Viral copy number quantification by TaqMan qRT-PCR

cDNA was synthesized from isolated RNA using the iScript Select cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). cDNA was diluted to a final concentration of 2.5 ng/μL for TaqMan qRT-PCR on the QuantStudio 5 real-time PCR system (Thermo Fisher) using TaqMan universal PCR master mix (Fisher Scientific) and ZIKV NS3-specific primers and probe (Table 4; IDT, Coralville, IA). The ZIKV NS3-specific probe has a 5′ 6-carboxyfluorescein (FAM) reporter dye and a 3′ Iowa Black FQ quencher. ZIKV NS3-specific primers target a conserved 88 nucleotide region in NS3. The ZIKV NS3 amplicon was amplified from ZIKV cDNA and cloned into the multiple cloning site in the pEF mammalian expression plasmid containing an N-terminal FLAG tag using the In-Fusion cloning kit (Table 4; TaKaRa Bio, Kusatsu, Japan) following the manufacturer’s instructions. Each sample was run in triplicate, and dilutions of known concentrations of plasmid containing the NS3 amplicon were included to generate a standard curve with copy number values plotted against threshold cycle (CT) values to derive a semi-log equation in GraphPad Prism to determine the viral copy number of each sample. Dilutions of cDNA synthesized from RNA extracted from ZIKV stocks were used to derive a log-log equation to convert viral copy numbers to their FFU equivalent in GraphPad Prism.