A nanoluciferase-expressing attenuated CHIKV vaccine strain enables rapid and visual drug screening in vitro and in vivo

Climate change has expanded the range of vectors for the chikungunya virus (CHIKV), thereby increasing its global threat. This situation underscores the urgent need for antiviral therapies. However, the development of drugs is limited by the need for Biosafety Level 3 (BSL-3) containment and the lack of tools for real-time infection monitoring. To address these challenges, we developed a replication-competent reporter virus called CHIKV-ΔnsP3-Nluc. We inserted the Nano-luciferase (Nluc) gene into the attenuation site of the FDA-approved live-attenuated vaccine strain VLA1553 (IXCHIQ). This modified virus retains an attenuated phenotype, replicating more slowly than wild-type CHIKV while maintaining high genetic stability. Nluc activity correlates well with viral titer ( R ² = 0.9689), enabling precise quantification of viral replication. In interferon-deficient A6 mice, CHIKV-ΔnsP3-Nluc caused a lethal yet attenuated infection. Real-time bioluminescence imaging revealed that the spleen and kidneys were the primary replication sites, with luminescence intensity closely matching viral titers ( R ² = 0.8388). We confirmed the platform’s potential for rapid antiviral screening using the nucleoside analog 4′-fluorouridine (4′-FlU). This compound inhibited viral replication in vitro (EC ₅₀ = 0.063 µM) and significantly reduced viremia and mortality in vivo at a dose of 10 mg/kg, resulting in 100% survival. CHIKV-ΔnsP3-Nluc is a BSL-2-compliant tool that combines the safety of a vaccine candidate with a sensitive reporter function. It has the potential to accelerate studies of CHIKV pathogenesis and assist in developing new antiviral therapies.