*2.4. ZIKV Shedding*

Daily ocular, vaginal and oral swabs were collected from 1–21 dpi, using pre-moistened FLOQswabs. Viral shedding was assessed by standard plaque assay on Vero E6 cells using ocular and vaginal swab samples collected daily 1–21 dpi. Oral swabs were unable to be assessed by plaque assay due to excessive outgrowth of oral microflora.

#### *2.5. ZIKV RNA Isolation and qPCR of Clinical Samples*

Tissues were homogenized on ice with a Bel-Art hand held tissue homogenizer with sterile pestles (Cole-Parmer, Vernon Hills IL, USA). RNA was extracted using an RNEasy Kit (Qiagen, Germantown, MD, USA) following manufacturer's protocol. The iScript cDNA Synthesis Kit was used to reverse

transcribe RNA into complementary DNA (cDNA) (Bio-Rad, Hercules, CA, USA). Briefly, 20 μL reactions were used for cDNA synthesis (4 μL 5× iScript reaction mix, 1 μL iScript reverse transcriptase, 10 μL nuclease-free water, and 5 μL RNA template). Thermocycler conditions were: 5 min 25 ◦C; reverse transcription for 30 min 42 ◦C; 5 min 85 ◦C. qPCR was conducted on a Viia 7 real-time PCR system (Applied Biosystems, v1.2.4, Foster City, CA, USA) using iTaq Universal SYBR Green Supermix (BioRad) and the previously synthesized cDNA. Ten μL reactions were used (5 μL iTaq supermix, 0.5 μL forward (5 AAR TAC ACA TAC CAR AAC AAA GTG GT) /reverse (5 TCC RCT CCC YCT YTG GTC TTG) primer mix (10 μM each), 0.5 μL rRNA primer mix, 1 μL nuclease-free water, 3 μL cDNA). Thermocycler conditions were on fast setting, 20 s 95 ◦C; 40 cycles of 1 sec 95 ◦C, 20 sec 60 ◦C; followed by melt-curve analysis. Resulting Ct values were used to determine number of ZIKV RNA copies/500 ng total RNA based on standards of known quantity.
