Absolute Quantification of ZIKV and CHIKV Genomic Copies

For the absolute quantification of the genomic copies, 76 pb of E and 125 pb of NSP4 of ZIKV/Col and CHIKV/Col were cloned, respectively [52 (link)]. The amplified PCR products were ligated into the pJET1.2/blunt vector and cloned using the CloneJET kit (Thermo Scientific, Waltham, MA, USA), following the previously described protocol [58 (link),59 (link)]. Total viral RNA extraction from the cell monolayers was conducted with the ZYMO^® Quick-RNA™ Viral kit, retrotranscription (from 500–1000 ng of RNA) with the High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific^®), and amplification with the Power Up™ SYBR Green Master Mix kit (Thermo Scientific^®), following the manufacturers’ instructions. The samples were amplified in a QuantStudio 3 thermocycler (Thermo Scientific^®); the thermal profile used was 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min.