The users may expect to see differences in organoid size by approximately 3 days after ZIKV infection, depending on the viral MOI applied to the organoids (Figure 5A-B). After these 3 days, there will also be an increase in cellular debris in the infected wells compared to the mock wells. This difference in organoid size will increase over the following week, until the infected organoids begin to break apart (Figure 5C-D). A variety of assays may be used at this point to investigate infection mechanisms, including viral RNA extraction and cryosectioning (recommended antibodies reported in the Table of Materials). Upon cryosectioning, users will observe a large presence of virus in the apical region of neuroepithelial structures, suggesting a susceptibility of neural progenitor cells (NPCs) to ZIKV infection (Figure 6). Immunofluorescence of sectioned organoids will also show an increase in cleaved caspase-3 expression in the infected organoids (Figure 7).
