Establishment and Characterization of Chikungunya Virus Replicon Cell Line

BHK-21 cells were purchased from The Global Bioresource Center (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 100U/mL of penicillin (Hyclone Laboratories), 100 mg/mL of streptomycin (Hyclone Laboratories), 1% dilution of stock of non-essential amino acids (Hyclone Laboratories) and 10% of fetal bovine serum (FBS, Hyclone Laboratories) in a humidified 5% CO2 incubator at 37 °C. BHK-21-Gluc-nSP-CHIKV-99659 cell line, harboring a replicative CHIKV replicon expressing Gaussia luciferase (Gluc) as a reporter gene, was maintained in DMEM 10% FBS with 500 µg/ml G418 (Sigma-Aldrich). The CHIKV replicon construct includes a T7 bacteriophage promotor followed by the viral 5' UTR region, the nsp1-4 coding sequence, the CHIKV subgenomic promoter (Sg) followed by the GLuc sequence and the expression cassette containing a ubiquitination sequence (Ubi) and the neomycin phosphotransferase gene (Neo-resistance gene), and the viral 3' UTR region. This construction and the development of this replicon cell line will be described elsewhere. The CHIKV expressing nanoluciferase reporter (CHIKV-nanoluc) used for the antiviral assays is based on the CHIKV isolate LR2006OPY1 (East/Central/South African genotype) and was produced, rescued, and titrated as previously described^{40 ,41 (link)}.