*2.8. RNA Extraction and Quantitative Real-Time PCR*

Trizol reagent (Invitrogen) was used to extract intracellular and extracellular RNA and transcribed into cDNA using a cDNA synthesis kit (Toyobo) according to the manufacturer's instructions. Quantitative real-time PCR was performed using Applied Biosystems 7500 real-time PCR system and TaqMan real-time PCR mix (NovoStart, China). The ZIKV MR-766 E gene or JEV-P3 E gene were used to generate the standard curve for the quantification of viral copy numbers. Each sample was analyzed in triplicate. ZIKV or JEV copy numbers were extrapolated from the generated standard curve using the Applied Biosystems protocol. Primers were as follows: ZIKV, 5'-CCGCTGCCCAACACAAG-3' (forward) and 5'-CCACTAACGTTCTTTTGCAGACAT-3' (reverse); ZIKV probe, 5'-AGCCTAACCTTGACAAGCAATCAGACACTCAA-3'; JEV, 5'- TGGTTTCATGACCTCGCTCTC-3' (forward) and 5'- CCATGAGGAGTTCTCTGTTTCT-3' (reverse); and JEV probe, 5'-CCTGGACGCCCCCTTCGAGCACAGCGT-3'.
