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Figures
- Fig. 1.
hiNSCs secrete virus, display an altered secretome and differentiation profile, and undergo apoptosis in response to ZIKV infection, which can be attenuated by Niclosamide treatment in vitro. (A) Infection rate of hiNSCs at different MOIs as determined by immunostaining against flavivirus envelope protein. Scale bar: 100 µM. (B) ELISA assay of secreted ZIKV NS1 protein in cell culture supernatants indicates that hiNSCs secrete increased ZIKV NS1 over time, which can be attenuated by pre- or concurrent NIC treatment. (C) Cytokine array data showing that hiNSCs secrete a variety of cytokines and growth factors. (D) qRT-PCR data indicating that NIC inhibits the expression of CXCL10, LIF and VEGF to varying degrees. (E) ZIKV affects the differentiation profile of hiNSCs. ZIKV infection causes hiNSCs to express higher levels of VGAT and lower levels of VGLUT2, and NIC partially restores the expression of VGAT to control levels. Scale bar: 100 µM. (F) ZIKV induces massive cell death in hiNSCs. By day 10, most hiNSCs are dead regardless of starting MOI. Panel shows immunostaining of mock or ZIKV-infected hiNSCs (MOI 0.0005). Pre- or concurrent NIC treatment can partially restore rate of Caspase3 expression to normal. Scale bar: 100 µM. *P≤0.05, **P≤0.01, ***P≤0.001; as determined by one-way ANOVA with post hoc Tukey test. Error bars show mean±s.d.
- Fig. 2.
Intracranial injection of ZIKV-infected hiNSCs into chick embryos results in microcephaly. Injection of ZIKV-infected hiNSCs results in microcephaly. (A) Schematic and image of intracranial injection. (B) Embryos injected with mock- (left) or ZIKV-infected (right) hiNSCs 10 days post-injection. (C) Coronal sections showing pan-neuronal Tuj1 immunostaining showing morphological characteristics. fb, forebrain; ot, optic tectum (midbrain); de, diencephalon; arrow, ventricle. Scale bar: 1 mM. (D) Immunostaining indicating presence of hiNSCs (HuNu+) in the developing brain of E12 chick embryos, which are also positive for Tuj1. Scale bar: 100 µM. (E) ZIKV-infected hiNSCs are also positive for anti-flavivirus marker (ZV). Scale bar: 100 µM.
- Fig. 3.
Systemic Niclosamide treatment can partially rescue ZIKV-induced reduction in cranial size in a humanized in vivo model. (A) Schematic diagram showing experimental timeline of injection of hiNSCs as well as systemic ZIKV infection and NIC treatment. Briefly, embryos were injected with hiNSCs intracranially and treated with the first dose of either vehicle or NIC via the CAM at E3. At E5, embryos were subjected to systemic ZIKV infection as well as the second dose of either vehicle or NIC via the CAM, and all embryos were harvested at E12. (B) Image of E12 embryos after treatment (left to right: Mock plus vehicle, ZIKV plus vehicle, ZIKV plus NIC). Quantification of body length (C), eye area (D) as well as morphological features of the cranium including length (E), width (F) and height (G) across treatments. *P≤0.05, **P≤0.01, ***P≤0.001; as determined by one-way ANOVA with post hoc Tukey test.
- Fig. 4.
Niclosamide treatment reduces ZIKV infection and altered differentiation of hiNSCs in vivo. (A) Tuj1 immunostaining through the intact cranium. fb, forebrain; arrow, 4th ventricle. Scale bar: 1 mM. Quantification of forebrain and 4th ventricle area across treatments. (B) Immunostaining showing co-localization of HuNu and ZV to quantify the number of ZIKV-infected hiNSCs. Scale bar: 50 µM. (C) Immunostaining showing co-localization of HuNu and CXCL10 with quantification. Scale bar: 50 µM. (D,E) Immunostaining showing co-localization of HuNu with either VGAT (D) to indicate the presence of GABAergic neurons or VGLUT2 (E) which indicates glutamatergic neurons, with respective quantification. Scale bar: 100 µM. *P≤0.05, **P≤0.01, ***P≤0.001; as determined by one-way ANOVA with post hoc Tukey test. Error bars show mean±s.d.
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