Circular polymerase extension reaction (CPER)

For generation of DNA fragments that can be assembled into infectious DNA, viral RNA was isolated from culture fluids of Vero cells infected with Zika MR766 at 3 dpi and used as a template for first strand cDNA synthesis with SuperScript IV reverse transcriptase (Invitrogen, USA) according to the manufacturer’s recommendations. Each RT reaction contained 11 μl of viral RNA, and 2 pmol of the reverse PCR primer for the corresponding fragment. RNA was denatured for 5 min at 95 °C, and annealed to the primers at 65 °C followed by cDNA synthesis at 55 °C for 1 h. DNA was then removed from RNA–DNA duplexes by incubation of RT mixture with 1 μl of E. coli RNase H (NEB, USA) for 20 min at 37 °C. RNase H-treated cDNA was used as a template for PCR with PrimeStar GXL Polymerase (Takara, Japan) and primers listed in Supplementary Table ¹ according to the manufacturer’s recommendations. The cycling conditions were 3 min at 98 °C; 40 cycles of 10 s at 98 °C, 15 s at 55 °C, 4 min at 68 °C; and a final extension for 5 min at 68 °C. PCR products were then separated in 1% agarose gel and DNA was extracted from the gel using Monarch DNA Gel Extraction Kit (NEB, USA).

PCR products that corresponded to the 3ʹ-end of the viral genome (fragment 4) were cloned into the SmaI digestion site of the pUC19 vector. Primers for mutagenesis (Supplementary Table ¹) were designed using NEBaseChanger online tool (NEB, USA [https://nebasechanger.neb.com]) and mutagenesis was performed using the Q5 Site-directed Mutagenesis Kit (NEB, USA) according to the manufacturer’s instructions. The resulting plasmid was used as a template for PCR amplification of mutated fragments using the same conditions as for amplification of viral cDNA fragments. All enzymes and DH5a E. coli competent cells were from NEB, USA. Plasmid isolation was performed using the Wizard Plus SV DNA Miniprep System (Promega, USA).

Assembly of the infectious viral cDNA was conducted using the circular polymerase extension reaction (CPER) assembly^18,20. PCR fragments 1–3 were mixed with the UTR-linker fragment (containing CMV promoter for mammalian cells or OpIE promoter for insect cells) and either WT ZIKV_MR766 fragment 4 or one of the three mutated fragments 4 (Supplementary Fig. ^1A,B). CPER mixtures contained 0.1 pmol of each DNA fragment and 2 μl of PrimeStar GXL DNA polymerase (Takara, Japan) in a total reaction volume of 50 μl. Buffer, MgCl₂ and dNTP concentrations were as recommended by the manufacturer. The cycling conditions were 2 min at 98 °C; 20 cycles of 10 s at 98 °C, 15 s at 55 °C, 12 min at 68 °C; and a final extension for 12 min at 68 °C.

CPER products (50 μl) were transfected directly into Vero (under CMV promoter) or C6/36 (under OpIE promoter) cells using Lipofectamine LTX Plus transfection reagent (Life Technologies, Inc.). Briefly, each CPER product was mixed with 3 μl of PLUS reagent and 100 μl of Opti-MEM, followed by addition of 12.5 μl of Lipofectamine LTX dissolved in 150 μl of Opti-MEM. DNA-lipid complexes were incubated for 5 min at room temperature before being added onto the cells grown to 80% confluence in the wells of 6-well plates. At 24 h after transfection, cell culture medium was replaced with fresh medium containing 2% FBS. At 5, 7, and 10 days post-transfection (dpt), cell culture supernatant containing passage (P0) viruses were harvested and virus titres were determined by IPA. Viable P0 viruses were then used to infect C6/36 cells at an MOI of 0.1. At 5 and 7 days post-infection (dpi) culture fluids were collected from infected cells and titres of P1 virus were determined.