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. 2021 Apr 16;12:2290. doi: 10.1038/s41467-021-22341-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Longevity of DVG-A in mosquitoes. Ae. aegypti mosquitoes were in vivo transfected with equimolar solutions of DVG-A or DVG-A out-of-frame RNA. At the indicated times post transfection, 5 mosquitoes were salivated and midgut, carcass, and head dissected. RNA was extracted from homogenates of a pool of the 5 mosquitoes and RT-PCR specific for the DVG was performed. Results from a representative experiment out of two is shown. Lanes 1, 3, 5, and 7 = DVG-A out-of-frame, Lanes 2, 4, 6, and 8 = DVG-A. Results for head and saliva samples are shown in Supplementary Fig. 11a. b Schematic representation of the experimental design. Ae. aegypti mosquitoes were injected with a transfection mix 0.02 pmoles of RNA. 2 days post-transfection, mosquitoes were fed a bloodmeal containing 2_x10⁶ PFU/mL of Zika virus, and dissected at 8 or 13 d p.i. c Prevalence of Zika virus infection in the midgut or carcass of mosquitoes pre-transfected with control (n = 7), DVG-A (n = 12) or DVG-A out-of-frame (n = 12) RNA 8 d p.i. ***p = 0.0009 (by Fisher’s exact test, two-sided). d Viral load in infected mosquitoes from the midgut (control: n = 6; DVG-A: n = 10; DVG-A out-of-frame: n = 10) or carcass (control: n = 6; DVG-A: n = 1; DVG-A out-of-frame: n = 1) of the mosquitoes from (c). Mean and SD are shown. For midgut data, a one-way ANOVA with Dunnet’s multiple comparison was performed. For carcass, no statistical test was performed because due to n = 1. e Prevalence of Zika virus infection in the midgut, carcass or heads of mosquitoes pre-transfected with control (n = 36), DVG-A (n = 35) or DVG-A out-of-frame (n = 33) RNA 13 d p.i. ****p ≤ 0.0001 (by Fisher’s exact test, two-sided). f Viral load in the organs of the same infected mosquitoes from (e): midgut (control: n = 21; DVG-A: n = 26; DVG-A out-of-frame: n = 25), carcass (control: n = 21; DVG-A: n = 13; DVG-A out-of-frame: n = 13), or heads (control: n = 21; DVG-A: n = 8; DVG-A out-of-frame: n = 8). Mean and SD are shown. ****p ≤ 0.0001 (by one-way ANOVA with Dunnet’s multiple comparison). g Percentage Zika virus-positive saliva in mosquitoes pre-transfected with control (n = 9), DVG-A (n = 16) or DVG-A out-of-frame (n = 13) RNA 13 d p.i. h Prevalence of Zika virus infection in the midgut or carcass of sucrose- or AZT-fed mosquitoes that were injected with control or DVG-A RNA. Prevalence was assessed at 7 d p.i. Control RNA, sucrose fed (n = 18); DVG-A RNA, sucrose fed (n = 14); control RNA, AZT fed (n = 18); DVG-A RNA, AZT fed (n = 22). **p = 0.0053 (by Fisher’s exact test, two-sided). i Viral load in the midgut (control, sucrose fed: n = 12; DVG-A, sucrose fed: n = 10; control, AZT fed: n = 14; DVG-A, sucrose fed: n = 19) or carcass (control: n = 7; DVG-A: n = 10<LOD; control, AZT fed: n = 8; DVG-A, sucrose fed: n = 4) of the same mosquitoes from (h). Mean and SD are shown. ****p ≤ 0.0001 (by one-way ANOVA with Dunnet’s multiple comparison). Data from two independent experiments is shown. LOD = limit of detection. Control RNA: pTRI-Xef RNA. Source data are provided as a Source Data file.