SUMMARY The long-term objective of this multi-PI project is to determine how host-pathogen interactions impact entry, infection, and spread of encephalitic alphaviruses in the central nervous system (CNS). The vector-borne neurotropic viruses, Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, WEEV), invade the CNS after subcutaneous inoculation and initial interaction with immune sentinel cells, such as macrophages and dendritic cells (DCs) (VEEV), or fibroblastic, osteoblastic and other cell types (EEEV, WEEV). Both EEEV and VEEV enter the brain via the hematogenous route but only VEEV is found in olfactory neurons. The CNS lacks intraparenchymal lymphoid tissues and cannot initiate adaptive immune responses; thus, it mainly relies on innate responses communicated through local and systemic cytokine secretion, which modulate the status of resident neural cells and limits viral neuroinvasion and the extent of infection. CNSresident cells may include multiple members of the neurovascular unit (NVU) such as brain microvascular endothelial cells (BMECs), pericytes, astrocytes, microglia and neurons themselves that may be infected directly or acted upon by regionally or systemically produced cytokines. VEEV is a highly lymphotropic virus eliciting robust serum cytokine responses after peripheral inoculation, while EEEV tropism in lymphoid tissues is highly restricted, and serum cytokine responses are much lower, and in the case of type I IFN, often undetectable. In published studies, two primary virulence factors for EEEV in human and murine models defined mechanisms that suppresses replication in immune sentinel myeloid cells and greatly limit innate immune (especially interferon) responses to EEEV infection in vitro and in vivo1. How these factors, which include the presence of binding sites for the hematopoietic cell-specific microRNA, mir142-3p, in the EEEV 3' untranslated region (UTR)1-3, and efficient binding of EEEV to heparan sulfate (HS) receptors on cells4,5, impact the differential neuroinvasion and CNS dissemination of EEEV versus VEEV is unknown. Supporting the idea that differences in EEEV versus VEEV cytokine induction may be critical to neuroinvasion, we found that type I IFN-dependent responses directly regulate transcytosis, preventing alphavirus entry across the BBB and modulating the level of infection and injury in cells of the NVU6. Thus, systemic and local cytokine responses during alphavirus infection induce BMECs and pericytes to regulate viral entry at the blood-brain barrier (BBB) and potentially other CNS sites. This is consistent with the relative extent of virus replication at terminal stages of disease as EEEV exhibits widespread infection of neurons throughout the CNS while VEEV replication is much more focal (unpublished). Using mutant VEEV and EEEV, novel viral vectors that express indicators of infection (e.g., eGFP, nanoLuciferase) in vivo, we have observed regional heterogeneity in dominant...