Recent observations highlight a notable surge in West Nile Virus (WNV) infections in Europe that can lead to neuroinvasive consequences associated with neurodegeneration, mainly triggered by WNV Non-Structural protein 1 (NS1). During viral replication, various protein-protein interactions take place, allowing viral proteins to interact with host factors. NS1 is actively secreted in the bloodstream by infected cells and is known to affect endothelial permeability and host immune response. Focusing on the recently discovered antimicrobial roles of Amyloid-Beta (Aβ) in the context Central Nervous System (CNS), we connected WNV late pathology to overlapping features encountered in neurodegenerative diseases. In fact, CNS viral infections, or presence of specific viral components, activate glial cells, which in turn increase Aβ expression as an antiviral mechanism, leading to Aβ accumulation and neuronal damage. Considering West Nile neuroinvasive disease (WNND) as a possible complication of WNV infection, we investigated the impact of soluble WNV (s)NS1 on glial and neuronal cells, in 2D and 3D in vitro models. We reported an increased Aβ deposition after WNV sNS1 treatment, particularly of Aβ-142 isoform, and increased glial activation with a subsequent neurotoxicity. These findings underscore the crucial role of sNS1 in CNS-related effects during WNV infection, suggesting a novel pathogenetic role.