Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.

: Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Reunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described.