HEV who developed fulminant hepatic failure (FHF) have higher transcriptional activity of NFkB compared to those from nonpregnant women or pregnant women infected HEV without the development of FHF (Prusty et al., 2007). Targeting NFkB signaling by niclosamide has been well studied. For example, a recent study showed that niclosamide abrogated the activation of the NFkB pathway induced by TNFα (Zhou et al., 2017a). Interestingly, we found exogenously adding TNFα to activate NFkB largely abrogated the anti-HEV activity of niclosamide. Using loss-of-function assay by knockdown NFkB p65, we demonstrated that NFkB is essentially required for the antiviral function of niclosamide.

In summary, we have identified that niclosamide potently inhibits HEV infection. The anti-HEV activity of niclosamide mainly through the inhibition of NFkB signaling, but the detailed mechanisms remain to be further characterized. Given that niclosamide is a safe and cheap drug that has been widely used as a single dose formulation to treat worm infections in the clinic, repurposing niclosamide provides a unique opportunity for treating HEV infection. However, further validation in HEV animal model is warranted, and the development of advanced niclosamide formulations with improved bioavailability is essential for future clinical applications.

## Funding

This study is supported by a VIDI grant (No. 91719300) from the Netherlands Organisation for Scientific Research (NWO) to Q. Pan, and the China Scholarship Council for funding PhD fellowship to Yunlong L (No. 201708530243), P.L (No. 201808370170), R.Z. (No. 201808530490), and Yang. L (No. 201703250073).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgement

The authors would like to thank Dr. Suzanne U. Emerson (National Institute of Allergy and Infectious Diseases, NIH, USA) for generously providing the plasmids to generate subgenomic and full-length HEV genomic RNA and Michael R. Beard (University of Adelaide) kindly provided STAT3 and STAT3-Luc plasmid.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.antiviral.2021.105228.

## References

* Andersen, P.I., Ianevski, A., Lysvand, H., Vitkauskiene, A., Oksenych, V., Bjorås, M., Telling, K., Lutsar, I., Dumpis, U., Irie, Y., Tenson, T., Kantele, A., Kainov, D.E., 2020. Discovery and development of safe-in-man broad-spectrum antiviral agents. Int. J. Infect. Dis. 93, 268-276.

* Andrews, P., Thyssen, J., Lorke, D., 1982. The biology and toxicology of molluscicides, Bayluscide. Pharmacol. Ther. 19, 245-295.

* Cassens, U., Lewinski, G., Samraj, A.K., von Bernuth, H., Baust, H., Khazaie, K., Los, M., 2003. Viral modulation of cell death by inhibition of caspases. Arch. Immunol. Ther. Exp. 51, 19-27.

* Chang, Y.E., Y., T., Lin, K.T., Chen, W.C., Yao, H.T., Lan, S.J., Wu, Y.S., Hsieh, H.P., Chen, C.M., Chen, C.T., 2006. Pharmacokinetics of anti-SARS-CoV agent niclosamide and its analogs in rats. J. Food Drug Anal. 14, 329-333.

* Chen, H., Yang, Z., Ding, C., Chu, L., Zhang, Y., Terry, K., Liu, H., Shen, Q., Zhou, J., 2013. Discovery of O-alkylamino tethered niclosamide derivatives as potent and orally bioavailable anticancer agents. ACS Med. Chem. Lett. 4, 180-185.

* Chen, W., Mook Jr., R.A., Premont, R.T., Wang, J., 2018. Niclosamide: beyond an antihelminthic drug. Cell. Signal. 41, 89-96.

* Debing, Y., Gisa, A., Dallmeier, K., Pischke, S., Bremer, B., Manns, M., Wedemeyer, H., Suneetha, P.V., Neyts, J., 2014. A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients. Gastroenterology 147, 1008-1011 e1007; quiz e1015-1006.

* Debing, Y., Moradpour, D., Neyts, J., Gouttenoire, J., 2016. Update on hepatitis E virology: implications for clinical practice. J. Hepatol. 65, 200-212.

* Devhare, P.B., Chatterjee, S.N., Arankalle, V.A., Lole, K.S., 2013. Analysis of antiviral response in human epithelial cells infected with hepatitis E virus. PLoS One 8, e63793.

* Diehl, N., Schaal, H., 2013. Make yourself at home: viral hijacking of the PI3K/Akt signaling pathway. Viruses 5, 3192-3212.

* Drave, S.A., Debing, Y., Walter, S., Todt, D., Engelmann, M., Friesland, M., Wedemeyer, H., Neyts, J., Behrendt, P., Steinmann, E., 2016. Extra-hepatic replication and infection of hepatitis E virus in neuronal-derived cells. J. Viral Hepat. 23, 512-521.

* Gassen, N.C., Niemeyer, D., Muth, D., Corman, V.M., Martinelli, S., Gassen, A., Hafner, K., Papies, J., Mösbauer, K., Zellner, A., Zannas, A.S., Herrmann, A., Holsboer, F., Brack-Werner, R., Boshart, M., Müller-Myhsok, B., Drosten, C., Müller, M.A., Rein, T., 2019. SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection. Nat. Commun. 10, 5770.

* Haagsma, E.B., Riezebos-Brilman, A., van den Berg, A.P., Porte, R.J., Niesters, H.G., 2010. Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b. Liver Transplant. 16, 474-477.

* Hakim, M.S., Ikram, A., Zhou, J., Wang, W., Peppelenbosch, M.P., Pan, Q., 2018. Immunity against hepatitis E virus infection: implications for therapy and vaccine development. Rev. Med. Virol. 28.

* Hakim, M.S., Wang, W., Bramer, W.M., Geng, J., Huang, F., de Man, R.A., Peppelenbosch, M.P., Pan, Q., 2017. The global burden of hepatitis E outbreaks: a systematic review. Liver Int. 37, 19-31.

* Huang, F., Yang, C., Zhou, X., Yu, W., Pan, Q., 2016. Rhesus macaques persistently infected with hepatitis E shed virus into urine. J. Hepatol. 64, 1446-1447.

* Huang, L., Yang, M., Yuan, Y., Li, X., Kuang, E., 2017. Niclosamide inhibits lytic replication of Epstein-Barr virus by disrupting mTOR activation. Antivir. Res. 138, 68-78.

* Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M.M., Ellis, E., van Wenum, M., Fuchs, S.A., de Ligt, J., van de Wetering, M., Sasaki, N., Boers, S.J., Kemperman, H., de Jonge, J., Ijzermans, J.N., Nieuwenhuis, E.E., Hoekstra, R., Strom, S., Vries, R.R., van der Laan, L.J., Cuppen, E., Clevers, H., 2015. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299-312.

* Hulseberg, C.E., Févéant, L., Szymańska-de Wijs, K.M., Kessler, N.P., Nelson, E.A., Shoemaker, C.J., Schmaljohn, C.S., Polyak, S.J., White, J.M., 2019. Arbidol and other low-molecular-weight drugs that inhibit Lassa and Ebola viruses. J. Virol. 93.

* Jin, Y., Lu, Z., Ding, K., Li, J., Du, X., Chen, C., Sun, X., Wu, Y., Zhou, J., Pan, J., 2010. Antineoplastic mechanisms of niclosamide in acute myelogenous leukemia stem cells: inactivation of the NF-kappaB pathway and generation of reactive oxygen species. Cancer Res. 70, 2516-2527.

* Jurgeit, A., McDowell, R., Moese, S., Meldrum, E., Schwendener, R., Greber, U.F., 2012. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects. PLoS Pathog. 8, e1002976.

* Kamar, N., Abravanel, F., Behrendt, P., Hofmann, J., Pageaux, G.P., Barbet, C., Moal, V., Couzi, L., Horvatitis, T., De Man, R.A., Cassuto, E., Elsharkawy, A.M., Riezebos-Brilman, A., Scemla, A., Hillaire, S., Donnelly, M.C., Radenne, S., Sayegh, J., Garrouste, C., Dumortier, J., Glowaki, F., Matignon, M., Coilly, A., Figueres, L., Mousson, C., Minello, A., Dharancy, S., Rerolle, J.P., Lebray, P., Etienne, I., Perrin, P., Choi, M., Marion, O., Izopet, J., Hepatitis. E.V.R.S.G., 2020. Ribavirin for hepatitis E virus infection after organ transplantation: a large European retrospective multicenter study. Clin. Infect. Dis. 71, 1204-1211.

* Kamar, N., Izopet, J., Tripon, S., Bismuth, M., Hillaire, S., Dumortier, J., Radenne, S., Coilly, A., Garrigue, V., D'Alteroche, L., Buchler, M., Couzi, L., Lebray, P., Dharancy, S., Minello, A., Hourmant, M., Roque-Afonso, A.M., Abravanel, F., Pol, S., Rostaing, L., Mallet, V., 2014. Ribavirin for chronic hepatitis E virus infection in transplant recipients. N. Engl. J. Med. 370, 1111-1120.

* Kamar, N., Rostaing, L., Abravanel, F., Garrouste, C., Esposito, L., Cardeau-Desangles, I., Mansuy, J.M., Selves, J., Peron, J.M., Otal, P., Muscari, F., Izopet, J., 2010. Pegylated interferon-alpha for treating chronic hepatitis E virus infection after liver transplantation. Clin. Infect. Dis. 50, e30-33.

* Kamar, N., Selves, J., Mansuy, J.M., Ouezzani, L., Péron, J.M., Guitard, J., Cointault, O., Esposito, L., Abravanel, F., Danjoux, M., Durand, D., Vinel, J.P., Izopet, J., Rostaing, L., 2008. Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N. Engl. J. Med. 358, 811-817.

* Kao, J.C., HuangFu, W.C., Tsai, T.T., Ho, M.R., Jhan, M.K., Shen, T.J., Tseng, P.C., Wang, Y.T., Lin, C.F., 2018. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Neglected Trop. Dis. 12, e0006715.

* Li, Y., Li, P., Li, Y., Zhang, R., Yu, P., Ma, Z., Kainov, D.E., de Man, R.A., Peppelenbosch, M.P., Pan, Q., 2020. Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation. Antivir. Res. 184, 104967.

* Li, Y., Li, P.K., Roberts, M.J., Arend, R.C., Samant, R.S., Buchsbaum, D.J., 2014. Multi-targeted therapy of cancer by niclosamide: a new application for an old drug. Cancer Lett. 349, 8-14.
Lin, C.K., Bai, M.Y., Hu, T.M., Wang, Y.C., Chao, T.K., Weng, S.J., Huang, R.L., Su, P.H., Lai, H.C., 2016. Preclinical evaluation of a nanoformulated antihelminthic, niclosamide, in ovarian cancer. Oncotarget 7, 8993–9006.

Marrugal-Lorenzo, J.A., Serna-Gallego, A., Berastegui-Cabrera, J., Pachón, J., Sánchez-Cespedes, J., 2019. Repositioning salicylanilide anthelmintic drugs to treat adenovirus infections. Sci. Rep. 9, 17.

Mook Jr., R.A., Wang, J., Ren, X.R., Chen, M., Spasojevic, I., Barak, L.S., Lyerly, H.K., Chen, W., 2015. Structure-activity studies of Wnt/β-catenin inhibition in the Niclosamide chemotype: identification of derivatives with improved drug exposure. Bioorg. Med. Chem. 23, 5829–5838.

Nimmerjahn, F., Dudziak, D., Dirmeier, U., Hobom, G., Riedel, A., Schlee, M., Staudt, L. M., Rosenwald, A., Behrends, U., Bornkamm, G.W., Mautner, J., 2004. Active NF-κappaB signalling is a prerequisite for influenza virus infection. J. Gen. Virol. 85, 2347–2356.

Organization, W.H., 2019. World health organization model list of essential medicines. 21st List. WHO REFERENCE NUMBER: WHO/MVP/ EMP/IAU/2019.06.

Prusty, B.K., Hedau, S., Singh, A., Kar, P., Das, B.C., 2007. Selective suppression of NF-κBp65 in hepatitis virus-infected pregnant women manifesting severe liver damage and high mortality. Mol. Med. 13, 518–526.

Pushpakom, S., Iorio, F., Eyers, P.A., Escott, K.J., Hopper, S., Wells, A., Doig, A., Guilliams, T., Latimer, J., McNamee, C., Norris, A., Sanseau, P., Cavalla, D., Pirmohamed, M., 2019. Drug repurposing: progress, challenges and recommendations. Nat. Rev. Drug Discov. 18, 41–58.

Qu, C., Li, Y., Li, Y., Yu, P., Li, P., Donkers, J.M., van de Graaf, S.F.J., de Man, R.A., Peppelenbosch, M.P., Pan, Q., 2019. FDA-drug screening identifies depropine inhibiting hepatitis E virus involving the NF-κB-RIPK1-caspase axis. Antivir. Res. 170, 104588.

Rehman, M.U., Khan, M.A., Khan, W.S., Shafique, M., Khan, M., 2018. Fabrication of Niclosamide loaded solid lipid nanoparticles: in vitro characterization and comparative in vivo evaluation. Artif Cells Nanomed Biotechnol 46, 1926–1934.

Ren, X., Duan, L., He, Q., Zhang, Z., Zhou, Y., Wu, D., Pan, J., Pei, D., Ding, K., 2010. Identification of niclosamide as a new small-molecule inhibitor of the STAT3 signaling pathway. ACS Med. Chem. Lett. 1, 454–459.

Rostaing, L., Izopet, J., Baron, E., Duffaut, M., Puel, J., Durand, D., 1995. Treatment of chronic hepatitis C with recombinant interferon alpha in kidney transplant recipients. Transplantation 59, 1426–1431.

Stachulski, A.V., Pidathala, C., Row, E.C., Sharma, R., Berry, N.G., Lawrenson, A.S., Moores, S.L., Iqbal, M., Bentley, J., Allman, S.A., Edwards, G., Helm, A., Hellier, J., Korba, B.E., Semple, J.E., Rossignol, J.F., 2011. Thiazolides as novel antiviral agents. 2. Inhibition of hepatitis C virus replication. J. Med. Chem. 54, 8670–8680.

Tai, D.I., Tsai, S.L., Chang, Y.H., Huang, S.N., Chen, T.C., Chang, K.S., Liaw, Y.F., 2000. Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma. Cancer 89, 2274–2281.

Todt, D., François, C., Anggakusuma Behrendt, P., Engelmann, M., Knegendorf, L., Vieyres, G., Wedemeyer, H., Hartmann, R., Pietschmann, T., Duverlie, G., Steinmann, E., 2016. Antiviral activities of different interferon types and subtypes against hepatitis E virus replication. Antimicrob. Agents Chemother. 60, 2132–2139.

Todt, D., Meister, T.L., Steinmann, E., 2018. Hepatitis E virus treatment and ribavirin therapy: viral mechanisms of nonresponse. Curr Opin Virol 32, 80–87.

Wang, Y., Chen, G., Pan, Q., Zhao, J., 2018. Chronic hepatitis E in a renal transplant recipient: the first report of genotype 4 hepatitis E virus caused chronic infection in organ recipient. Gastroenterology 154, 1199–1201.

Wang, Y., Zhou, X., Debing, Y., Chen, K., Van Der Laan, L.J., Neyts, J., Janssen, H.L., Metselaar, H.J., Peppelenbosch, M.P., Pan, Q., 2014. Calcineurin inhibitors stimulate and mycophenolic acid inhibits replication of hepatitis E virus. Gastroenterology 146, 1775–1783.

Wang, Y.M., Lu, J.W., Lin, C.C., Chin, Y.F., Wu, T.Y., Lin, L.I., Lai, Z.Z., Kuo, S.C., Ho, Y. J., 2016. Antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission. Antivir. Res. 135, 81–90.

Wedemeyer, H., Pischke, S., Manns, M.P., 2012. Pathogenesis and treatment of hepatitis E virus infection. Gastroenterology 142, 1388–1397 e1381.

Wen, C.C., Kuo, Y.H., Jan, J.T., Liang, P.H., Wang, S.Y., Liu, H.G., Lee, C.K., Chang, S.T., Kuo, C.J., Lee, S.S., Hou, C.C., Hsiao, P.W., Chien, S.C., Shyur, L.F., Yang, N.S., 2007. Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus. J. Med. Chem. 50, 4087–4095.

Xu, M., Lee, E.M., Wen, Z., Cheng, Y., Huang, W.K., Qian, X., Tcw, J., Kouznetsova, J., Ogden, S.C., Hammack, C., Jacob, F., Nguyen, H.N., Itkin, M., Hanna, C., Shinn, P., Allen, C., Michael, S.G., Simeonov, A., Huang, W., Christian, K.M., Goate, A., Brennand, K.J., Huang, R., Xia, M., Ming, G.L., Zheng, W., Song, H., Tang, H., 2016. Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen. Nat. Med. 22, 1101–1107.

Zhou, J., Jin, B., Jin, Y., Liu, Y., Pan, J., 2017a. The antihelminthic drug niclosamide effectively inhibits the malignant phenotypes of uveal melanoma in vitro and in vivo. Theranostics 7, 1447–1462.

Zhou, X., de Man, R.A., de Knegt, R.J., Metselaar, H.J., Peppelenbosch, M.P., Pan, Q., 2013. Epidemiology and management of chronic hepatitis E infection in solid organ transplantation: a comprehensive literature review. Rev. Med. Virol. 23, 295–304.

Zhou, X., Huang, F., Xu, L., Lin, Z., de Vrij, F.M.S., Ayo-Martin, A.C., van der Kroeg, M., Zhao, M., Yin, Y., Wang, W., Cao, W., Wang, Y., Kushner, S.A., Marie Peron, J., Alric, L., de Man, R.A., Jacobs, B.C., van Eijk, J.J., Aronica, E.M.A., Sprengers, D., Metselaar, H.J., de Zeeuw, C.I., Dalton, H.R., Kamar, N., Peppelenbosch, M.P., Pan, Q., 2017b. Hepatitis E virus infects neurons and brains. J. Infect. Dis. 215, 1197–1206.