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Characterization of reemerging chikungunya virus.

Sourisseau M, Schilte C, Casartelli N, Trouillet C, Guivel-Benhassine F, Rudnicka D, Sol-Foulon N, Le Roux K, Prevost MC, Fsihi H, Frenkiel MP, Blanchet F, Afonso PV, Ceccaldi PE, Ozden S, Gessain A, Schuffenecker I, Verhasselt B, Zamborlini A, Saïb A, Rey FA, Arenzana-Seisdedos F, Desprès P, Michault A, Albert ML, Schwartz O - PLoS Pathog. (2007)

Bottom Line: CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells.CHIKV was highly sensitive to the antiviral activity of type I and II interferons.These results provide a general insight into the interaction between CHIKV and its mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Unité Virus et Immunité, Institut Pasteur, Paris, France.

ABSTRACT
An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

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CHIKV Entry Pathway(A and B) CHIKV infection requires a low endosomal pH for entry. HeLa cells were pretreated 1 h before infection with chloroquine (10 μM) or bafilomycin-A1 (25 nM) and exposed to CHIKV (moi 10), or treated with the two drugs 3 h after viral exposure.(A) Inhibition of CHIKV replication by chloroquine and bafilomycin-A1. At 24 h pi, HeLa cells were stained with mouse anti-CHIKV antibodies and analyzed by flow cytometry. Ctrl, control.(B) Inhibition of CHIKV CPE by chloroquine and bafilomycin-A1. Cell viability was measured in a colorimetric assay (MTT cell viability test) 24 h pi.(C) Downregulation of Dyn-2 expression by shRNAs. HeLa cells were transduced with a lentiviral vector expressing GFP and a shRNA against Dyn-2 or an unrelated protein as a control (Ctrl). Upper panel: The Western blot shows the downregulation of Dyn-2 expression. Lower panel: surface levels of transferrin receptor (Tf-R) were upregulated in the absence of Dyn-2. Flow cytometry analysis was gated on transduced GFP+ cells.(D) Dyn-2 is required for CHIKV replication. HeLa cells lacking Dyn-2 are resistant to CHIKV. Infected HeLa cells (moi 10, 24 h pi) were stained with anti-CHIKV antibodies and analyzed by flow cytometry. The percentage of CHIKV+ cells among GFP+ and GFP− cells is depicted. One representative experiment out of three is shown in the upper panel. A mean ±SD of four independent experiments is shown in the lower panel.NI, noninfected cells.
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ppat-0030089-g007: CHIKV Entry Pathway(A and B) CHIKV infection requires a low endosomal pH for entry. HeLa cells were pretreated 1 h before infection with chloroquine (10 μM) or bafilomycin-A1 (25 nM) and exposed to CHIKV (moi 10), or treated with the two drugs 3 h after viral exposure.(A) Inhibition of CHIKV replication by chloroquine and bafilomycin-A1. At 24 h pi, HeLa cells were stained with mouse anti-CHIKV antibodies and analyzed by flow cytometry. Ctrl, control.(B) Inhibition of CHIKV CPE by chloroquine and bafilomycin-A1. Cell viability was measured in a colorimetric assay (MTT cell viability test) 24 h pi.(C) Downregulation of Dyn-2 expression by shRNAs. HeLa cells were transduced with a lentiviral vector expressing GFP and a shRNA against Dyn-2 or an unrelated protein as a control (Ctrl). Upper panel: The Western blot shows the downregulation of Dyn-2 expression. Lower panel: surface levels of transferrin receptor (Tf-R) were upregulated in the absence of Dyn-2. Flow cytometry analysis was gated on transduced GFP+ cells.(D) Dyn-2 is required for CHIKV replication. HeLa cells lacking Dyn-2 are resistant to CHIKV. Infected HeLa cells (moi 10, 24 h pi) were stained with anti-CHIKV antibodies and analyzed by flow cytometry. The percentage of CHIKV+ cells among GFP+ and GFP− cells is depicted. One representative experiment out of three is shown in the upper panel. A mean ±SD of four independent experiments is shown in the lower panel.NI, noninfected cells.

Mentions: Alphaviruses like SINV and SFV penetrate target cells through clathrin-mediated endocytosis, the low pH of the endosomal compartment promoting conformational changes of envelope glycoprotein and viral fusion [15,16,18]. We examined whether CHIKV infection requires a low endosomal pH for entry. HeLa cells were treated with two compounds that impair intracellular vesicle acidification, bafilomycin-A1, an inhibitor of vacuolar proton-ATPases [35], or the weak base chloroquine. Both compounds potently inhibited the appearance of CHIKV-positive cells and CHIKV-associated CPE (Figure 7A and 7B). Bafilomycin-A1 was efficient at doses from 2.5 to 250 nM, without obvious cytotoxicity, whereas the active concentration range of chloroquine was much narrower: this compound fully inhibited CHIKV infection at 10 μM, but was toxic at 100 μM (Figure 7B). Of note, no inhibitory effect was observed if addition of these two compounds was delayed until 3 h after CHIKV exposure (Figure 7A). This suggests that the requirement for an acidic compartment is a feature of an early step of the viral cycle.


Characterization of reemerging chikungunya virus.

Sourisseau M, Schilte C, Casartelli N, Trouillet C, Guivel-Benhassine F, Rudnicka D, Sol-Foulon N, Le Roux K, Prevost MC, Fsihi H, Frenkiel MP, Blanchet F, Afonso PV, Ceccaldi PE, Ozden S, Gessain A, Schuffenecker I, Verhasselt B, Zamborlini A, Saïb A, Rey FA, Arenzana-Seisdedos F, Desprès P, Michault A, Albert ML, Schwartz O - PLoS Pathog. (2007)

CHIKV Entry Pathway(A and B) CHIKV infection requires a low endosomal pH for entry. HeLa cells were pretreated 1 h before infection with chloroquine (10 μM) or bafilomycin-A1 (25 nM) and exposed to CHIKV (moi 10), or treated with the two drugs 3 h after viral exposure.(A) Inhibition of CHIKV replication by chloroquine and bafilomycin-A1. At 24 h pi, HeLa cells were stained with mouse anti-CHIKV antibodies and analyzed by flow cytometry. Ctrl, control.(B) Inhibition of CHIKV CPE by chloroquine and bafilomycin-A1. Cell viability was measured in a colorimetric assay (MTT cell viability test) 24 h pi.(C) Downregulation of Dyn-2 expression by shRNAs. HeLa cells were transduced with a lentiviral vector expressing GFP and a shRNA against Dyn-2 or an unrelated protein as a control (Ctrl). Upper panel: The Western blot shows the downregulation of Dyn-2 expression. Lower panel: surface levels of transferrin receptor (Tf-R) were upregulated in the absence of Dyn-2. Flow cytometry analysis was gated on transduced GFP+ cells.(D) Dyn-2 is required for CHIKV replication. HeLa cells lacking Dyn-2 are resistant to CHIKV. Infected HeLa cells (moi 10, 24 h pi) were stained with anti-CHIKV antibodies and analyzed by flow cytometry. The percentage of CHIKV+ cells among GFP+ and GFP− cells is depicted. One representative experiment out of three is shown in the upper panel. A mean ±SD of four independent experiments is shown in the lower panel.NI, noninfected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1904475&req=5

ppat-0030089-g007: CHIKV Entry Pathway(A and B) CHIKV infection requires a low endosomal pH for entry. HeLa cells were pretreated 1 h before infection with chloroquine (10 μM) or bafilomycin-A1 (25 nM) and exposed to CHIKV (moi 10), or treated with the two drugs 3 h after viral exposure.(A) Inhibition of CHIKV replication by chloroquine and bafilomycin-A1. At 24 h pi, HeLa cells were stained with mouse anti-CHIKV antibodies and analyzed by flow cytometry. Ctrl, control.(B) Inhibition of CHIKV CPE by chloroquine and bafilomycin-A1. Cell viability was measured in a colorimetric assay (MTT cell viability test) 24 h pi.(C) Downregulation of Dyn-2 expression by shRNAs. HeLa cells were transduced with a lentiviral vector expressing GFP and a shRNA against Dyn-2 or an unrelated protein as a control (Ctrl). Upper panel: The Western blot shows the downregulation of Dyn-2 expression. Lower panel: surface levels of transferrin receptor (Tf-R) were upregulated in the absence of Dyn-2. Flow cytometry analysis was gated on transduced GFP+ cells.(D) Dyn-2 is required for CHIKV replication. HeLa cells lacking Dyn-2 are resistant to CHIKV. Infected HeLa cells (moi 10, 24 h pi) were stained with anti-CHIKV antibodies and analyzed by flow cytometry. The percentage of CHIKV+ cells among GFP+ and GFP− cells is depicted. One representative experiment out of three is shown in the upper panel. A mean ±SD of four independent experiments is shown in the lower panel.NI, noninfected cells.
Mentions: Alphaviruses like SINV and SFV penetrate target cells through clathrin-mediated endocytosis, the low pH of the endosomal compartment promoting conformational changes of envelope glycoprotein and viral fusion [15,16,18]. We examined whether CHIKV infection requires a low endosomal pH for entry. HeLa cells were treated with two compounds that impair intracellular vesicle acidification, bafilomycin-A1, an inhibitor of vacuolar proton-ATPases [35], or the weak base chloroquine. Both compounds potently inhibited the appearance of CHIKV-positive cells and CHIKV-associated CPE (Figure 7A and 7B). Bafilomycin-A1 was efficient at doses from 2.5 to 250 nM, without obvious cytotoxicity, whereas the active concentration range of chloroquine was much narrower: this compound fully inhibited CHIKV infection at 10 μM, but was toxic at 100 μM (Figure 7B). Of note, no inhibitory effect was observed if addition of these two compounds was delayed until 3 h after CHIKV exposure (Figure 7A). This suggests that the requirement for an acidic compartment is a feature of an early step of the viral cycle.

Bottom Line: CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells.CHIKV was highly sensitive to the antiviral activity of type I and II interferons.These results provide a general insight into the interaction between CHIKV and its mammalian host.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Unité Virus et Immunité, Institut Pasteur, Paris, France.

ABSTRACT
An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

Show MeSH
Related in: MedlinePlus