FIG 7.

Analysis of anterograde axonal transport of HSV-1 capsids. Neonatal rat DRG neurons were dissociated, pelleted through a 35% Percoll gradient, and plated into the somal compartment of three-chamber microfluidic devices. Cultures were incubated for 5 to 6 days to allow axons to grow into the axonal compartment via the middle grooves. Vero cells were added to the axonal compartment 24 h prior to the addition of either HSV-1pUS9KBDM or HSV-1pUS9KBDR (5 PFU/cell) to the somal compartment. Foscarnet (100 μg/ml) was added to the axonal compartment 8 h p.i. to prevent secondary virus spread in Vero cells. The cultures were fixed at 22 h p.i., immunostained for C capsids (PTNC), and examined using a Leica SP5 confocal microscope. (A) Representative confocal micrographs of axons (indicated by arrows) emerging from the middle grooves (visible on the left side of the lower images) into the axonal compartment. These axons were counted from 10 randomly selected fields of view and scored as positive or negative for the presence of HSV-1 capsid (red in the upper images). Scale bars, 25 μm. (B) The percentage of capsid-positive (+ve) axons versus capsid-negative (−ve) axons in either case is shown for three separate experiments. A significant reduction in capsid transport along axons was observed for HSV-1pUS9KBDM compared to that of HSV-1pUS9KBDR.