PMC

(A) Both the flavivirus Dengue-2 virus (DENV) and human Cytomegalovirus (HCMV) infect NCX-NES cells and induce aCASP3-mediated cell death, but only HCMV can induce relocalization of pTBK1 at DPI 3.5.
(B) Bar plot indicating the percentage of pTBK1 that was localized in centrosomes or mitochondria in Mock, DENV, or HCMV conditions (number of pTBK1⁺ cells=10 out of 1074 total cells for mock; 7 out of 1194 cells for DENV and 392 out of 441 cells for HCMV, respectively). Fisher’s exact test *** p<0.001.
(C) Bar plot of the percentage of aCASP3⁺ cells in Mock and DENV- or HCMV-infected NCX-NES cells at DPI 3.5 (number of aCASP3⁺ cells=1 out of 1041 total cells for mock; 21 out of 842 cells for DENV and 10 out of 472 cells for HCMV). Two-tailed t-test * p<0.05, ** p<0.01.
(D) Treatment with the agonist of RIG-I-like receptors KIN1408 induces relocalization of pTBK1 from centrosomes to mitochondria and increased aCASP3-mediated cell death.
(E) Bar plot indicating the percentage of pTBK1 that was localized in centrosomes or mitochondria in vehicle- or KIN1408-treated NES cells after 2 day-treatment (number of pTBK1⁺ cells=10 out of 1074 total cells for vehicle and 19 out of 934 cells for KIN1408). Fisher’s exact test *** p<0.001.
(F) Bar plot indicating the percentage of aCASP3⁺ cells in vehicle- or KIN1408-treated NES cells (number of aCASP3⁺ cells=3 out of 1236 total cells for vehicle, and 23 out of 546 cells for KIN1408). Two-tailed t-test ** p<0.01.
Scale bars, 50µm.

(A) Representative pictures showing 2´-C-methyladenosine and Sofosbuvir treatment on ZIKV-infected NES cells at DPI 3.5. At different concentrations, both drugs show a potent anti-ZIKV activity, as demonstrated by NS1 staining. pTBK1 localization is centrosomal in infected, drug-treated cells. aCASP3 staining shows apoptotic cell death rate in the different conditions.
(B) Bar plot indicating the percentage of NS1⁺ cells in ZIKV-infected NES cells after vehicle- or drug-treatment at DPI 3.5 [number of NS1⁺ cells=2961 out of 3501 total cells for vehicle; 7 out of 2118 total cells for 2´-C-methyladenosine (20µM); 2 out of 1959 total cells for 2´-C-methyladenosine (40µM), 136 out of 2248 total cells for Sofosbuvir (20µM ) and 25 out of 2099 total cells for Sofosbuvir (100µM)]. Two-tailed t-test * p<0.05, *** p<0.001. n.s., not significant.
(C) Bar plot of the percentage of aCASP3⁺ cells in ZIKV-infected NES cells after vehicle- or drug-treatment at DPI 3.5 [number of aCASP3⁺ cells=27 out of 1403 total cells for vehicle; 14 out of 955 total cells for 2´-C-methyladenosine (20µM); 24 out of 502 total cells for 2´-C-methyladenosine (40µM); 5 out of 657 total cells for Sofosbuvir (20µM ) and 4 out of 727 total cells for Sofosbuvir (100mµM)].Two-tailed t-test * p<0.05, ** p<0.01. n.s., not significant.
(D) Bar plot indicating the percentage of pTBK1 that was localized in centrosomes or mitochondria in ZIKV-infected NES cells after vehicle- or drug-treatment at DPI 3.5 (number of pTBK1⁺ cells=32 out of 2098 total cells for vehicle; 5 out of 1163 total cells for 2´-C-methyladenosine (20µM); 4 out of 1457 cells for 2´-C-methyladenosine (40µM); 13 out of 1591 cells for Sofosbuvir (20µM) and 17 out of 1373 total cells for Sofosbuvir (100µM). Fisher’s exact test *** p<0.001. Scale bars, 20µm.

(A) Tissue section of a 30 pcw microcephalic fetus from a ZIKV-infected mother. Higher magnification shows protrusions (asterisk) in ventricular zone (A’). LV, Lateral ventricle; STR, striatum; NCX, neocortex.
(B and C) Immunostaining of an uninfected brain show no NS1⁺ cells in NCX (B) and no ZIKVE⁺ cells in the NCX VZ/SVZ (C).
(D and E) In the infected brain, most of the infected cells (arrowhead) are in VZ/SVZ (64.2% of the total cells) (D). Only 1.2% of CP cells are ZIKV-infected (D) (number of total analyzed cells in the single microcephalic fetal human brain reported=1050). Filled arrowhead depicts red blood cells (D).
(F and G) Immunostaining for vimentin (VIM) in both VZ/SVZ and NCX shows a normal radial orientation of basal processes of RGCs (arrow, F and G).
(H) Upper layers of the NCX show an impaired organization and lower nuclei density compared to uninfected control NCX. Arrow depicts a defective radial process.
(I) Periventricular region showing dysplastic intraventricular protrusions with lower density of RGCs (asterisk) and disorganized RG morphology (arrow) and processes.
(J) NCX of the infected brain displays disorganized arrangement of dysplastic TUBB3⁺ neurons.
(K) Central canal of the spinal cord immunostained for ZIKVE. Filled arrowhead shows positive red blood cells. Ependymal cells are not labeled.
(L) Co-labelling of nestin (NES) and aCASP3 shows positive cells (arrowheads) in the infected brain with no radial fibers.
(M) ZIKVE and aCASP3 staining shows positive cells (arrowheads) in the infected NCX. Filled arrowhead depicts red blood cells.
Scale bars, 50µm.

(A) Schematic of experimental procedure. NES cells were derived from human developing neocortex (NCX) or spinal cord (SC). Donor-matched brain tissues and NCX-NES cells were subjected to single-cell RNA-seq. SC-NES cells are described in .
(B) NCX-NES cells form rosette structures after 24h in culture. Scale bars, 50µm.
(C–E) NCX-NES cells are positive for nestin (NES), SOX2 (99.4±0.8%, n=973), SOX1 (87.8±1.9%, n=1265), CTNNB1 and FOXG1 (96.5±1.1%, n=1277). Scale bars, 50µm.
(F) Violin plots showing expression of cell type-specific marker genes in NCX-NES cells and donor-matched NCX tissue single cells. pan-NSC, pan-neural stem cell; vRGC, ventricular radial glia cell; oRGC outer radial glia cell; IPC, intermediate progenitor cell; Intern., interneuron; Ast., astrocyte; Olig., oligodendrocyte; Micro., microglia. Y-axis, log₂ transformed RPKM.
(G) As in F, violin plots of ventral forebrain marker genes in NCX-NES and donor-matched NCX single cells.
(H and I) t-SNE plots of all NES cell lines and brain single cells colored by origin of the cells (H) or by cell type clusters identified with SNN-Cliq clustering algorithm (I).
(J and K) Electron microscopy images of mock (J) or ZIKV-infected (K) NCX-NES cells. Nucleus (N) appears to be normal (J’) and cytoplasm is clear of ZIKV viral particle (J”) in mock condition.
Nuclear fragmentation is evident in ZIKV-infected condition. Viral particles are evident around nuclear envelope (red arrows in K’, K”). Scale bars, 1µm.
See also .

(A) Schematic representation of the experimental procedure.
(B) Schematic illustration of the early fetal neocortex (NCX). Vimentin (VIM) and DAPI immunostaining of pre-infection slices shows a normal architecture of neocortical wall. Higher magnification of CP and VZ reveals the radial organization of VIM⁺ RG fibers reaching the pial surface. MZ, marginal zone; CP, cortical plate; IZ/SP, intermediate zone/subplate; SVZ, subventricular zone; VZ, ventricular zone; PN, projection neuron; oRGC, outer radial glia cell; IPC, intermediate progenitor cell; vRGC, ventricular radial glia cell.
(C) Organotypic brain slices show ZIKV infection in RGCs at 1.5 days post infection (DPI), 80.6±0.6% of the infected cells localized in VZ/SVZ, 19.4±4% in the IZ/SP and none in CP (total number of infected cells=20). VZ/SVZ are significantly more infected than CP (paired t-test p<0.05). The slices maintain a normal structure, with radial glial fibers reaching the pial surface and a normal orientation in VZ and SVZ.
(D and E) Non-infected neocortical slices show normal morphology and RG scaffold at both DPI 3.5 (D) and 6.5 (E).
(F and G) ZIKV-infected slices show severe defects in RG scaffolding at DPI 3.5 (F) and 6.5 (G). Most of the infected cells are located in VZ/SVZ and IZ/SP regions (73.3±28.7% at DPI 3.5; 89.3±3.4% at DPI 6.5) with fewer CP neurons showing infection (26.7±28.7% at DPI 3.5 and 10.7±3.4% DPI at DPI 6.5, respectively; total number of infected cells=456 at DPI 3.5 and 1315 at DPI 6.5). At DPI 6.5, CP was significantly less infected than the other zones of the NCX (paired two-tailed t-test p<0.01). Regions with high numbers of infected cells, in between arrowheads, show a more pronounced defect in radial orientation of RG fibers. Furthermore, it is possible to observe balloon-like cells that express VIM.
Arrowheads indicate cells that are infected with ZIKV, immunopositive for NS1. Arrows show RG fibers. Scale bars, 50µm. See also .

(A and B) Violin plots of candidate entry factor gene expression distributions in NCX-NES and donor-matched NCX single cells (A) or adult NCX (B). Y-axis, log₂ transformed RPKM (A) or log₂ transformed RPM (B). Proj. neuro., projection neuron; Interneuro., interneuron; Oligo., oligodendrocyte; Endo. cells, endothelial cells.
(C and D) Expression trajectories of AXL (C) and TYRO3 (D) during human neurodevelopment in different brain regions. Y-axis represents gene expression quantified by log₂ transformed signal intensities and the X-axis represents the developmental ages quantified by the log₁₀ transformed post-conceptional days. The dash vertical lines indicate the 15 developmental periods as defined by . The trajectory curves are smoothed by lowess function. The shading areas represent mean±SEM. NCX, neocortex; HIP, hippocampus; AMY, amygdala; STR, striatum; MD, mediodorsal nucleus of the thalamus; CBC, cerebellar cortex.
(E, F) Immunostaining showing that NES cells co-express AXL and VIM.
(G) Immunostaining of AXL and VIM in a 16 pcw fetal NCX shows that AXL is predominantly low expressed in the CP (G’), where it is detected mainly in blood vessels. Contrarily, higher expression of AXL is detected in the VZ/SVZ (G”).
(H) Fetal organotypic slices infected with ZIKV are NS1⁺ in VZ/SVZ RGCs that are also AXL⁺. Arrows point to AXL⁺ cells. Arrowheads indicate the presence of ZIKV infection. Asterisks indicate signal blood vessels.
(I) Expression of AXL in the adult NCX is restricted mainly to the astrocyte ribbon, which is also strongly labeled by GFAP, endothelial cells and some astrocyte processes in CP.
MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone; LV, lateral ventricle; EL, ependymal layer. Scale bars, 50µm See also .

(A) Violin plots of TBK1 gene expression level in single cells from NCX-NES cells, fetal neocortical slices and adult NCX. Y-axis, log₂ transformed RPKM for NCX-NES cells and fetal NCX cells, or log₂ transformed RPM for adult NCX single cells. (B) Digital droplet PCR (n=3 biological replicates) shows higher TBK1 expression in NCX-NES cells compared to organotypic slices. No differences in TBK1 levels between ZIKV (Cambodian or Brazilian strains, ZIKV^C and ZIKV^B, respectively) infected and Mock conditions, in NCX-NES cells and fetal neocortical organotypic slices at DPI 3.5 (as well as at DPI 1.5, not shown).
(C) Western blot of TBK1 and pTBK1 in VZ/SVZ and CP.
(D) Immunostaining for pTBK1 and phosphorylated VIM (pVIM) in NES cells, labeling centrosome and midbody (arrows). pTBK1 and PAX6 are expressed in fetal NCX. pTBK1 and pVIM can be detected in NES cells that are dividing. PAX6 is co-expressed with pTBK1 in VZ and SVZ of fetal NCX and labels cells that are in mitosis. Neither pTBK1 nor PAX6 are detected in CP.
(E – H) NES cells immunostaining shows that pTBK1 is colocalized with the centrosome marker TUBG (G), but not with the mitochondria marker ATP5A1 (E), in non-infected cells. Contrarily, in ZIKV-infected NES cells, pTBK1 is relocalized to mitochondria, as can be observed by co-staining of pTBK1 with ATP5A1 (F), but not with TUBG (H). Infected cells have an abnormal number of centrosomes, as pointed by arrows. Arrowheads indicate mitochondrial staining and asterisks multiple nuclei.
(I and J) Electron microscopy confirms the subcellular localization of pTBK1 in the centrosome (red arrows) and cytoplasm (asterisks) of non-infected cells (I). In infected cells, the signal is detected mainly in mitochondria (J), as pointed by arrowheads. ZIKV particles are also visible in the cytoplasm of infected cells (green arrows).
(K – N) Immunostaining of pTBK1 in neocortical slices shows its localization in the centrosomes and colocalization with TUBG, but not with ATP5A1 in non-infected cells (K and M). In infected cells pTBK1 is present in mitochondria (L). Infected cells in mitosis also show an abnormal number of centrosomes (N). In some infected cells with multiple centrosomes pTBK1 and TUBG colocalize.
(O) Bar plot indicating the percentage of pTBK1 localized in centrosomes or mitochondria in Mock or ZIKV conditions. pTBK1 is mainly localized in centrosomes in Mock or mitochondria in ZIKV-infected cells (number of pTBK1⁺ NES cells=23 out of 1364 total cells for Mock and 56 out of 673 total cells for ZIKV; number of pTBK1⁺ brain slice cells=50 from 2 sections for Mock and 31 from 4 sections for ZIKV). Data are presented as mean±SEM. Fisher’s exact test *** p<0.001.
(P) Jitter plot showing the number of centrosomes in pTBK1⁺ cells, in both NES cells and slice cultures. While in non-infected mitotic cells the number of centrosomes is predominantly 2, in infected cells it ranges from 1 to 5 (number of pTBK1⁺ NES cells=40 for Mock and 80 for ZIKV condition; number of pTBK1⁺ brain slice cells=76 from 2 sections for Mock and 21 from 4 sections for ZIKV). Fisher’s exact test ** p<0.01, *** p<0.001.
(Q) Bar plot of mitochondrial area and mitochondrial density in non-infected and infected NES cells. There is a statistical significant increase in the mitochondrial area between Mock and ZIKV conditions. No difference is present between Mock and ZIKV for mitochondrial density. Two-tailed t-test * p≤0.05.
(R) Representative immunofluorescence staining of vehicle-, Amlexanox- and BX795-treated NCX-NES cells shows supernumerary centrosomes induced by TBK1 inhibition.
(S) Quantification of pTBK1⁺ mitotic NCX-NES cells with or without TBK1 inhibition. Scale bars, 20µm (D–N) and 50µm (R). Two-tailed t-test ** p<0.01, *** p<0.001. See also .