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Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

View Article: PubMed Central - PubMed

ABSTRACT

--: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Timeofaddition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

No MeSH data available.


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Virus yield reduction by CW-33. BHK-21 (A,B) and TE671 (C,D) cells were infected with JEV and immediately treated with indicated CW-33 concentration. Supernatant was harvested 36, 48, or 72 h post-infection, virus yield measured by plaque assay. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
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ijms-17-01386-f004: Virus yield reduction by CW-33. BHK-21 (A,B) and TE671 (C,D) cells were infected with JEV and immediately treated with indicated CW-33 concentration. Supernatant was harvested 36, 48, or 72 h post-infection, virus yield measured by plaque assay. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.

Mentions: Initially, cytotoxicity of CW-33 was evaluated using MTT assays (Figure 1B,C). The 50% cytotoxicity concentration (CC50) values of CW-33 were greater 500 μM to BHK-21 cells and 189 μM to TE671 cells. Inhibitory assays of cytopathic effect (CPE) and apoptosis were subsequently used for determining the in vitro antiviral activity of CW-33 against JEV (Figure 2 and Figure 3). JEV-infected cells were treated with or without the various concentrations of CW-33 (2.5, 25 and 125 μM). Microscopic photography indicated CW-33 concentration-dependently reducing the JEV-induced cytopathic effect of BHK-21 cells 48 and 72 h post-infection, as well as TE671 cells 36 and 48 h post-infection, respectively (Figure 2). Annexin-V/PI apoptosis analysis using flow cytometry showed CW-33 significantly decreasing early and late apoptosis of JEV-infected cells in a dose-dependent manner (Figure 3A,B). Western blotting also showed CW-33 diminishing active form of caspase-3 in JEV-infected cells (Figure 3C). Supernatant virus yield using the plaque assay demonstrated CW-33 markedly inhibiting JEV production in BHK-21 and TE671 cells (Figure 4). The 50% inhibitory concentration (IC50) of CW-33 on inhibiting supernatant virus yield ranged from 12.7 to 38.5 μM based on the cell type and the harvest time. Notably, the therapeutic index value (CC50/IC50) of CW-33 against JEV exceeded 5. The results revealed the antiviral potential of CW-33 against JEV.


Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload
Virus yield reduction by CW-33. BHK-21 (A,B) and TE671 (C,D) cells were infected with JEV and immediately treated with indicated CW-33 concentration. Supernatant was harvested 36, 48, or 72 h post-infection, virus yield measured by plaque assay. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037666&req=5

ijms-17-01386-f004: Virus yield reduction by CW-33. BHK-21 (A,B) and TE671 (C,D) cells were infected with JEV and immediately treated with indicated CW-33 concentration. Supernatant was harvested 36, 48, or 72 h post-infection, virus yield measured by plaque assay. * p value < 0.05; ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
Mentions: Initially, cytotoxicity of CW-33 was evaluated using MTT assays (Figure 1B,C). The 50% cytotoxicity concentration (CC50) values of CW-33 were greater 500 μM to BHK-21 cells and 189 μM to TE671 cells. Inhibitory assays of cytopathic effect (CPE) and apoptosis were subsequently used for determining the in vitro antiviral activity of CW-33 against JEV (Figure 2 and Figure 3). JEV-infected cells were treated with or without the various concentrations of CW-33 (2.5, 25 and 125 μM). Microscopic photography indicated CW-33 concentration-dependently reducing the JEV-induced cytopathic effect of BHK-21 cells 48 and 72 h post-infection, as well as TE671 cells 36 and 48 h post-infection, respectively (Figure 2). Annexin-V/PI apoptosis analysis using flow cytometry showed CW-33 significantly decreasing early and late apoptosis of JEV-infected cells in a dose-dependent manner (Figure 3A,B). Western blotting also showed CW-33 diminishing active form of caspase-3 in JEV-infected cells (Figure 3C). Supernatant virus yield using the plaque assay demonstrated CW-33 markedly inhibiting JEV production in BHK-21 and TE671 cells (Figure 4). The 50% inhibitory concentration (IC50) of CW-33 on inhibiting supernatant virus yield ranged from 12.7 to 38.5 μM based on the cell type and the harvest time. Notably, the therapeutic index value (CC50/IC50) of CW-33 against JEV exceeded 5. The results revealed the antiviral potential of CW-33 against JEV.

View Article: PubMed Central - PubMed

ABSTRACT

--: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 &mu;M. Timeofaddition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 &mu;M, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

No MeSH data available.


Related in: MedlinePlus