*2.8. RT-qPCR*

Total RNA including genomic viral RNA was extracted from cells (Qiagen, Hilden, Germany) and reverse transcription was performed using 500 ng of total RNA, random hexamer primers (intracellular viral RNA) or E reverse primer (virus particles) and moloney mouse leukemia virus reverse transcriptase (Life Technologies, Carlsbad, CA, USA) at 42 ◦C for 50 min. Quantitative PCR was performed on a CFX96 qPCR System (Bio-Rad, Hercules, CA, USA). Briefly, 10 ng of cDNA was amplified using 0.2 μM of each primer and 1X GoTaq Master Mix (Promega, Madison, WI, USA). When appropriate, data were normalised to the internal standard GAPDH. For each single-well amplification reaction, a threshold cycle (Ct) was calculated using the CFX96 qPCR program (Bio Rad, Hercules, CA, USA) in the exponential phase of amplification. Relative changes in gene expression were determined using the 2ΔΔCt method and reported relative to the control. Primers used in this study are listed in [31]. ZIKV E primers were designed to match both MR766-NIID and BeH819015 sequences (forward 5-gtcttggaacatggagg-3 and reverse 5-ttcaccttgtgttgggc-3).
