Imaging the Transport Dynamics of Single Alphaherpesvirus Particles in Intact Peripheral Nervous System Explants from Infected Mice

Alphaherpesvirus particles travel long distances in the axons of neurons using host microtubule molecular motors. The transport dynamics of individual virions in neurons have been assessed in cultured neurons, but imaging studies of single particles in tissue from infected mice have not been reported. We developed a protocol to image explanted, infected peripheral nervous system (PNS) ganglia and associated innervated tissue from mice infected with pseudorabies virus (PRV). This ex vivo preparation allowed us to visualize and track individual virions over time as they moved from the salivary gland into submandibular ganglion neurons of the PNS. We imaged and tracked hundreds of virions from multiple mice at different time points. We quantitated the transport velocity, particle stalling, duty cycle, and directionality at various times after infection. Using a PRV recombinant that expressed monomeric red fluorescent protein (mRFP)-VP26 (red capsid) and green fluorescent protein (GFP)-Us9 (green membrane protein), we corroborated that anterograde transport in axons occurs after capsids are enveloped. We addressed the question of whether replication occurs initially in the salivary gland at the site of inoculation or subsequently in the neurons of peripheral innervating ganglia. Our data indicate that significant amplification of infection occurs in the peripheral ganglia after transport from the site of infection and that these newly made particles are transported back to the salivary gland. It is likely that this reseeding of the infected gland contributes to massive invasion of the innervating PNS ganglia. We suggest that this “round-trip” infection process contributes to the characteristic peripheral neuropathy of PRV infection.