Zika Virus Infection at Different Pregnancy Stages: Anatomopathological Findings, Target Cells and Viral Persistence in Placental Tissues

Lucia de Noronha; Camila Zanluca; Marion Burger; Andreia Akemi Suzukawa; Marina Azevedo; Patricia Z. Rebutini; Iolanda Maria Novadzki; Laurina Setsuko Tanabe; Mayra Marinho Presibella; Claudia Nunes Duarte dos Santos

Viral RNA was isolated from 140 μ L of biological fluids (serum, cerebrospinal fluid, lacrimal fluid, saliva or urine) using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. RNA extraction from unfixed placenta or viscera was performed with an RNeasy Mini Kit (Qiagen), while RNA from formalin-fixed paraffin-embedded (FFPE) tissues was extracted using the ReliaPrep TM FFPE Total RNA Miniprep System (Promega, Madison, USA). RNA was stored at -80°C until use. The primers and probes were synthesized and purified by Integrated DNA Technologies (IDT, Coralville, USA). The reporter dye 5-FAM was used for the probes. All real-time assays were performed with the GoTaq Probe 1-Step RT-qPCR System (Promega) with amplification by a LightCycler 96 instrument (Roche, Mannheim, Germany). The ZIKV genome was detected according to the protocol of Lanciotti et al. (Lanciotti et al., 2008) , which used 5 µL of RNA per 20 µL reaction, while the dengue virus (DENV) serotypes were detected according to the protocol of Johnson et al. (Johnson et al., 2005) . Human RNAse P was used as an endogenous control (Emery et al., 2004) . Positive controls for ZIKV and DENV were obtained by virus isolation from Brazilian patients sera in C6/36 or Vero E6 cells.