Competitive Passage of Chikungunya Virus Variants

As described previously for chikungunya virus WT_IC virus was competed with NS5_Reenc_IC virus [27 (link)]: five initial TCID50 ratios (WT_IC/NS5_Reenc_IC virus: 1/99, 20/80, 50/50, 80/20, 99/1) were used to infect a 25cm² culture flask of confluent BHK21 cells at a calculated moi of 0.5. Cells were washed twice with HBSS and then incubated for 48h after addition of 7mL of medium. Recovered infectious cell supernatant was then sequentially passaged 10 times in the same manner with the clarified cell supernatant medium from the previous passage. At each passage, a calculated moi of 1 was used. Aliquots of cell supernatant from each passage were clarified by centrifugation and stored at -80°C. Viral RNA was extracted from clarified culture supernatant medium using the EZ1 Virus Mini Kit v2 on the EZ1 Biorobot (both from Qiagen). Using two specific quantitative real time RT-PCR assays targeting the re-encoded NS5 coding region (see the quantitative real time RT_PCR assays section for more details), the amount of viral RNA was assessed for each virus (WT_IC and NS5_Reenc_IC) and the ratio of the two values (WT_IC/NS5_Reenc_IC) was calculated.