Combined Glycoprotein Mutations in Rabies Virus Promote Astrocyte Tropism and Protective CNS Immunity in Mice

Rabies virus (RABV) causes fatal encephalitis once it invades the central nervous system (CNS), and treatment options are extremely limited at this stage. We investigated the recombinant RABV variants SPBN, SPBNGA (glycoprotein substitution R333E), SPBNGAK (R333E plus N194K), SPBNGAS (R333E plus N194S), and TriGAS (three copies of the R333E/N194S glycoprotein). We evaluated their cellular tropism and immune activation in an intracerebral mouse infection model using immunohistochemistry and confocal immunofluorescence. SPBNGAK (R333E/N194K) resulted in mixed neuronal and astrocytic infection and lethal disease. In contrast, the R333E/N194S mutations in the GAS variants were associated with reduced neuronal infection and apparent astrocyte-restricted infection patterns. This tropism shift coincided with microglial activation (allograft inflammatory factor 1, amoeboid transformation) and astrocytic activation (nestin), along with T-cell infiltration and endothelial activation that persisted beyond viral clearance. SPBNGAK-infected astrocytes expressed nestin, while GAS variant-infected astrocytes remained nestin-negative and were rapidly cleared. Intracerebral co-inoculation of astrocytotropic TriGAS with the lethal neurotropic DOG4 strain was associated with survival and a marked reduction in detectable DOG4 neuronal infection. These findings suggest that glycoprotein-mediated astrocyte tropism may be associated with altered immune responses after rabies CNS invasion. While mechanistic causality cannot be inferred, these observations may inform the design of future studies exploring astrocyte-restricted RABV infection in therapeutic-related contexts.