Fig. 3.

Infection of hESC-derived neurons with GFP-expressing recombinant VZV observed in living cultures. (A) Low-magnification view of an hESC-derived neural culture before addition of virus. A large clump of neurons (white asterisk) at the center of the image has multiple radiating neurites (arrows). Many individual and small clusters of neurons (arrowheads) are also apparent. (B) Infection of human neurons 2 days after seeding VZV GFP66. A GFP-expressing MeWo cell (white arrow) and a single GFP-expressing neuron (bold white arrow) are indicated. At higher magnification (inset), this neuron can be seen to have a distinct neurite that is GFP negative. (C) At 9 days postinfection, most of the neurons express GFP. (D) Higher magnification image of cells shown in panel C, showing neurites (arrowheads); GFP fluorescence is evident in the somata but not in the neurites. Merge of GFP fluorescence and phase contrast images is shown. Some small ORF66-GFP-negative nuclei are present (arrowheads). (E) Same field as in panel D but of GFP-fluorescence only, showing that almost all of the cells in the field are VZV infected. (F) At 22 days postinfection, most cells are GFP⁺ but extend few neurites, and many neurons have detached from the substrate. In contrast, parallel cultures of MeWo cells that were not infected with VZV (G) contain healthy neurons with an extensive plexus of neurites. (H) Merged images of phase-contrast and fluorescence images of live hESC-derived neurons infected with VZV_BAC. The GFP diffusely fills the entirety of the neurons, including the full extent of the axons. (I) Same field as in panel H, showing only GFP fluorescence. Most of the neurons in the field are infected. Scale bars, 120 μm (A), 100 μm (C and G; applies also to B and F), 20 μm (E) and 40 μm (I).