ABSTRACT The maintenance of homeostasis in the central nervous system (CNS) during neurotropic viral infections is critical for host survival. Inflammation of the CNS is characterized by the recruitment of leukocytes to the brain and the activation of resident CNS cells, including astrocytes. The pivotal in vivo function of astrocytes during viral encephalitis is poorly defined. Toll like receptor 3 (TLR3) is an interferon-inducing double stranded RNA sensor that senses viral infections. Although it is well known that astrocytes express functional TLR3, its role in response to viral RNA infections of the CNS remains poorly understood. The interplay between transcription factors and microRNAs, and their upstream pattern recognition receptors during viral encephalitis has not been extensively studied. MicroRNA-155 (miR-155) has a key role of in the fine-tuning of TLR responses, and exerts both positive and negative regulation during inflammation. We and others have identified miR-155 as one of the most over- expressed miRNAs after stimulation of TLR3 with the synthetic dsRNA agonist Poly (I:C) in macrophages. In the CNS, miR-155 is also expressed in glial cells. However, a CNS-intrinsic role of miR-155 has yet to be defined in vivo. Signal transducers and activators of transcription (STAT) proteins and their negative regulators, the suppressors of cytokine signaling (SOCS) protein family play central roles in regulating many cytokines and growth factors. Recently, two STAT3 binding sites have been identified in the promoter of miR-155. Furthermore, miR-155 targets SOCS1 and SOCS3 suppressing their negative regulatory effect on JAK/STAT signaling pathway. Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded, polyadenylated RNA viruses. Mouse hepatitis virus JHM (MHV-JHM) is a CNS-tropic strain that induces fatal encephalitis, associated with non-protective, enhanced macrophage and neutrophil infiltration that correlates with exacerbated chemokine and Th1 response and no Th17 involvement. We have identified a TLR3-dependent, miR-155 /STAT3 regulatory loop that promotes exacerbated, detrimental neuroinflammation and blood brain barrier (BBB) disruption in response to MHV-JHM fatal infection. Intriguingly, TRIF, the only TLR3 adaptor known, is not required for TLR3- dependent activation of STAT3 and induction of miR-155 both in vitro and in vivo. We have identified astrocytes and inflammatory monocytes infiltrating the brain as the cell sources driving exacerbated STAT3 activation. Our findings prompted us to investigate the following hypotheses: AIM 1 MHV-JHM infection in astrocytes and macrophages induces STAT3 activation through a TRIF-independent, TLR3-dependent non-canonical axis that is negatively regulated by its adaptor TRIF; AIM 2 Cell type specific genetic ablation of STAT3 and miR-155 expression in astrocytes or myeloid cells will reduce detrimental neuroinflammation and increase host survival after fatal MHV-JHM infection; an...