DM and NDM differ in their ability to translocate from gray matter to white matter during acute infection. DM- and NDM-infected mice were sacrificed at day 7 p.i.; brain and spinal cord tissues were harvested and processed for frozen sections. Cryostat sections were post fixed with 95% ice-cold ethanol for 20 minutes and observed by fluorescent microscopy for viral antigen (EGFP fluorescence). (a) In DM-infected spinal cord, viral antigen positive EGFP-expressing cells are present in both gray and white matters. (b) Area of EGFP positive cells from (a) is marked in a corresponding LFB-stained section. (c) In NDM-infected mice, EGFP positive cells with viral antigen are predominantly restricted to the gray matter. (d) Area of EGFP positive cells from (c) is marked in corresponding LFB-stained section. (e) Axonal distribution of the EGFP fluorescent viral antigen is evident in DM-infected brain. (f) NDM-infected neurons in the brain show considerably reduced axonal transport of viral antigen. (g–i) to further confirm the ability of DM and NDM to infect neurons and spread from neuron to neuron, 4-day-old primary hippocampal neuronal cultures were infected with DM and NDM at MOI of 2. Representative fields containing an equivalent density of cells are shown in each image. (g) At 24 hours p.i. DM infect a small percentage of the neurons in culture. (h) By 72 hours p.i. DM-infected neurons were observed to spread from one neuron to the next, as nearly all cells in the culture contained viral antigen despite initial infection of only a small percentage of cells. (i) NDM-infected neurons, on the other hand, demonstrate limited axonal transport efficiency at 72 hours p.i., with only a small number of cells containing a bright concentrated signal that is retained after primary infection. (adapted from the work of [73]).
