Quiescent infection and reactivation of VZV in hESC-derived neurons without the use of ACV.

<p>hESC-derived neurons were infected via their axons in compartmentalized microfluidic chambers as detailed in the methods. A) Quantification of VZV genomes and transcripts from ORF63 and ORF31 were quantified by digital qPCR from nuclei acids extracted from the cell-body compartment two weeks after infection. B) & C) Reactivation of VZV in axonally-infected neurons. (B) Reactivation of VZV in hESC-derived neurons by LY 2 weeks after quiescent axonal infection. Neurons infected as in A were treated for 4 days with LY at 37°C and DNA and RNA extracted. qPCR revealed an increase in VZV genomes and transcripts from ORF62 and ORF31. No GFP-positive neurons were observed under these reactivation conditions. (C) Reactivation of neurons axonally-infected by VZV at 34°C. A photomicrograph of a microfluidic chamber where the cell body compartment (CB) containing the somata of axonally infected neurons was treated for 4 days with LY at 34 degrees is shown. A cluster of neurons (upper box) expressing ORF66GFP as a result of the treatment (higher magnification image in upper inset). The axons in the axonal compartment (Ax) are coated by the GFP-fluorescent debris (lower box, higher magnification image in the lower inset) used to infect the axons. Ch = microfluidic channels connecting the cell body and axonal compartments. Scale Bars = 100μm.