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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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Detection of viral NS3 protein in NTZ-treated cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at 3 μg/ml. The cells were harvested at 24 or 60 h post-infection (hpi). (A) The same amount (20 μg) of cell lysate was loaded in each lane. The expression of viral NS3 protein was examined by western blot. (B) The viral NS3 protein (green fluorescence) was detected at 24 hpi by immunofluorescence analysis. The cells were also stained for DNA with 4′, 6′-diamidino-2-phenylindole (DAPI, blue fluorescence). The Merge panels show the superimposed images. (C) Fluorescence intensity of NS3 and DAPI was quantified using the software ImageJ. The ratio of fluorescence intensity between NS3 and DAPI was calculated and plotted. The data are means with standard errors from three independent experiments. *, p < 0.01 between the groups tested.
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Figure 4: Detection of viral NS3 protein in NTZ-treated cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at 3 μg/ml. The cells were harvested at 24 or 60 h post-infection (hpi). (A) The same amount (20 μg) of cell lysate was loaded in each lane. The expression of viral NS3 protein was examined by western blot. (B) The viral NS3 protein (green fluorescence) was detected at 24 hpi by immunofluorescence analysis. The cells were also stained for DNA with 4′, 6′-diamidino-2-phenylindole (DAPI, blue fluorescence). The Merge panels show the superimposed images. (C) Fluorescence intensity of NS3 and DAPI was quantified using the software ImageJ. The ratio of fluorescence intensity between NS3 and DAPI was calculated and plotted. The data are means with standard errors from three independent experiments. *, p < 0.01 between the groups tested.

Mentions: The inhibitory effect of NTZ on the replication of JEV was further confirmed at viral protein level. The expression of viral NS3 protein in the NTZ-treated and DMSO-treated cells was detected by western blot and immunofluorescence analysis. As shown in Figure 4A, the abundance of viral NS3 protein in the NTZ-treated cells (JEV + NTZ) at 24 h post-infection was remarkably less than that in the DMSO-treated cells (JEV + DMSO). In the immunofluorescence analysis, although the numbers of NS3-positive cells (green fluorescence) among the NTZ-treated cells (JEV + NTZ panels) were similar to those detected among the DMSO-treated cells (JEV + DMSO panels), the strength of the fluorescence signals in the NTZ-treated cells was remarkably lower than that in the DMSO-treated cells (Figure 4B and 4C). Taken together, these data suggested that NTZ inhibited the replication of JEV.


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)

Detection of viral NS3 protein in NTZ-treated cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at 3 μg/ml. The cells were harvested at 24 or 60 h post-infection (hpi). (A) The same amount (20 μg) of cell lysate was loaded in each lane. The expression of viral NS3 protein was examined by western blot. (B) The viral NS3 protein (green fluorescence) was detected at 24 hpi by immunofluorescence analysis. The cells were also stained for DNA with 4′, 6′-diamidino-2-phenylindole (DAPI, blue fluorescence). The Merge panels show the superimposed images. (C) Fluorescence intensity of NS3 and DAPI was quantified using the software ImageJ. The ratio of fluorescence intensity between NS3 and DAPI was calculated and plotted. The data are means with standard errors from three independent experiments. *, p < 0.01 between the groups tested.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927656&req=5

Figure 4: Detection of viral NS3 protein in NTZ-treated cells. BHK-21 cells were infected with JEV at a MOI of 0.001 and treated with NTZ at 3 μg/ml. The cells were harvested at 24 or 60 h post-infection (hpi). (A) The same amount (20 μg) of cell lysate was loaded in each lane. The expression of viral NS3 protein was examined by western blot. (B) The viral NS3 protein (green fluorescence) was detected at 24 hpi by immunofluorescence analysis. The cells were also stained for DNA with 4′, 6′-diamidino-2-phenylindole (DAPI, blue fluorescence). The Merge panels show the superimposed images. (C) Fluorescence intensity of NS3 and DAPI was quantified using the software ImageJ. The ratio of fluorescence intensity between NS3 and DAPI was calculated and plotted. The data are means with standard errors from three independent experiments. *, p < 0.01 between the groups tested.
Mentions: The inhibitory effect of NTZ on the replication of JEV was further confirmed at viral protein level. The expression of viral NS3 protein in the NTZ-treated and DMSO-treated cells was detected by western blot and immunofluorescence analysis. As shown in Figure 4A, the abundance of viral NS3 protein in the NTZ-treated cells (JEV + NTZ) at 24 h post-infection was remarkably less than that in the DMSO-treated cells (JEV + DMSO). In the immunofluorescence analysis, although the numbers of NS3-positive cells (green fluorescence) among the NTZ-treated cells (JEV + NTZ panels) were similar to those detected among the DMSO-treated cells (JEV + DMSO panels), the strength of the fluorescence signals in the NTZ-treated cells was remarkably lower than that in the DMSO-treated cells (Figure 4B and 4C). Taken together, these data suggested that NTZ inhibited the replication of JEV.

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