Quantification of Zika Virus Infection

RNA was extracted from whole blood, plasma, and tissue using RNeasy Protect Animal Blood, QIAamp Viral RNA Mini, and RNeasy Mini kits (Qiagen). Tissue samples were homogenized using a FastPrep^®-24 homogenizer and Lysing Matrix D (MP Biomedicals). qPCR primers and probes ZIKV_1086, ZIKV_1162c, ZIKV_1107_P were synthesized by Integrated DNA Technologies [48 (link)]. Viral loads were quantified by qRT-PCR using the SuperScript^™ III Platinum^™ One-Step qRT-PCR Kit (Thermo Fisher) and a LightCycler 480 (Roche) instrument as previously described [10 (link)]. To determine the concentration of ZIKV RNA in a sample, a synthetic DNA fragment corresponding to the H/PF/2013 genome from positions 1086 to 1107 was amplified and subcloned into pCR4-TOPO (Thermo Fisher). RNA standards were synthesized using a MAXIscript^™ T7/T3 Transcription Kit (Thermo Fisher), quantified by absorbance and Quant-iT Ribogreen RNA Assay Kit (Life Technologies), and serially diluted to generate in-run standard curves for qRT-PCR analysis. To quantify ZIKV NS1-antigenemia, plasma from guinea pigs was subjected to a Zika Virus NS1 ELISA (BioFront Technologies).