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. 2017 Oct;24:189-194.
doi: 10.1016/j.ebiom.2017.09.034. Epub 2017 Sep 28.

Chloroquine, a FDA-approved Drug, Prevents Zika Virus Infection and Its Associated Congenital Microcephaly in Mice

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Free PMC article

Chloroquine, a FDA-approved Drug, Prevents Zika Virus Infection and Its Associated Congenital Microcephaly in Mice

Chunfeng Li et al. EBioMedicine. .
Free PMC article

Abstract

Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public.

Keywords: Antiviral effects; Chloroquine; FDA-approved drug; Microcephaly; ZIKV entry.

Figures

Fig. 1
Fig. 1
CQ inhibits ZIKV entry in vitro and in vivo. (A) BHK-21 cells were treated for 12 h with 10 μM of 16 individual compounds known to inhibit entry by EBOV (25HC was used as positive control; Ribavirin was used as a negative control). The cells were then infected by ZIKV (GZ01/2016 strain, 200 PFU/well) for 1 h and plaque assay was performed to compare the levels of infection. 5 molecules (chloroquine, clomiphene, amodiaquine, alverine and sertraline) were identified to inhibit ZIKV infection. (B–D) Cells were pretreated with CQ for 12 h and then infected with ZIKV for 1 h. ZIKV in cell supernatants were detected with qRT-PCR at 48 hpi. The IC50 of CQ on ZIKV (GZ01) in Vero (B) or Huh7 cells (C), or on ZIKV (FSS13025) in Huh7 cells (D) are indicated. (E–G) Inhibitory effects of CQ on binding to Vero cells (E), ZIKV internalization (F) and RNA synthesis (G) are shown. (E-F) Cells were pretreated with CQ for 12 h, then were infected with ZIKV (E, 200 PFU/well; F, MOI = 1) at 4 °C for 1 h. (F) Then cells were washed once by PBS and incubated at 37 °C for 1 h. The bound ZIKV (E) and internalized ZIKV (F) were detected using plaque assay and qRT-PCR, respectively. (G) ZIKV replicon RNA was transfected into BHK-21 cells. 6 h later, CQ or NITD008 were used to treat cells for 48 h. Renilla luciferase activity was measured with a microplate reader. All data are shown as mean ± SEM from three independent experiments, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, unpaired student t-test.
Fig. 2
Fig. 2
CQ protects against ZIKV infection in mouse models. (A) Antiviral effect of CQ on ZIKV infection in BALB/c mice (A) and A129 mice (B–C). 100 mg/kg of CQ was administrated to mice i.g. 6 h before ZIKV infection i.p. (105 PFU/mouse for BALB/c, 103 PFU/mouse for A129 experiment). Viremia was determined at 1 (A), 3 or 5 (B–C) dpi as indicated. (D) Improved survival of ZIKV-infected suckling neonatal mice after CQ was given to breastfeeding mothers. 1-day-old neonatal mice were infected intracerebrally with ZIKV (100 PFU/mouse, GZ01 strain). Kunming mother mice were administrated CQ subcutaneously (s.c.) at 0, 12 or 24 hpi. CQ continued to be given daily to the mothers for another 10 days. Survival of ZIKV-infected neonatal mice was followed until 30 dpi. Median values for 4–7 mice for experiments in A–C are shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, unpaired student t-test. Log-Rank (Mantel-Cox) test were used in D (PBS, n = 10; 0 hpi, n = 20; 12 hpi, n = 18; 24 hpi, n = 8).
Fig. 3
Fig. 3
CQ protects embryonic brains from ZIKV infection and microcephaly. Littermate embryonic brains were injected with ZIKV or medium at E13.5, then the pregnant mice were treated daily with CQ or vehicle until E18.5. (A) Images of brains and Nissl staining of coronal sections. (B) Measurements of cortical layer thickness. Mock + Veh: n = 9/4, Mock + CQ: n = 6/3, ZIKV + Veh: n = 6/3, ZIKV + CQ: n = 8/4. CP: cortical plate, SP: subplate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone. (C) Images of coronal sections stained with ZIKV antiserum (green) and DAPI (blue). Right panel: Quantification of relative intensity. n = 7/3 for each. (D) Images of cortices stained for the activated form of Caspase3 (white) and DAPI (blue). Right panel: quantification of relative intensity. N = 7/3 for each. (E) Images of cortices stained with phosphorylated Histone H3 (P–H3, red). Right panel: quantification of P-H3 + cells. Mock + Veh: n = 7/4, Mock + CQ: n = 8/3, ZIKV + Veh: n = 5/3, ZIKV + CQ: n = 5/3. (F) Coronal sections stained for ZIKV (green), Sox2 (red) and Tbr2 (white). Right panel: quantification of the density of Sox2 + cells and Tbr2 + cells in the cortices. Mock + Veh: n = 8/4 (Sox2 +), 9/4 (Tbr2 +); Mock + CQ: n = 6/3 (Sox2 +), 6/3 (Tbr2 +); ZIKV + Veh: n = 10/5 (Sox2 +), 10/5 (Tbr2 +); ZIKV + CQ: n = 10/5 (Sox2 +), 10/5 (Tbr2 +). All data are means ± SEM. Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. #P < 0.05, ##P < 0.01, ###P < 0.001. ns: not significant. n: number of slices/different brains. Scale bar for 1 mm (A), 100 μm (B–F).

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