Quantitative PCR for Viral RNA Detection

Total RNA was extracted from tissues or cultured cells by using TRIreagent (Molecular Research Center, Inc.) and converted to cDNA using iSCRIPT cDNA synthesis kit (Bio-Rad). QPCR assays were performed using iTAQ polymerase supermix for probe-based assays (Bio-Rad) or iQ SYBR Green Supermix (Bio-Rad). Viral RNA of ZIKV envelope (E) (Acharya et al., 2016 (link)) was measured by qPCR and infectious viruses were measured by plaque assay as we previously described (Acharya et al., 2015 (link)). Threshold cycle values that were ≥39 cycles were excluded from the qPCR results, and 1 PFU per volume of sample was set as the limit of viral detection for the plaque assays. WNV-envelope (E) gene primers and probes sequences were adapted according to a previous publication (Town et al., 2009 (link)). All additional gene primer sequences are described in Supplementary Table S1.