Infectious cDNA Clone of Chikungunya Virus 181/25 for Antiviral Assay Development.

Chikungunya is a mosquito-borne viral disease in humans and remains a significant threat in many parts of the world. Although Chikungunya fever is self-limiting, it often progresses to a chronic phase marked by debilitating arthralgia and joint pain that can persist for months or even years. The Chikungunya virus (CHIKV) vaccine strain 181/25 is a prototypical strain of the virus that has been extensively used to develop second-generation vaccine candidates. In this study, we report the construction of an infectious cDNA clone of CHIKV-181/25 and a GFP-expressing variant designed to facilitate a high-throughput and robust antiviral assay for compound screening. A full-length cDNA copy of the viral genome was cloned downstream of the SP6 RNA polymerase promoter. An infectious virus was successfully recovered through electroporation of in vitro transcribed RNA, yielding high titers. Furthermore, a GFP-expressing CHIKV-181/25 variant was engineered by inserting GFP under a duplicated subgenomic promoter. This recombinant virus remained stable in both GFP expression and genomic integrity over serial passages. Using this reporter virus, we developed a reliable, high-throughput capable cell-based assay for antiviral compound screening, which was validated with known CHIKV inhibitors and demonstrated strong reproducibility over repeated runs. Overall, the infectious cDNA clone of CHIKV-181/25 provides a valuable platform for antiviral drug discovery, vaccine development, and studies of viral replication dynamics under evolutionary pressures.