West Nile virus (WNV) is an emerging pathogen that has the potential to cause severe neuropathologies, including encephalitis, acute flaccid paralysis, and meningitis. The severity of WNV infection is highly strain dependent, ranging from avirulent to highly neuropathogenic. Here, we assessed both viral and host-specific factors that contribute to WNV-mediated neuropathogenesis. We initially observed that despite inoculating equivalent levels of virus directly into the blood, mice inoculated with a nonvirulent strain of WNV, WNV-MAD78, exhibited increased survival compared to mice inoculated with a highly pathogenic strain, WNV-NY. Thus, one limitation for nonneuropathogenic WNV may exist at the blood-brain barrier (BBB). Therefore, we investigated the strain-specific contributions to establishing infection in the CNS by comparing the replication of WNV-MAD78 and WNV-NY in neurons, astrocytes, and microvascular endothelial cells, which comprise the neurovascular unit within the CNS. WNVMAD78 replicated in and traversed brain microvascular endothelial cells as efficiently as WNV-NY. Similar levels of replication for both strains were detected in neuronal cells. Thus, WNV-MAD78's nonneuropathogenic phenotype is not due to an intrinsic inability to invade the CNS or replicate within neurons. In contrast, WNV-MAD78 replicated to lower levels than WNV-NY in astrocytes. Reduced susceptibility of astrocytes to WNV-MAD78 was a result of a delay in initiating viral replication and an interferon-independent reduction in cell-to-cell spread. Further characterization indicated that the restriction to WNV-MAD78 cell-to-cell spread in astrocytes was due in part to the higher production of defective particles compared to WNV-NY infected astrocytes. To identify regions of the genome that contribute to this restriction, we generated recombinant viruses of WNV-NY and WNV-MAD78. While replication of a recombinant virus consisting of the structural proteins of WNV-NY and the nonstructural proteins of

WNV-MAD78 was delayed in astrocytes, the virus reached peak viral titers similar to WNV-NY. In contrast, a recombinant virus comprised of the structural proteins from WNV-MAD78 and nonstructural proteins from WNV-NY replicated at a similar rate and level as WNV-MAD78 in astrocytes. Thus, protein composition of the virion is a determining factor in the level of WNV replication in astrocytes. Therefore, viral processing pathways within astrocytes may be attractive targets for managing WNV-induced neuropathologies.