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. 2022 Oct 6;29(10):1475–1490.e6. doi: 10.1016/j.stem.2022.09.008

Figure 3.

Figure 3

Risk alleles of rs917172 and rs12386620 cause increased sensitivity to ZIKV infection

(A) Relative luciferase activity of rs917172 (risk: G, non-risk: T) and rs12386620 (risk: C, non-risk: T) in 293T cells.

(B) Relative NDUFA4 mRNA expression in hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles. The value was normalized to ACTB.

(C) Western blotting analysis of NDUFA4 protein expression levels in hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles. β-Actin was used as a loading control.

(D and E) Representative confocal images (D) and the quantification (E) of ZIKV-E staining in ZIKV-infected hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles at 72 hpi (ZIKVPR, MOI = 1). Scale bar, 50 μm.

(F) qRT-PCR analysis of (+) and (−) ZIKV vRNA strands in ZIKV-infected hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles at 72 hpi (ZIKVPR, MOI = 1). The value was normalized to ACTB.

(G) Multiple step growth curve of ZIKV in the supernatant of ZIKV-infected hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles (ZIKVPR, MOI = 1).

(H and I) Representative confocal images (H) and the quantification (I) of ZIKV-E staining in cerebral organoids derived from hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles (ZIKVPR, 3 × 106 PFU/mL). Cerebral organoids were age-matched and collected at day 20, then infected with ZIKV for 24 h. After removal of virus-containing medium, organoids were maintained in organoid medium for an additional 3 days. Scale bar = 50 μm.

(J) qRT-PCR analysis of (+) and (−) ZIKV vRNA strands of cerebral organoids derived from hiPSCs carrying risk (G/G; C/C) and non-risk (T/T; T/T) alleles (ZIKVPR, 3 × 106 PFU/mL). Cerebral organoids were age-matched and collected at day 20, then infected with ZIKV for 24 h. After removal of virus-containing medium, organoids were maintained in organoid medium for an additional 3 days. The value was normalized to ACTB.

(K) The percentage of IgG+ or IgM+ patients carrying risk or non-risk allele of rs917172 and rs12386620.

Data are representative of at least three independent experiments. Data are shown as mean ± SD. For comparison with more than two samples, p values were calculated by two-way ANOVA analysis. For comparison with two samples, p values were calculated by unpaired two-tailed Student’s t test; ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. See also Figure S3.