Superscript 3 reverse transcriptase kit

(a) In vitro selection: 2 × 10⁵ PFU of CHIKV-37997 was incubated with 10 µg/ml of C9 and 1 × 10⁶ PFU of CHIKV-37997 was incubated with 5 µg/ml of IM-CKV063 for one hour at 37°C. Virus-NAb mixtures were added to Vero cells in 96-well-plate for 24 hours of infection. At each passage, half of the supernatant was mixed with 20 µg/ml of C9 or 10 µg/ml of IM-CKV063 for one hour at 37°C. The mixtures were added to fresh Vero cells to infect 2 hours. After three passages for C9 resistant virus selection and five passages for IM-CKV063 resistant virus selection, escape mutant viruses were selected. Viral RNA was extracted from the supernatant using a QIAamp Viral RNA Mini kit (Qiagen) and reverse transcribed into cDNA with random hexamer primer using the Superscript III Reverse Transcriptase kit (Invitrogen). CHIKV structural gene was amplified by PCR and sequenced. (b) In vivo selection: Ifnαr−/− mice were infected with 10 PFU of CHIKV 37997 strain subcutaneously and received 100 ug/mouse C9 or 50 ug/mouse IM-CKV063 at 24 hours post infection. Brain tissue was collected when mice were moribund at 2 or 3 days post infection and viral RNA was extracted from the tissue using RNeasy Mini kit (Qiagen). cDNA was synthesized using the Superscript III Reverse Transcriptase kit (Invitrogen) and CHIKV structure gene was amplified by PCR and sequenced.