2.1 Chikungunya Virus Stocks, Titration, and Ultraviolet Light Inactivation of Chikungunya Virus

A clinical isolate of CHIKV was obtained following the protocol described in (Pastorino et al., 2005) from a CHIKF patient (kindly gifted by Professor Francisco Javier Díaz, University of Antioquia). CHIKV was amplified from a Colombian patient’s serum and propagated in Vero cells (ATTC CCL-81), as we previously reported (Valdés-López et al., 2020) (Valdés-López et al., 2021). Briefly, cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, United States) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Massachusetts, United States), 4 mM L-glutamine (Sigma-Aldrich), 0.3% (v/v) sodium carbonate (NaCO3; Sigma-Aldrich) and 1% (v/v) antibiotic-antimycotic solution (Corning-Cellgro, New York, United States), and incubated at 37°C and 5% CO₂ in cell culture flasks, to a cell density of 1 × 10⁵-1 × 10⁶ cells/ml. Vero cells were inoculated with CHIKV at 0.1 multiplicity of infection (MOI), incubated at 37°C and 5% CO₂ for 2 days, or until an advanced cytopathic effect was observed. Next, supernatants were collected, precleared by centrifugation (1,650 × g for 10 min), and stored at −80°C. CHIKV stocks were titrated by plaque assay on Vero cells, as previously described (Valdés-López et al., 2020). The virus titer was determined to be 2.1 × 10⁸ PFU/ml. CHIKV inactivation using ultraviolet (UV) radiation was performed by exposing 200 µL volumes of virus preparation at 10 cm of UV light (∼365 nm, 60 min, ∼145 mW/cm²) as was described in (Valdés-López et al., 2020). Inactivated CHIKV was stored to −80°C. The inactivation efficacy was confirmed by plaque assay in Vero cells and the plaques were counted and expressed as plaque-forming units per mL (PFU/ml).