Longitudinal in vivo imaging reveals asynchronous, incomplete Nipah virus clearance with prolonged focal CNS involvement in IFNAR-/- mice.

Nipah virus (NiV) infection causes fatal acute respiratory and neurological disease. Survivors may develop long-term sequelae or experience relapsing encephalitis months to years after recovery. The basis of divergent disease trajectories and incomplete viral clearance remains poorly defined. Here, we establish a red-shifted bioluminescent resonance energy transfer reporter system to enable longitudinal tracking of NiV infection in vivo and apply it in mouse models of infection. We demonstrate route-, strain-, and immune-dependent differences in dissemination following NiV infection and reveal spatially heterogeneous and asynchronous viral clearance. While intranasal infection remains largely confined to the respiratory tract, intraperitoneal infection results in systemic spread followed by prolonged, fluctuating focal viral signal detectable up to 42 days post-infection in IFNAR-/- mice. Integration with RT-qPCR, histopathology, and in situ hybridization showed persistent viral RNA in discrete brain regions after apparent clinical recovery, indicating anatomically restricted persistence. Together, these findings reveal the spatiotemporal dynamics of incomplete NiV clearance in vivo and provide a framework to study relapsing disease.