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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 effect of NTZ on JEV replication in BHK-21 cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at the indicated concentrations. (A) The cells were incubated for 48 h and harvested for virus titration. The reduction in virus titer was calculated and plotted. The x-axis is in a base 2 logarithmic scale. (B) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The virus titers in the cells and supernatants were determined by a plaque assay, respectively. (C) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The number of RNA copies of viral C gene in the cells was determined by qRT-PCR. The data are means with SD from three independent experiments. *, p < 0.05 compared with the DMSO-treated cells. hpi, hours post-infection.
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Figure 3: Analysis of the effect of NTZ on JEV replication in BHK-21 cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at the indicated concentrations. (A) The cells were incubated for 48 h and harvested for virus titration. The reduction in virus titer was calculated and plotted. The x-axis is in a base 2 logarithmic scale. (B) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The virus titers in the cells and supernatants were determined by a plaque assay, respectively. (C) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The number of RNA copies of viral C gene in the cells was determined by qRT-PCR. The data are means with SD from three independent experiments. *, p < 0.05 compared with the DMSO-treated cells. hpi, hours post-infection.

Mentions: To determine whether NTZ inhibits the replication of JEV, the JEV-infected BHK-21 cells were treated with NTZ at various concentrations ranging from 0.01 to 10 μg/ml for 48 h. The JEV-infected cells that were parallelly treated with DMSO were used as a control. The titers of virus present in the cell lysates and supernatants were determined by a plaque assay and the reduction in the virus titer was calculated. An inhibition curve of JEV in the presence of a serial dilutions of NTZ is shown in Figure 3A. The 50% effective concentration (EC50) of NTZ was 0.12 ± 0.04 μg/ml. The chemotherapeutic index (CTI) calculated as a ratio СC50/EC50 was 154.92.


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 effect of NTZ on JEV replication in BHK-21 cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at the indicated concentrations. (A) The cells were incubated for 48 h and harvested for virus titration. The reduction in virus titer was calculated and plotted. The x-axis is in a base 2 logarithmic scale. (B) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The virus titers in the cells and supernatants were determined by a plaque assay, respectively. (C) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The number of RNA copies of viral C gene in the cells was determined by qRT-PCR. The data are means with SD from three independent experiments. *, p < 0.05 compared with the DMSO-treated cells. hpi, hours post-infection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Analysis of the effect of NTZ on JEV replication in BHK-21 cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at the indicated concentrations. (A) The cells were incubated for 48 h and harvested for virus titration. The reduction in virus titer was calculated and plotted. The x-axis is in a base 2 logarithmic scale. (B) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The virus titers in the cells and supernatants were determined by a plaque assay, respectively. (C) The JEV-infected cells were treated with NTZ at 3 μg/ml and incubated for the indicated times. The number of RNA copies of viral C gene in the cells was determined by qRT-PCR. The data are means with SD from three independent experiments. *, p < 0.05 compared with the DMSO-treated cells. hpi, hours post-infection.
Mentions: To determine whether NTZ inhibits the replication of JEV, the JEV-infected BHK-21 cells were treated with NTZ at various concentrations ranging from 0.01 to 10 μg/ml for 48 h. The JEV-infected cells that were parallelly treated with DMSO were used as a control. The titers of virus present in the cell lysates and supernatants were determined by a plaque assay and the reduction in the virus titer was calculated. An inhibition curve of JEV in the presence of a serial dilutions of NTZ is shown in Figure 3A. The 50% effective concentration (EC50) of NTZ was 0.12 ± 0.04 μg/ml. The chemotherapeutic index (CTI) calculated as a ratio СC50/EC50 was 154.92.

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