A long-term goal of my laboratory is to determine the molecular mechanisms by which neuroinvasive alpha-herpesviruses invade and spread in the mammalian nervous system. Work in this proposal centers on using new imaging technology (e.g., two-photon laser scanning microscopy and serial block face scanning electron microscopy) to reveal how virion components move inside and between neurons. Experiments are divided between two aims focusing on using imaging technology to visualize how infection spreads from one neuron to another in vitro (dissociated neurons; aim 1) and in vivo (living animals and tissues; aim 2). The primary hypothesis to be tested is that trans-neuronal spread is mediated by an enveloped capsid released from the infected cell that is subsequently taken up by the synaptically-opposed uninfected neuron. Two contrasting hypotheses are also considered. In aim 1, virion structures will be labeled with fluorescent fusion proteins, neuronal structures will be labeled with specific dyes and antibodies, and imaged using two-photon microscopy and conventional confocal microscopy in live and fixed dissociated peripheral nervous system neurons. To aid in visualizing sites where trans-neuronal infection occurs, selected viral mutants defective in spread and marked with various fluorescent reporters will be used. In aim 2, I will study in vivo viral invasion and spread of infection from the peripheral nervous system (the submandibular ganglia) to the central nervous system in living animals, tissue explants, and fixed tissues. Serial block face scanning electron microscopy will enable collection of stacks of in-register 50nm sections to reveal 3D images of PRV-infected submandibular gland ganglia. These images will complement and expand the observations made using two-photon laser scanning microscopy. The knowledge obtained from these studies enables not only a better understanding of herpesvirus pathogenesis, it also provides the means to reveal aspects of neuronal cell biology and neural circuitry that are key to viral invasion and spread. Despite modern medicine and antiviral drugs, viral infections of the nervous system are devastating and exceedingly difficult to manage. Understanding the host and viral interactions involved in neuroinvasion and resulting pathogenesis has relevance to human health by revealing new targets for prevention and therapy. On the other hand, the same properties of neuroinvasion provide opportunities to use such viruses and mechanisms to understand the organization of the nervous system using viruses as tracers of neural circuitry. PUBLIC HEALTH RELEVANCE: Despite considerable progress over the years, we still do not have a comprehensive understanding of the molecular mechanisms of trans-neuronal spread of herpesviruses, even in simple outline. Understanding the mechanisms of axonal and dendritic transport, assembly, release, and uptake will provide targets for manipulation that could substantially expand our understanding of viral spread. The information obtained from these studies will be important for at least three reasons. First, it will advance our understanding of alpha- herpesvirus infection of the nervous system and trans-neuronal spread of infection by providing an entirely new imaging-based perspective. Second, it will inform us about other diseases involving axonal transport and function such as Charcot-Marie-Tooth disease type II, hereditary spastic paraplegia, amyloid lateral sclerosis, and other peripheral neuropathies such as post-herpetic neuralgia. These diseases are likely to affect similar pathways as those engaged by viruses for their replication and spread in the nervous system. Third, it will provide critical information for the construction of new genetically engineering strains of virus for neural tracing. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.