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Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model.

Shi Z, Wei J, Deng X, Li S, Qiu Y, Shao D, Li B, Zhang K, Xue F, Wang X, Ma Z - Virol. J. (2014)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No, 518, Ziyue Road, Shanghai 200241, PR China. zhiyongma@shvri.ac.cn.

ABSTRACT

Background: 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.

Methods: 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.

Findings: 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.

Conclusions: 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.

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Analysis of the protective effect of NTZ on mice challenged with a lethal dose of JEV. Mice (10 mice/group) were infected intraperitoneally with 6×104 PFU of JEV and treated with NTZ from 1 day post-infection. NTZ was administered daily at the indicated doses (50, 75 or 100 mg/kg/day) by intragastric administration for up to 25 days. (A) The mice were monitored daily for morbidity and mortality. The daily survival rates were plotted. *, p < 0.0001 between the group JEV + DMSO and group JEV + NTZ tested by the Gehan-Breslow-Wilcoxon test. (B) The brain tissues were collected from the mice euthanized on the indicated days after JEV infection. The virus titer in each group was determined by a plaque assay and plotted. The data are means with SD from three independent experiments. *, p < 0.05 compared with the NTZ-treated groups.
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Figure 6: Analysis of the protective effect of NTZ on mice challenged with a lethal dose of JEV. Mice (10 mice/group) were infected intraperitoneally with 6×104 PFU of JEV and treated with NTZ from 1 day post-infection. NTZ was administered daily at the indicated doses (50, 75 or 100 mg/kg/day) by intragastric administration for up to 25 days. (A) The mice were monitored daily for morbidity and mortality. The daily survival rates were plotted. *, p < 0.0001 between the group JEV + DMSO and group JEV + NTZ tested by the Gehan-Breslow-Wilcoxon test. (B) The brain tissues were collected from the mice euthanized on the indicated days after JEV infection. The virus titer in each group was determined by a plaque assay and plotted. The data are means with SD from three independent experiments. *, p < 0.05 compared with the NTZ-treated groups.

Mentions: To evaluate the protective effect of NTZ on mice challenged with a lethal dose of JEV, NTZ was administered intragastrically at the indicated doses from 1 day post-infection, daily, for up to 25 days. The mice that were infected with JEV and received a placebo (DMSO) treatment (group JEV + DMSO) started to show the clinical signs of JE including limb paralysis, restriction of movements, piloerection, body stiffening and whole body tremors, from 5 days post-infection, and all mice (10/10 mice) died within 9 days post-infection. In contrast, the mice that were infected with JEV and received NTZ treatment (group JEV + NTZ, 100 mg/kg/day) showed the clinical signs of JE from 11 days post-infection, among these 10 mice, 1 died within 12 days post-infection and 9 survived the experimental period (25 days) (Figure 6A). The NTZ-mediated protection appeared to be dose dependent, as the infected mice receiving 50 mg/kg/day, 75 mg/kg/day and 100 mg/kg/day NTZ led to 30%, 70% and 90% mice survival, respectively (Figure 6A). These data suggested that NTZ treatment reduced the mortality of JEV-infected mice and protected mice from a lethal dose challenge of JEV. The mice that were mock-infected with JEV and received NTZ treatment (group Mock + NTZ) showed no detectable signs of abnormal behavior, similarly to the mice that were mock-infected and received DMSO treatment (group Mock + DMSO) (Figure 6A). Analysis of JEV titers in the brain samples from the experimental mice indicated that NTZ treatment significantly reduced the virus load in the brain from the group JEV + NTZ compared with that from group JEV + DMSO (Figure 6B). The brain samples from the experimental mice were examined for the presence of viral NS3 protein by immunohistochemistry. The viral NS3 protein was stained as brown deposits in the cytoplasm of neuronal cells (Additional file 1, arrowed cells). The brain sections from the JEV + DMSO group of mice showed remarkably higher numbers of NS3-stained positive cells than the sections from the JEV + NTZ group. No NS3-stained positive cells were detected in the sections from the Mock + NTZ or Mock + DMSO groups of mice.


Nitazoxanide inhibits the replication of Japanese encephalitis virus in cultured cells and in a mouse model.

Shi Z, Wei J, Deng X, Li S, Qiu Y, Shao D, Li B, Zhang K, Xue F, Wang X, Ma Z - Virol. J. (2014)

Analysis of the protective effect of NTZ on mice challenged with a lethal dose of JEV. Mice (10 mice/group) were infected intraperitoneally with 6×104 PFU of JEV and treated with NTZ from 1 day post-infection. NTZ was administered daily at the indicated doses (50, 75 or 100 mg/kg/day) by intragastric administration for up to 25 days. (A) The mice were monitored daily for morbidity and mortality. The daily survival rates were plotted. *, p < 0.0001 between the group JEV + DMSO and group JEV + NTZ tested by the Gehan-Breslow-Wilcoxon test. (B) The brain tissues were collected from the mice euthanized on the indicated days after JEV infection. The virus titer in each group was determined by a plaque assay and plotted. The data are means with SD from three independent experiments. *, p < 0.05 compared with the NTZ-treated groups.
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Related In: Results  -  Collection

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Figure 6: Analysis of the protective effect of NTZ on mice challenged with a lethal dose of JEV. Mice (10 mice/group) were infected intraperitoneally with 6×104 PFU of JEV and treated with NTZ from 1 day post-infection. NTZ was administered daily at the indicated doses (50, 75 or 100 mg/kg/day) by intragastric administration for up to 25 days. (A) The mice were monitored daily for morbidity and mortality. The daily survival rates were plotted. *, p < 0.0001 between the group JEV + DMSO and group JEV + NTZ tested by the Gehan-Breslow-Wilcoxon test. (B) The brain tissues were collected from the mice euthanized on the indicated days after JEV infection. The virus titer in each group was determined by a plaque assay and plotted. The data are means with SD from three independent experiments. *, p < 0.05 compared with the NTZ-treated groups.
Mentions: To evaluate the protective effect of NTZ on mice challenged with a lethal dose of JEV, NTZ was administered intragastrically at the indicated doses from 1 day post-infection, daily, for up to 25 days. The mice that were infected with JEV and received a placebo (DMSO) treatment (group JEV + DMSO) started to show the clinical signs of JE including limb paralysis, restriction of movements, piloerection, body stiffening and whole body tremors, from 5 days post-infection, and all mice (10/10 mice) died within 9 days post-infection. In contrast, the mice that were infected with JEV and received NTZ treatment (group JEV + NTZ, 100 mg/kg/day) showed the clinical signs of JE from 11 days post-infection, among these 10 mice, 1 died within 12 days post-infection and 9 survived the experimental period (25 days) (Figure 6A). The NTZ-mediated protection appeared to be dose dependent, as the infected mice receiving 50 mg/kg/day, 75 mg/kg/day and 100 mg/kg/day NTZ led to 30%, 70% and 90% mice survival, respectively (Figure 6A). These data suggested that NTZ treatment reduced the mortality of JEV-infected mice and protected mice from a lethal dose challenge of JEV. The mice that were mock-infected with JEV and received NTZ treatment (group Mock + NTZ) showed no detectable signs of abnormal behavior, similarly to the mice that were mock-infected and received DMSO treatment (group Mock + DMSO) (Figure 6A). Analysis of JEV titers in the brain samples from the experimental mice indicated that NTZ treatment significantly reduced the virus load in the brain from the group JEV + NTZ compared with that from group JEV + DMSO (Figure 6B). The brain samples from the experimental mice were examined for the presence of viral NS3 protein by immunohistochemistry. The viral NS3 protein was stained as brown deposits in the cytoplasm of neuronal cells (Additional file 1, arrowed cells). The brain sections from the JEV + DMSO group of mice showed remarkably higher numbers of NS3-stained positive cells than the sections from the JEV + NTZ group. No NS3-stained positive cells were detected in the sections from the Mock + NTZ or Mock + DMSO groups of mice.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No, 518, Ziyue Road, Shanghai 200241, PR China. zhiyongma@shvri.ac.cn.

ABSTRACT

Background: 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.

Methods: 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.

Findings: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus