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Virocidal activity of Egyptian scorpion venoms against hepatitis C virus.

El-Bitar AM, Sarhan MM, Aoki C, Takahara Y, Komoto M, Deng L, Moustafa MA, Hotta H - Virol. J. (2015)

Bottom Line: Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed.S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step.To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Faculty of Science, Al-Azhar University, Assiut, Egypt. sci.elbitar@gmail.com.

ABSTRACT

Background: Hepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions.

Methods: The possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities.

Results: S. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC₅₀) being 6.3 ± 1.6 and 88.3 ± 5.8 μg/ml, respectively. S. maurus palmatus venom (30 μg/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60°C. The antiviral activity was directed preferentially against HCV.

Conclusions: S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

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Related in: MedlinePlus

Analysis of mode-of-action ofS. maurus palmatusvenom. Huh7.5 cells were infected with HCV and treated with S. maurus palmatus venom (30 μg/ml) at different time points as indicated (A, B) or left untreated as a control. (A) Amounts of HCV infectious particles in the supernatants. Data represent means ± SEM of the data obtained from two independent experiments. *, P <0.05; †, P <0.001, compared with the untreated control; §, below the detection limit; dpi, days post-infection. (B) HCV NS3 protein accumulation in the cells. Virus-infected cells were subjected to immunoblot analysis using monoclonal antibody against the HCV NS3 protein at 1 and 2 days post-infection. GAPDH served as an internal control to verify equal amounts of sample loading. Signal intensities of NS3 were normalized to the corresponding GAPDH signal. (C) Amounts of HCV RNA in the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. HCV RNA amounts were normalized to GAPDH mRNA expression. (D) Amounts of HCV infectious particles inside the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. Virus-infected cells were subjected to 3 cycles of freezing and thawing at −80°C and 37°C, respectively, and HCV infectivity inside the cells was measured.
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Fig2: Analysis of mode-of-action ofS. maurus palmatusvenom. Huh7.5 cells were infected with HCV and treated with S. maurus palmatus venom (30 μg/ml) at different time points as indicated (A, B) or left untreated as a control. (A) Amounts of HCV infectious particles in the supernatants. Data represent means ± SEM of the data obtained from two independent experiments. *, P <0.05; †, P <0.001, compared with the untreated control; §, below the detection limit; dpi, days post-infection. (B) HCV NS3 protein accumulation in the cells. Virus-infected cells were subjected to immunoblot analysis using monoclonal antibody against the HCV NS3 protein at 1 and 2 days post-infection. GAPDH served as an internal control to verify equal amounts of sample loading. Signal intensities of NS3 were normalized to the corresponding GAPDH signal. (C) Amounts of HCV RNA in the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. HCV RNA amounts were normalized to GAPDH mRNA expression. (D) Amounts of HCV infectious particles inside the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. Virus-infected cells were subjected to 3 cycles of freezing and thawing at −80°C and 37°C, respectively, and HCV infectivity inside the cells was measured.

Mentions: To further explore the mode of action of the S. maurus palmatus venom, time-of-addition experiments were performed. In brief, S. maurus palmatus crude venom (30 μg/ml) was added to the virus and/or cells at different time points relative to virus inoculation, as described in the Methods section: (i) pre-treatment of cells for 2 hr (−2 hr cells). This experiment examines whether there is any interaction between the venom and the cells. (ii) pre-treatment of virus for 2 hr (−2 hr virus). This experiment examines the possible virocidal activity of the venom. (iii) co-treatment of cells and virus during virus inoculation for 2 hr (0 hr). This experiment examines the antiviral effect at the entry step. (iv) treatment of virus-infected cells during post-inoculation for 46 hr (+2 hr). This experiment examines the antiviral effect during the post-entry step. (v) co-treatment and post-inoculation (0 hr & +2 hr) as a positive control. For each set of experiments, HCV infectivity in culture supernatants was determined and compared with each other. The result revealed that pre-treatment of virus for 2 hr (−2 hr virus) markedly suppressed production of HCV infectious particles in culture supernatants while pre-treatment of cells for 2 hr (−2 hr cells) only marginally suppressed it (Figure 2A). This suggested the possibility that S. maurus palmatus venom had direct virocidal activity. Also, treatment during post-inoculation for 46 hr (+2 hr) suppressed HCV infectivity in culture supernatants, though to a lesser extent compared to pre-treatment of virus for 2 hr (−2 hr virus). The result suggested that this treatment inhibited either HCV replication in the cell or HCV infectivity outside the cells (in culture supernatants). On the other hand, treatment during post-inoculation for 46 hr (+2 hr) did not significantly inhibit HCV NS3 protein accumulation while pre-treatment of virus for 2 hr (−2 hr virus) completely inhibited it at 1 and 2 days post-infection (Figure 2B). We further examined HCV RNA replication and virus infectivity inside the cells. The result revealed that the post-inoculation treatment (+2 hr) did not significantly inhibit HCV RNA replication in the cells (Figure 2C). Moreover, the venom treatment (+2 hr) did not reduce HCV infectivity inside the cells (Figure 2D). Taken together, these results suggest that the S. maurus palmatus venom acts directly on HCV particles in culture medium to impair the viral infectivity and that it does not exert its antiviral effect inside the cells.Figure 2


Virocidal activity of Egyptian scorpion venoms against hepatitis C virus.

El-Bitar AM, Sarhan MM, Aoki C, Takahara Y, Komoto M, Deng L, Moustafa MA, Hotta H - Virol. J. (2015)

Analysis of mode-of-action ofS. maurus palmatusvenom. Huh7.5 cells were infected with HCV and treated with S. maurus palmatus venom (30 μg/ml) at different time points as indicated (A, B) or left untreated as a control. (A) Amounts of HCV infectious particles in the supernatants. Data represent means ± SEM of the data obtained from two independent experiments. *, P <0.05; †, P <0.001, compared with the untreated control; §, below the detection limit; dpi, days post-infection. (B) HCV NS3 protein accumulation in the cells. Virus-infected cells were subjected to immunoblot analysis using monoclonal antibody against the HCV NS3 protein at 1 and 2 days post-infection. GAPDH served as an internal control to verify equal amounts of sample loading. Signal intensities of NS3 were normalized to the corresponding GAPDH signal. (C) Amounts of HCV RNA in the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. HCV RNA amounts were normalized to GAPDH mRNA expression. (D) Amounts of HCV infectious particles inside the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. Virus-infected cells were subjected to 3 cycles of freezing and thawing at −80°C and 37°C, respectively, and HCV infectivity inside the cells was measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig2: Analysis of mode-of-action ofS. maurus palmatusvenom. Huh7.5 cells were infected with HCV and treated with S. maurus palmatus venom (30 μg/ml) at different time points as indicated (A, B) or left untreated as a control. (A) Amounts of HCV infectious particles in the supernatants. Data represent means ± SEM of the data obtained from two independent experiments. *, P <0.05; †, P <0.001, compared with the untreated control; §, below the detection limit; dpi, days post-infection. (B) HCV NS3 protein accumulation in the cells. Virus-infected cells were subjected to immunoblot analysis using monoclonal antibody against the HCV NS3 protein at 1 and 2 days post-infection. GAPDH served as an internal control to verify equal amounts of sample loading. Signal intensities of NS3 were normalized to the corresponding GAPDH signal. (C) Amounts of HCV RNA in the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. HCV RNA amounts were normalized to GAPDH mRNA expression. (D) Amounts of HCV infectious particles inside the cells. The venom treatment was done only during the post-inoculation (+2 hr) step. Virus-infected cells were subjected to 3 cycles of freezing and thawing at −80°C and 37°C, respectively, and HCV infectivity inside the cells was measured.
Mentions: To further explore the mode of action of the S. maurus palmatus venom, time-of-addition experiments were performed. In brief, S. maurus palmatus crude venom (30 μg/ml) was added to the virus and/or cells at different time points relative to virus inoculation, as described in the Methods section: (i) pre-treatment of cells for 2 hr (−2 hr cells). This experiment examines whether there is any interaction between the venom and the cells. (ii) pre-treatment of virus for 2 hr (−2 hr virus). This experiment examines the possible virocidal activity of the venom. (iii) co-treatment of cells and virus during virus inoculation for 2 hr (0 hr). This experiment examines the antiviral effect at the entry step. (iv) treatment of virus-infected cells during post-inoculation for 46 hr (+2 hr). This experiment examines the antiviral effect during the post-entry step. (v) co-treatment and post-inoculation (0 hr & +2 hr) as a positive control. For each set of experiments, HCV infectivity in culture supernatants was determined and compared with each other. The result revealed that pre-treatment of virus for 2 hr (−2 hr virus) markedly suppressed production of HCV infectious particles in culture supernatants while pre-treatment of cells for 2 hr (−2 hr cells) only marginally suppressed it (Figure 2A). This suggested the possibility that S. maurus palmatus venom had direct virocidal activity. Also, treatment during post-inoculation for 46 hr (+2 hr) suppressed HCV infectivity in culture supernatants, though to a lesser extent compared to pre-treatment of virus for 2 hr (−2 hr virus). The result suggested that this treatment inhibited either HCV replication in the cell or HCV infectivity outside the cells (in culture supernatants). On the other hand, treatment during post-inoculation for 46 hr (+2 hr) did not significantly inhibit HCV NS3 protein accumulation while pre-treatment of virus for 2 hr (−2 hr virus) completely inhibited it at 1 and 2 days post-infection (Figure 2B). We further examined HCV RNA replication and virus infectivity inside the cells. The result revealed that the post-inoculation treatment (+2 hr) did not significantly inhibit HCV RNA replication in the cells (Figure 2C). Moreover, the venom treatment (+2 hr) did not reduce HCV infectivity inside the cells (Figure 2D). Taken together, these results suggest that the S. maurus palmatus venom acts directly on HCV particles in culture medium to impair the viral infectivity and that it does not exert its antiviral effect inside the cells.Figure 2

Bottom Line: Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed.S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step.To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Faculty of Science, Al-Azhar University, Assiut, Egypt. sci.elbitar@gmail.com.

ABSTRACT

Background: Hepatitis C virus (HCV) is a major global health problem, causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Development of well-tolerated regimens with high cure rates and fewer side effects is still much needed. Recently, natural antimicrobial peptides (AMPs) are attracting more attention as biological compounds and can be a good template to develop therapeutic agents, including antiviral agents against a variety of viruses. Various AMPs have been characterized from the venom of different venomous animals including scorpions.

Methods: The possible antiviral activities of crude venoms obtained from five Egyptian scorpion species (Leiurus quinquestriatus, Androctonus amoreuxi, A. australis, A. bicolor and Scorpio maurus palmatus) were evaluated by a cell culture method using Huh7.5 cells and the J6/JFH1-P47 strain of HCV. Time-of-addition experiments and inactivation of enzymatic activities of the venoms were carried out to determine the characteristics of the anti-HCV activities.

Results: S. maurus palmatus and A. australis venoms showed anti-HCV activities, with 50% inhibitory concentrations (IC₅₀) being 6.3 ± 1.6 and 88.3 ± 5.8 μg/ml, respectively. S. maurus palmatus venom (30 μg/ml) impaired HCV infectivity in culture medium, but not inside the cells, through virocidal effect. The anti-HCV activity of this venom was not inhibited by a metalloprotease inhibitor or heating at 60°C. The antiviral activity was directed preferentially against HCV.

Conclusions: S. maurus palmatus venom is considered as a good natural source for characterization and development of novel anti-HCV agents targeting the entry step. To our knowledge, this is the first report describing antiviral activities of Egyptian scorpion venoms against HCV, and may open a new approach towards discovering antiviral compounds derived from scorpion venoms.

Show MeSH
Related in: MedlinePlus