Figure 1. Zika virus (ZIKV) productively infects cranial neural crest cells (CNCCs).
A. Model of in vitro human neural development.
B. Embryonic stem cells (ESCs), neurospheres, and CNCCs were infected with ZIKV at MOI 0.1 (or mock infected in PBS) for 1 hour, and incubated for 23 hours at 37C. Infection was detected by intracellular staining for ZIKV with anti-flavivirus group surface antigen and analyzed by flow cytometry. See also Figure S1A.
C. Viral titers in culture supernatants as determined by plaque assay on Vero cells. Bars represent mean titer from two technical replicates of supernatants from two independent biological experiments. Plaque formation was not detected (N.D.) in supernatants from mock-infected controls. Error bars are S.D.
D. CNCCs were infected with ZIKV or DENV at matched MOI (or mock infected in PBS) for 1 hour, followed by 23 hours of incubation at 37°C. Infection was detected by intracellular staining for ZIKV with anti-flavivirus group surface antigen and flow cytometry. Data is from a representative experiment.
E. Summary of infections of CNCCs. Points represent independent biological experiments. ZIKV vs. mock, p = 0.0078, Wilcoxon paired signed-rank test. ZIKV vs. DENV, p = 0.044, Mann-Whitney test. (Mock and ZIKV n=10, DENV n=2). *p < 0.05; **p < 0.01.
F–G. Quantifications of flow cytometry analysis of apoptotic death at 24 hours (F) and 72 hours (G) after infection. Cells were stained with Annexin V and propidium iodide to mark early apoptosis (Annexin V+/PI−), late apoptosis (Annexin V+/PI+) and non-apoptotic dead cells (Annexin V−/PI+). See also Figure S1E.
H. Confocal immunofluorescent images of neurospheres after 72 hours of transwell culture. Neurospheres were incubated alone (left), or with uninfected (center) and ZIKV-infected (right) CNCCs on the transwell membrane. Neurospheres were stained for cleaved caspase-3 (green), Tuj1 (red), and DAPI (blue). See Figure S1F for ZIKV-infected neurospheres alone.
- Example of non-neuronal migratory cellular outgrowth
- Example of neuronal projections
I. Quantification of neurosphere morphology after exposure to transwell CNCCs. Spheres alone vs. +ZIKV-inf CNCCs, p = 0.0041, chi-squared test of equal proportions; uninfected CNCCs vs. ZIKV-inf CNCCs, p = 0.0139, chi-squared test of equal proportions. *p < 0.05; **p < 0.01.
J. Quantification of apoptotic activity in neurospheres. Confocal image z-stacks were analyzed for the intensity of Tuj1 and caspase signals. Caspase-3 intensities were normalized by Tuj1 signal (in the central bolus of the neurosphere) to account for differences in neurosphere size. Spheres alone vs. +ZIKV-inf CNCCs, p = 0.0260, Mann-Whitney test; uninfected CNCCs vs. ZIKV-inf CNCCs, p = 0.1320, Mann-Whitney test.
*p < 0.05.