Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

Abstract

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

Keywords: Zika virus; antiviral; chloroquine; microcephaly; neural stem cell.

Figure 1

Inhibition of Zika virus (ZIKV) infection by chloroquine in Vero cells. (A) Vero cells were infected with ZIKV MR766 at a multiplicity of infection (MOI) of 2, treated with chloroquine for 5 days, and were then stained for the viral envelope protein and analyzed by flow cytometry; (B) Vero cells were infected, treated for 2 days, and ZIKV infection was evaluated by immunofluorescence staining with 4G2 antibody (green) and DAPI (blue); Infectious (C) or total (D) virus particles were quantified on the supernatant 48 h post-infection. Titers are expressed as plaque forming units (PFU) per milliliter. The dashed line represents no reduction in RNA levels; (E) Cell viability of uninfected cells treated with increasing concentrations of chloroquine was evaluated by fluorescence measurement at 560/590 nm after viability dye incubation; (F) Protection against ZIKV infection was evaluated through cell viability in ZIKV-infected Vero cells treated with chloroquine for 5 days. Data are represented as mean fluorescent intensity (MFI) ± standard deviation (SD) from two to four independent experiments. Statistical analysis was performed with the Kruskal–Wallis test and multiple comparisons with infected and untreated control corrected by Dunn’s test (* p < 0.05; ** p < 0,005).

Figure 2

Chloroquine inhibits infection by Asian lineage. (A) Vero cells were infected with Brazilian ZIKV strain at an MOI of 2, treated with chloroquine at the indicated concentrations for 5 days, and the frequency of infected cells was evaluated by flow cytometry; (B) Vero cells were infected with Brazilian strain at an MOI of 2 and exposed to chloroquine for 48 h. The supernatant was collected and viral RNA was relatively quantified over the untreated infected control (B) or infectivity was analyzed by immunofluorescence with 4G2 antibody (C). The dashed line represents fold reduction on virus production of 1. Data are represented as mean ± SD from two independent experiments. Statistical significance was assessed by Kruskal–Wallis test and multiple comparisons with infected and untreated control corrected by Dunn’s test (* p < 0.05).

Figure 3

Early stages of infection are inhibited by chloroquine. Vero cells were infected with ZIKV MR766 at an MOI of 10 and 50 μM chloroquine was added at different times post-infection. At 30 h post-infection, the supernatant was collected and viral RNA (A) or infectious particles (B) were quantified. Viral RNA reduction is represented in fold change (2^∆Ct). The dashed line represents no reduction in RNA levels. Infectious particles are depicted as PFU/mL. Data are represented as mean ± SD from two independent experiments. Statistical analysis was performed with Kruskal–Wallis test and multiple comparisons with infected and untreated control corrected by Dunn’s test (* p < 0.05).

Figure 4

Chloroquine reduces the number of ZIKV-infected human brain microvascular endothelial cells (hBMECs). (A) hBMECs were infected with ZIKV MR 766 at an MOI of 2 followed by chloroquine treatment for 5 days. Cells were stained with the 4G2 antibody and analyzed by flow cytometry; (B) Uninfected hBMECs were incubated with chloroquine for 5 days and cell viability was analyzed; (C) Protection against ZIKV infection was measured through cell viability in chloroquine-treated ZIKV-infected cells; (D) Immunofluorescence with 4G2 antibody (green) and DAPI (blue) of ZIKV-infected cells treated with chloroquine for 5 days. Data are represented as mean ± SD from two independent experiments. Statistical significance was assessed by Kruskal–Wallis test and multiple comparisons with infected and untreated control corrected by Dunn’s test (* p < 0.05).

Figure 5

Chloroquine inhibits ZIKV infection in human neural stem cells (hNSCs). hNSCs were infected with ZIKV MR766 at an MOI of 2 and incubated with increasing concentrations of chloroquine for 4 days. (A) The frequency of ZIKV-infected cells was analyzed by 4G2 staining and flow cytometry; (B) Chloroquine cytotoxicity was assessed by the viability of uninfected hNSCs treated with chloroquine; (C) Chloroquine treatment protection from ZIKV infection was evaluated by cell viability measurement at 4 days post-infection. Immunofluorescence NSCs infected with ZIKV MR766 (D) or ZIKV BR (E) at an MOI of 2 and treated with chloroquine for 4 days with 4G2 antibody (green) and DAPI (blue). Data are represented as mean ± SD from two to three independent experiments. Statistical analysis was performed with Kruskal–Wallis test and multiple comparisons with infected and untreated control corrected by Dunn’s test (* p < 0.05).

Figure 6

Chloroquine inhibits ZIKV infection in mouse neurospheres. Mouse neurospheres were infected with ZIKV MR766 (2.5 × 10⁵ PFU and were treated with chloroquine for 3 days. Neurospheres were analyzed by phase contrast microscopy (A–C), and triple stained for envelope viral protein (green), microtubule-associated protein 2 (Map-2, red), a neuron-specific protein, and DAPI (blue) (D–F).