Viral encephalitis is an important cause of morbidity and mortality worldwide. Key gaps in knowledge about the pathogenesis of viral encephalitis include how neurotropic viruses target the central nervous system (CNS), transit neurons to disseminate in the brain, and elicit neural injury. The proposed research uses reovirus, a genetically tractable double-stranded RNA virus, to dissect viral tropism, spread, and damage in the CNS. Following primary infection in the murine intestine, reovirus disseminates to the brain, where it exhibits serotype-specific differences in producing neurologic disease. Serotype 3 reovirus is exquisitely neurotropic, spreads via both anterograde and retrograde axonal transport, and elicits apoptosis by activating NF-?B- dependent transcriptional networks. Junctional adhesion molecule-A (JAM-A) is required for reovirus infection of endothelial cells and hematogenous dissemination, but this receptor is dispensable for infection in the CNS. Nogo receptor-1 (NgR1) is a recently identified reovirus receptor required for infection of some but not all populations of CNS neurons. Three integrated specific aims are proposed to enhance knowledge of reovirus neural targeting, axonal transport, and brain injury.
In Specific Aim 1, the function of NgR1 in reovirus neurotropism will be determined. Wild-type (WT) and NgR1-/- mice will be inoculated intracranially with reovirus and monitored for infection of distinct brain regions and cell types. NgR1 co-receptors and NgR1-dependent signaling molecules activated by reovirus infection will be identified using pharmacologic inhibitors, RNA interference, and reconstitution of receptor complexes in nonsusceptible cells. Additional CNS receptors for reovirus will be identified by screening a cDNA library prepared from susceptible neural tissues of JAM-A-/- ? NgR1-/- mice.
In Specific Aim 2, mechanisms of reovirus neural dissemination will be defined. The pathway of reovirus spread to and within the murine CNS will be determined using quantitative immunohistochemistry. The molecular basis of reovirus neuronal entry, axonal transport, and progeny particle release will be determined using microfluidic chambers, pharmacologic inhibitors of axonal transport and synaptic transmission, live-cell imaging, and immunofluorescence and electron microscopy.
In Specific Aim 3, the process of NF-?B- dependent cell killing by reovirus in the CNS will be elucidated. Reovirus-induced changes in the expression of NF-?B-dependent genes in the brain will be defined by comparing transcriptional profiles in infected WT mice and mice lacking the NF-?B p65 subunit specifically in neurons. NF-?B-dependent mediators of reovirus encephalitis will be identified by testing NF-?B-dependent candidates using complementary gain-of-function and loss-of-function approaches. Functions of reovirus encephalitis mediators in neural injury caused by enterovirus-71 and herpes simplex virus-1, which also induce NF-?B, will be determined using mediator- deficient mice. These studies will enhance an understanding of mechanisms used by neurotropic viruses to infect specific regions in the CNS, disseminate within and between neurons, and cause lethal encephalitis.
The proposed research will enable a better understanding of mechanisms used by viruses to spread within nerves to infect specific regions of the brain and produce damage to neural tissue. This information can be used to identify new drug targets for viral encephalitis and develop precision therapies for nervous system cancers using oncolytic viruses.
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