Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model
- Zixue Shi1Email author,
- Jianchao Wei1Email author,
- Xufang Deng1,
- Shuqing Li2,
- Yafeng Qiu1,
- Donghua Shao1,
- Beibei Li1,
- Keyu Zhang1,
- Feiqun Xue1,
- Xiaodu Wang3 and
- Zhiyong Ma1Email author
© Shi et al.; licensee BioMed Central Ltd. 2014
Received: 10 July 2013
Accepted: 15 January 2014
Published: 23 January 2014
Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk’ regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model.
JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated.
NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 μg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 μg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV.
Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.
KeywordsJapanese encephalitis virus Nitazoxanide (NTZ) Antiviral
50% effective concentration
50% cytotoxic concentration
3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Multiplicity of infection
Plaque forming units.
The mechanism of activity of NTZ against non-viral anaerobic microorganisms is attributed to its interference with pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reactions, which are essential for anaerobic energy metabolism . By contrast, the mechanisms that underlie the antiviral activity of NTZ are not well understood. NTZ has been described to induce PKR (double-stranded-RNA-activated protein kinase) phosphorylation, which leads to the elevation of phosphorylated eIF2α eukaryotic translation initiation factor 2 alpha, an antiviral intracellular protein, in HCV-infected cells . While in influenza virus-infected cells, NTZ prevents the maturation of viral hemagglutinin (HA) protein possibly by blocking HA trafficking between the endoplasmic reticulum and the Golgi complex .
Japanese encephalitis (JE), previously known as Japanese B encephalitis, is caused by Japanese encephalitis virus (JEV), an enveloped arbovirus of the Flavivirus genus in the family Flaviviridae. JE is most prevalent in Southeast Asia and the Far East, and domestic pigs and wild birds are the natural reservoirs of JEV. Transmission of JEV to humans, which occurs mainly via the bite of infected mosquitoes, may cause severe symptoms typically including fever, headache and other incapacitating manifestations . Although two types of JEV vaccines have long been used, JE still causes 10,000-15,000 encephalitic deaths throughout the world annually . As an arthropod-borne infectious disease, JE has recently spread its geographic footprint into previously non-endemic areas . An estimated three billion people in 'at-risk’ regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing because of population growth, intensified rice farming, pig rearing, climate change and the lack of vaccination programs and surveillance . Consequently, there is an urgent need for the development of novel therapeutic agents against JE. Although attempts to develop new antiviral drug are ongoing , no effective antiviral drug against JE is available at present.
JEV and HCV belong to the family Flaviviridae, and NTZ inhibits the replication of HCV both in cell culture  and in patients . The emergence of NTZ with broad-spectrum antiviral properties prompted us to evaluate its antiviral activity against JEV. We found that NTZ inhibited the replication of JEV in cultured cells and reduced mortality in mice challenged with a lethal dose of JEV.
NTZ inhibits the JEV-induced cytopathic effect (CPE)
NTZ inhibits the replication of JEV in BHK-21 cells
To further characterize the antiviral property of NTZ, the JEV-infected BHK-21 cells were treated with NTZ at a concentration of 3 μg/ml and incubated for the indicated times (Figure 3B). The NTZ concentration of 3 μg/ml was chosen because at this concentration NTZ cytotoxicity had no significance and approximately 100% of virus titer reduction was observed (Figure 2A and Figure 3A). The JEV-infected cells that were parallelly treated with DMSO were used as a control. The titers of virus in the cells and in the supernatants were measured by a plaque assay, respectively. In the DMSO-treated cells, the infectious progeny viruses, both in the cells and the supernatants, were detectable from 12 h post-infection, while they were not detectable until 24 h post-infection in the NTZ-treated cells. The viral yields from the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the DMSO-treated cells (Figure 3B). A decline in viral titer was observed 60 h post-infection in the DMSO-treated cells. This decrease was probably due to serious necrocytosis/apoptosis of the cells induced by rapid viral replication, which contributes to the decline in virus infectivity in addition to temperature that influences the infectivity and stability of extracellular viruses. The number of intracellular RNA copies of the viral C gene in the NTZ-treated and DMSO-treated cells was determined by a quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). As shown in Figure 3C, treatment of JEV-infected cells with 3 μg/ml NTZ significantly reduced the number of copies of viral RNA compared with the DMSO-treated cells.
NTZ inhibits JEV replication at the early-mid stage of viral infection
NTZ reduces the mortality of mice challenged with a lethal dose of JEV
NTZ is an anti-infective licensed in the United States for the treatment of parasitic gastroenteritis [1–3]. This drug has been demonstrated to be safe and effective even when administered over a year . NTZ has been considered to be a new class of broad-spectrum antiviral drugs . Given the fact that there is an urgent need for an effective drug against JEV and that there is currently no effective antiviral agent available for the treatment of JE, we tested the antiviral effect of NTZ on JEV infection in cell and mouse models. We found that NTZ was able to inhibit the replication of JEV in BHK-21 cells in a dose-dependent manner (Figure 2 and Figure 3). NTZ also reduced the mortality of mice challenged with a lethal dose of JEV (Figure 6).
We observed that NTZ significantly inhibited the replication of JEV in BHK-21 cells in a dose dependent manner with EC50 value of 0.12 ± 0.04 μg/ml (Figure 3A), whereas the EC50 for influenza A virus (PR8 strain) is 1.0 μg/ml . As a new class of broad-spectrum antiviral drugs, NTZ has been speculated to target host functions that are essential for viral replication [9, 14]. In the case of HCV infection, NTZ increases eIF2α phosphorylation, a modification known to mediate host antiviral defenses . However, in the case of influenza virus infection, NTZ prevents the maturation of viral HA possibly by blocking HA trafficking between the endoplasmic reticulum and the Golgi complex . In our study, we found that NTZ exerted its antiviral activity in vitro at the early-mid stage of viral infection (Figure 5), indicating that NTZ does not affect JEV infectivity, adsorption or entry into target cells. However, the exact mechanism of how NTZ inhibited JEV replication needs to be further explored.
Although there are currently no effective drugs available for the treatment of JE, attempts to develop new antiviral drugs are ongoing. For example, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have recently been reported which are antisense agents targeting the 3′ cyclization sequence of JEV, they have been shown to significantly inhibit the replication of JEV in cells and in a mouse model . In comparison with previously proposed therapeutic agents, such as PPMOs, siRNA [20, 21], arctigenin  and isatis indigotica extracts , NTZ is a licensed, safe drug and is therefore a promising candidate for use as an anti-JEV therapeutic agent. Further studies into the mechanism of action and the efficacy of NTZ are currently underway in our laboratory.
The anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. NTZ significantly inhibited the replication of JEV in BHK-21 cells in a dose dependent manner with EC50 value of 0.12 ± 0.04 μg/ml. NTZ was found to exert its anti-JEV effect in vitro at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated with NTZ were protected from a lethal challenge dose of JEV. Both our in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of JE.
Materials and methods
Virus, cells and NTZ administration
JEV strain (SH-JEV01)  was grown in 3-day-old BALB/c mice and titrated by a plaque assay using BHK-21 cells as described below. BHK-21 (C-13, ATCC number CCL-10) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO2.
NTZ (purity ≥ 98%) was purchased from Sigma–Aldrich (Sigma, St. Louis, MO, USA) and was dissolved in culture-grade DMSO (Sigma) at a concentration of 50 μg/μl. NTZ solution was added immediately after a 1 h adsorption period and was kept in the culture medium for the duration of the experiment, unless specified otherwise. Controls received equal amounts of DMSO (final concentration ≤ 0.06%), which did not affect cell viability or virus replication.
BHK-21 cells were seeded in a 96-well plate at a density of 5 × 103 cells per well. Following 24 h of incubation, the cells were treated with NTZ at various concentrations ranging from 0.1 to 32 μg/ml at 37°C for 48 h. Cells treated with DMSO alone were used as a control. The cellular toxicity of NTZ was assessed using MTT assay (Roche, Mannheim, Germany). Cell viability was calculated as a percentage of the total number of viable DMSO-treated control cells. The CC50, which is defined as the concentration that inhibits the proliferation of exponentially growing cells by 50%, was calculated as described .
Viral infection and titration
BHK-21 cells were seeded at 1 × 106 cells per well on six-well plates. Cultures were inoculated with JEV at a multiplicity of infection (MOI) of 0.001 when the cells reached approximately 80–90% confluence. The inoculum was removed following a 1 h incubation period, and cells were washed twice with phosphate-buffered saline (PBS). Cultures were further incubated in DMEM with 2% FBS for the indicated times. Following incubation, the cells and the culture supernatants were collected and stored at -70 °C until further use.
The plaque assay for JEV infectivity was performed as described . Briefly, BHK-21 cells were seeded in six-well plates and incubated overnight. The medium was replaced with PBS containing serially diluted JEV. The cells were incubated at 37°C for 4 h. After the adsorption period, the inoculum was replaced with DMEM containing 2% FBS and 1% low-melting-point agarose and incubated for 5 days. On 5 days post-inoculation, the cells were fixed with 1% formaldehyde overnight to penetrate the agarose. The cells were stained with 0.05% neutral red for 1 h at room temperature after removing the overlaid agarose. The plaques were then counted, and infectivity was expressed as plaque forming units (PFU)/ml.
Total RNA was isolated from cells using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. One microgram of total RNAs isolated from JEV-infected cells was used for synthesis of cDNA using AMV reverse transcriptase (TaKaRa, Otsu, Japan). The qRT-PCR for analysis of JEV replication was performed using Premix Ex Taq™ (TaKaRa), according to the manufacturer’s protocol. Briefly, reactions were prepared in 20 μl containing 1 μl of cDNA, 10 μl of SYBR Premix Ex Taq™ (2×), and 0.2 μM of specific primers. The amplification parameters were: 2 min at 95°C, followed by 40 cycles of 15 sec at 95°C, and 60 sec at 60°C. The sequences of the primers used to amplify 181 bp of the gene encoding core protein C (C) of JEV were: 5′-AAAAACCAGGAGGGCCCGG-3′(nucleotide position 103–121) and 5′-TTGGTCGGGGCTAATGCTGTAAA-3′(nucleotide position 283–261). These primers were designed according to the sequence of JEV strain SA14 (GenBank accession No. U14163). Quantification was achieved by relating the cycle threshold (Ct) value of virus to the Ct value on a standard curve of a measured number of copies of the JEV C gene cloned into pMD18-T vector which the resulting plasmid was named pM-C. The quantitative standard curve for the determination of JEV RNAs copy number was created by quantitative real-time PCR of standard plasmid pM-C preparations at serial dilutions of 106, 105, 104, 103, 102 and 101 copies/μl. The number of JEV RNAs copies was calculated by Eppendorf Mastercycler ep realplex 2.2 software automaticlly.
Analysis of the antiviral effect of NTZ in BHK-21 cells
BHK-21 cells in six-well plates were infected with JEV at a MOI of 0.001. After a 1 h adsorption period, the cells were treated with NTZ at concentrations ranging from 0.01 to 10 μg/ml and incubated at 37°C for 48 h or the indicated times. The virus yield was determined by a plaque assay and qRT-PCR. The reduction in the virus titer was calculated as follows:% virus titer reduction = [1-(PFUJEV+NTZ/PFUJEV+DMSO)] × 100. The EC50 that is defined as the concentration offering 50% inhibition of viral yield in cells was calculated as described .
Western blot, immunofluorescence and immunohistochemistry analysis
The expression of viral NS3 in JEV-infected cells was examined by western blot and immunofluorescence analysis as described [24, 27]. The fluorescence intensity of cells was quantified using the software ImageJ that is a Java-based image processing program developed at the National Institutes of Health (http://rsbweb.nih.gov/ij/index.html). The immunohistochemistry for analysis of viral NS3 in brain of JEV-infected mice was performed as described .
Analysis of the antiviral effect of NTZ in a mouse model
Three-week old female Chinese Kunming mice (12–14 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China) and were randomly divided into six groups (10 mice/group). Group JEV + NTZ was infected with JEV and received NTZ treatment (50, 75 or 100 mg/kg/day). Group JEV + DMSO was infected with JEV and received a placebo (DMSO) treatment. Group Mock + NTZ was mock-infected with JEV and received NTZ treatment. Group Mock + DMSO was mock-infected with JEV and received a placebo (DMSO) treatment. For infection, mice were infected intraperitoneally with 6×104 PFU of JEV (containing 50 × LD50 of JEV). For NTZ treatment, NTZ was dissolved in DMSO and administered intragastrically by gavage, in which a feeding needle was introduced into the esophagus and NTZ was delivered directly into the stomach. NTZ was tested at a total dose of 50, 75 or 100 mg/kg/day, and was consecutively administered from 1 day post-infection, daily, for up to 25 days. The mice were monitored daily for morbidity and mortality. The mice that showed neurological signs were euthanized according to the Guidelines on the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China, Policy No. 2006 398). The animal experiments were in compliance with the Guidelines on the Humane Treatment of Laboratory Animals (Policy No. 2006 398) and were approved by the Institutional Animal Care and Use Committee at the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science.
All experiments were carried out in triplicate. The measured values are expressed as the means with standard deviations (SD). Significance was analyzed using the Student’s t-test or the Gehan-Breslow-Wilcoxon test. Value of p < 0.05 was considered significant.
We thank the Key Open Laboratory of Animal Parasitology, Ministry of Agriculture of China, for the provision of laboratory equipment. This research was sponsored by the Project of International Science and Technology Cooperation (No.2010DFB33920), the Zhejiang Provincial Natural Science Foundation (No.Y3110124) and the National Natural Science Foundation of China (No.81371814).
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