HEK-293T cells were transfected with the DVG-encoding clones along with the WT Zika virus clone at different molar ratios (1:10, 1:1 and 10:1 DVG:WT) using LT-1 transfection reagent (Mirus Bio). As controls, additional WT virus clone or a GFP-encoding clone (control clone unrelated to Zika virus) were used. 72 h p.t. WT virus titers were determined by plaque assay.

Due to the low efficacy of the CMV promoter in invertebrate cells58, we tested DVG-induced inhibition in mosquito cells by transfection of DVG RNA. Briefly, PCR amplicons of the full DVG sequences were generated using primers with an SP6 promoter at the 5’ end. SP6 in vitro transcription was then performed using the PCR product as a template (mMessage mMachine SP6 Transcription kit, ThermoFisher). Following DNase treatment, the reaction was cleaned up (RNeasy MinElute Cleanup kit, QIAGEN) and RNA quantified with a Qubit fluorimeter (Invitrogen). 0.062, 0.125, or 0.25 pmoles DVG or pTRI Xef control RNA were transfected into sub-confluent C6/36 or U4.4 cells seeded in a 48-well plate (Lipofectamine LTX, ThermoFisher). The next day, cells were infected with Zika virus at an MOI 0.1 PFU/cell and the medium replaced. 5 days p.i., infectious virus in the cell culture medium was quantified by plaque assay. For assessing the effect of DVG RNA in Ago-2 knockdown cells, co-transfection of the DVG or control RNA was carried out with Ago-2 or control dsRNA.

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