Activatable Fluorescent Probes for In Vivo and Ex Vivo Dynamic Profiling of Virus-Infected Macrophages

Immune cells are critical for viral clearance, yet they can also be infected by viruses, transforming them into reservoirs that promote viral replication and dissemination. Macrophages, as the primary phagocytic cells, have been found to exhibit contradictory roles. However, due to the lack of analytical methods capable of real-time detection of virus-infected macrophages, the detailed mechanisms underlying macrophage polarization, migration, and dissemination following viral infection remain less understood. In this study, we develop two activatable near-infrared fluorescent probes, ZIP (a single-locked probe) for broadly detecting Zika virus (ZIKV)-infected cells and ZIMP (a dual-locked probe) for specifically identifying ZIKV-infected macrophages. ZIP activates its fluorescence in response to the ZIKV NS3 protease, enabling broad detection of ZIKV-infected cells. ZIMP, in contrast, requires both NS3 protease activity and the presence of caspase-1, which is overexpressed in macrophages, ensuring the selective detection of ZIKV-infected macrophages. Using both probes for in vivo imaging and ex vivo analysis reveals that ZIKV infection induces macrophage polarization toward the M1 phenotype in the lymph nodes of living mice, followed by M1 macrophage-mediated viral transportation to the brain at later time points. Furthermore, ZIMP can be employed to evaluate the efficacy of anti-ZIKV therapeutics treatment. This study highlights the potential of activatable optical probes for in vivo and ex vivo dynamic profiling of virus-infected immune cells, providing new insights into the complexities of viral immunity and antiviral therapeutics screening.