Tragacanth

For the first round of inoculations, whole mosquito samples were individually titrated by Vero cell plaque assay using 24-well plates in which 150 μL of each 10-fold dilution from undiluted through 10⁻⁵ was added to each well. Samples were incubated for 1h at 37°C in 5% CO₂ then covered with 1 mL of semi-solid medium (4% EF-DMEM, 2X Pen-Strep and 4 μg/mL Amphotericin B mixed with an equal volume of 1.2% Tragacanth gum) (Tragacanth; MP Biomedicals, Santa Ana, USA) per well. Plates were incubated at 37°C in 5% CO₂ for 4–5 days. Semi-solid medium was removed and 0.1% crystal violet in 20% ethanol was added to each well in a volume sufficient to cover the monolayers. After 30 min, cells were washed in tap water. Plates were allowed to dry for 24h and plaques were counted to calculate PFU/mL.
In the second round of inoculations, in order to confirm the infectivity of RNA-positive samples and validate qRT-PCR results, a subset of saliva samples that were positive by qRT-PCR were plaque-titrated on Vero cells. Approximately 38% (N=91) of qRT-PCR-positive saliva samples were selected from the three replicates for each virus for plaque assay on Vero cells at dilutions of 1:5, 1:50 and 1:500.
Infectious blood-meals were titrated on Vero cells as described for whole mosquito samples, except that sample dilutions ranged from 10⁻³ to 10⁻⁷ (in 10-fold serial dilutions).

In addition to RT-qPCR, salivary secretions from ZIKV-infected Ae. aegypti were assayed by plaque assay to confirm the presence of infectious virus particles. Briefly, saliva from individual mosquitoes were diluted in cell culture medium and plated on sub-confluent monolayers of Vero cells. Cells were incubated for 1 hr. at 37°C after which a semi-solid medium overlay (Dulbecco’s Modified Eagle Medium with 4% EquaFETAL, 2X Pen-Strep and 4 μg/mL Amphotericin B, mixed with an equal volume of 1.2% Tragacanth gum) (EquaFETAL, Atlas Biologicals, Fort Collins, USA) (Tragacanth, MP Biomedicals, Santa Ana, USA) was added. Cells were incubated for 4–5 days at 37^oC in 5% CO₂, stained with crystal violet, and plaques enumerated.