Sensitive Zika Virus Quantification

Virus detection from leg, wing and body samples was carried out using 1/10 and 1/100 dilutions in 96-well plates containing a Vero cell monolayer. Saliva samples were titrated directly in 96-well plates in a Vero cell monolayer. Vero cells were maintained with DMEM supplemented with 2% FCS and 2% of penicillin/streptomycin/nystatin (1000 U/ml, 10 mg/ml and 500 U/ml, respectively; Sigma-Aldrich) and incubated for seven days at 37 °C and 5% CO₂ until cytopathic effect observation.
Prior to viral RNA extraction, the samples were homogenized using a TissueLyser II (Qiagen GmbH, Hilden, Germany) at 30 Hz for 1 min. Viral RNA was extracted from the samples using NucleoSpin^® RNA Virus (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. Zika RNA was detected by reverse-transcription quantitative PCR (RT-qPCR) using the primers ZIKA 1086 and ZIKA 1162c defined previously [32 (link)] and AgPath-ID™ One-Step RT-PCR reagents (Applied Biosystems, Foster City, CA, USA). The nucleic acids were detected with a Real-Time PCR 7500 Fast System (Applied Biosystems) with the following amplification protocol: 45 °C for 10 min; 95 °C for 10 min; then 45 cycles at 95 °C for 15 s and at 60 °C for 45 s. The RT-qPCR sensibility was 0.451 TCID₅₀/reaction for detection of MR766 and 0.035 TCID₅₀/reaction for Suriname ZIKV strains.