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, 21 (2), 274-283.e5

High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates That Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain

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High-Content Screening in hPSC-Neural Progenitors Identifies Drug Candidates That Inhibit Zika Virus Infection in Fetal-like Organoids and Adult Brain

Ting Zhou et al. Cell Stem Cell.

Abstract

Zika virus (ZIKV) infects fetal and adult human brain and is associated with serious neurological complications. To date, no therapeutic treatment is available to treat ZIKV-infected patients. We performed a high-content chemical screen using human pluripotent stem cell-derived cortical neural progenitor cells (hNPCs) and found that hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ) can inhibit ZIKV infection in hNPCs. Further validation showed that HH also rescues ZIKV-induced growth and differentiation defects in hNPCs and human fetal-like forebrain organoids. Finally, HH and AQ inhibit ZIKV infection in adult mouse brain in vivo. Strikingly, HH suppresses viral propagation when administered to adult mice with active ZIKV infection, highlighting its therapeutic potential. Our approach highlights the power of stem cell-based screens and validation in human forebrain organoids and mouse models in identifying drug candidates for treating ZIKV infection and related neurological complications in fetal and adult patients.

Keywords: Zika virus; high content chemical screen; human cortical neuron progenitor cells; human forebrain organoids; human pluripotent stem cells.

Figures

Figure 1
Figure 1. A high content chemical screen identifies anti-ZIKV compounds in hNPCs
(A) Scheme of high content chemical screen. (B) Chemical structure of hit compounds. (C) Inhibitory curve of HH or AQ on hNPCs (n=3) based on infectious particles in supernatant as determined by the Vero reinfection assay. (D–H) Immunocytochemistry analysis (D), the quantification of the percentage of ZIKV E+ cells (E), proliferation rate (F), cell apoptosis rate (G) and the normalized cell viability (H) of hNPCs at 72 h after ZIKV (MR766) infection. Scale bars, 100 μm. (n=6). (I) qRT-PCR analysis to monitor the total ZIKV vRNA in the supernatant of hNPC cultures at 72 h after ZIKV infection. (J and K) Vero cell reinfection assay (J) and the quantification of ZIKV infectious particles produced by ZIKV-infected hNPCs (K). Scale bars, 200 μm. (n=3). (L) Heatmap of mock-infected hNPCs+DMSO (mock); mock-infected hNPCs+25 μM HH (HH), mock-infected hNPCs+15 μM AQ (AQ), ZIKV infected hNPCs+DMSO (ZIKV+DMSO); ZIKV-infected hNPCs+25 μM HH (ZIKV+HH), and ZIKV-infected hNPCs+15 μM AQ (ZIKV+AQ). Genes were selected from the up/down-regulated genes (fold change >4) in ZIKV-infected hNPCs+DMSO compared to mock-infected hNPCs+DMSO. (M and N) Immunocytochemistry analysis (M) and quantification of the percentage of ZIKV E+ cells (N) of hNPCs at 72 h after ZIKV (PRVABC59, MOI=0.1) infection. Scale bars, 100 μm. p values were calculated by one-way repeated measures ANOVA with a Bonferroni test. *p<0.05, **p<0.01 and ***p<0.001. Related to Figure S1, and Table S1.
Figure 2
Figure 2. HH eliminates virus in ZIKV-infected hNPCs
(A) Scheme to evaluate the elimination of ZIKV. (B) Inhibitory curve of HH calculated based on infectious particles in the supernatant as determined by the Vero reinfection assay (n=3). (C) Effective curve of HH calculated based on normalized cell viability (n=3). (D and E) Immunocytochemistry analysis (D) and the quantification of the percentage of ZIKV E+ cells, cell apoptosis rate, and total cell number (E) of hNPCs on day 3 and day 6. Scale bars, 100 μm. (n=3). p values were calculated by one-way repeated measures ANOVA with a Bonferroni test. **p<0.01 and ***p<0.001. (F) qRT-PCR analysis to monitor the (+) strand ZIKV vRNA in the supernatant of hNPC cultures on day 6. Scale bars, 200 μm. (G and H) Vero cell reinfection assay (G) and the quantification of ZIKV infectious particles produced by ZIKV-infected hNPCs at day 6. Scale bars, 200 μm. (n=3). (I) hNPCs with or without ZIKV MR766 infection were treated with 25 μM HH or DMSO for 3 days and then stained for hNPC markers: SOX2 (green) and NESTIN (red), and with DAPI (blue), or were differentiated to cortical neurons. Scale bars, 100 μm. (J) qRT-PCR analysis to quantify the (+) strand and replicating (−) strand ZIKV vRNAs in the hNPCs at different time points post infection. The dash line shows detection limit. p values were calculated by one-way repeated measures ANOVA with a Bonferroni test. ***p<0.001. UD: undetectable. (K) qRT-PCR analysis of hNPCs, on which 25 μM HH or DMSO were added at 24, 36 and 48 hpi (MR766) infection and maintained for additional 3 days. p values were calculated by unpaired two-tailed Student’s t-test. *p<0.05, **p<0.01 and ***p<0.001, if not mentioned specifically. Related to Figure S2.
Figure 3
Figure 3. HH inhibits ZIKV infection in short and long term forebrain organoid cultures
(A) Scheme to evaluate HH using forebrain organoids. (B and C) Immunocytochemistry analysis (B) and the quantification of the percentage of ZIKV E+ cells (C) in mock, DMSO or 25 μM HH treated ZIKV-infected D20 organoids at D20+4 (n=3). Scale bars, 100 μm. (D) qRT-PCR analysis of (+) strand ZIKV vRNA in the supernatant of DMSO or 25 μM HH treated ZIKV-infected D20 organoids (n=3). (E) HH rescues ZIKV-related proliferation and apoptosis defects in D20+4 organoids. (n=3). Scale bars, 100 μm. (F) D20+18 organoids are highly infiltrated by ZIKV (red), while HH suppressed any detectable ZIKV infection (n=3). Scale bars, 50 μm. (G and H) Brightfield images of whole organoids (G) and the quantification of average cross sectional area of D20+18 organoids (H). Values represent mean ± SEM (n=3). Scale bars, 1 mm. (I) HH rescues ZIKV-induced structural defects in forebrain organoids. (n=3). Scale bars, 100 μm. p values were calculated by unpaired two-tailed Student’s t-test. *p<0.05, **p<0.01 and ***p<0.001 Related to Figure S3.
Figure 4
Figure 4. HH suppresses ZIKV infection in vivo
(A) Scheme of prophylactic treatment of HH in vivo. (B) qRT-PCR analysis of (+) strand ZIKV vRNA in liver, spleen, kidney and brain of ZIKV infected mice. n=13 for liver, spleen and kidney and n=30 for brain. (C) qRT-PCR analysis of (+) strand ZIKV vRNA in brains of ZIKV-infected mice treated with vehicle (n=14) or HH (n=8). UD, undetectable. (D–F) Immunohistochemical analysis of adult cortex using antibodies against ZIKV E (D), βIII-tubulin (E) and GFAP (F). Scale bar, 100 μm in (D) and 10 μm in (E, F). (G–I) Immunohistochemical analysis of adult hippocampus including the post-mitotic CA regions using antibodies against ZIKV E (G), SOX2 (H) and DCX (I). Scale bar, 100 μm in (G) and 10 μm in (H, I). (J–L) Immunohistochemical analysis of adult striatum using antibodies against ZIKV E (J), SOX2 (K) and DCX (L). Scale bar, 100 μm in (J) and 10 μm in (K, L). (M and N) Immunohistochemical analysis of cellular apoptosis in adult cortex antibodies against CAS3. Scale bar, 100 μm in (M) and 10 μm in (N). (O, P) Kinetics of ZIKV (MR766). Mice were infected with (1 x106 PFU) through intraperitoneal injection. Mice were euthanized at 24 hpi, 4 and 5 dpi. vRNA level was analyzed by qRT-PCR (O). Infectivity of viral particles was analyzed by Vero assay (P). n ≥ 4 mice at each time point. (Q) Scheme of therapeutic intervention with HH 24 hpi (MR766) in (R). (R) qRT-PCR analysis of ZIKV vRNA in brains of mice treated with vehicle (n=10) or HH (n=8) for 5 days starting at 24 hpi. (S) Scheme of therapeutic intervention with HH 5 dpi (MR766) in (T–V). (T) qRT-PCR analysis of ZIKV vRNA in the brains of mice treated with vehicle (n=8) or 100 mg/kg HH (n=8) for 5 days starting at 5 dpi. (U) qRT-PCR analysis of ZIKV vRNA in the brains of mice treated with vehicle (n=15) or 200 mg/kg HH (n=8) for 5 days starting at 5 dpi. (V) Quantification of ZIKV infectious particles in serum of ZIKV-infected mice (MR766) treated with vehicle (n=8) or 200 mg/kg HH (n=8) for 5 days starting at 5 dpi. p values were calculated by one-way repeated measures ANOVA or two-way repeated measures ANOVA with a Bonferroni test. *p<0.05, **p<0.01, ***p<0.001. Related with Figure S4

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