ZIKV-specific primers to generate 5 overlapping amplicons were designed based on an alignment of the reference sequence ZIKV strain MR766 and other complete genomes present in GenBank in February 2016 (Table S1 ).
ZIKV RNA was isolated from infected cell culture medium using the Qiamp Viral mini kit (Qiagen) and cDNA was generated by reverse transcription (5 separate reactions) with Superscript III polymerase (Thermo Fisher Scientific) and primers A1R, A2R, A3R, A4R and A5R (TableS1 ) according to the supplier’s instructions. Subsequently, 5 µl cDNA was used in 50 µl PCR reactions with Q5 high-fidelity DNA Polymerase (NEB) with GC enhancer to generate 5 amplicons with sizes of 2.4, 3.3, 3.1, 2.5 and 0.54 kb in 5′ to 3′ order, using primer combinations A1F & A1R, A2F & A2R, A3F & A3R, A4F & A4R and A5F & A5R. An initial step of 30 s at 98 °C was followed by 35 cycles consisting of a 10 s denaturation step at 98 °C, a 30-s annealing step at 64 °C and a 140 s extension at 72 °C for amplicons 1–4, while a 66 °C annealing temperature and 40 s extension time was used to generate amplicon 5. After cleanup with the ISOLATE II PCR Kit (Bioline), DNA was quantified using the Qubit Fluorometer and dsDNA BR Assay (Thermo Fisher Scientific). It was analyzed on agarose gels and the quality was checked with a Bioanalyzer DNA 12000 Kit (Agilent).
ZIKV RNA was isolated from infected cell culture medium using the Qiamp Viral mini kit (Qiagen) and cDNA was generated by reverse transcription (5 separate reactions) with Superscript III polymerase (Thermo Fisher Scientific) and primers A1R, A2R, A3R, A4R and A5R (Table