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, 103 (11), 4198-203

Engineering RNA Interference-Based Resistance to Dengue Virus Type 2 in Genetically Modified Aedes Aegypti

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Engineering RNA Interference-Based Resistance to Dengue Virus Type 2 in Genetically Modified Aedes Aegypti

Alexander W E Franz et al. Proc Natl Acad Sci U S A.

Abstract

Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Southern blot analysis of total DNA extracted from whole body mosquitoes. (A) Schematic representation of the Mos-carb/Mnp+/i/Mnp−/svA construct with restriction sites of endonucleases used for DNA digestions. ma. left/ma. right, left/right arms of mariner MosI; AeCPA promoter, A. aegypti carboxypeptidase A promoter; Mnp+, Mnp−, 578-bp cDNAs of the DENV-2 prM protein encoding region in sense and antisense orientations, respectively; i, minor intron of A. aegypti sialokinin I (44); svA, polyadenylation signal of Simian virus 40 VP1 gene. Numbers below indicate the sizes of the DNA fragments in base pairs. Black solid bars show the positions of the probes “mariner left,” “Mnp+,” and “mariner right/3xP3.” (B) Southern blots after hybridization of EcoRI (E), KpnI (K), or PstI (P) digested total DNA of Carb77 (C77) and HWE with [α32P] dCTP-labeled random-primed DNA probes complementary to the mariner MosI right arm/3xP3 fragment (blot on the left), Mnp+ (blot in the center), or mariner left arm (blot on the right). Blots were hybridized overnight at 45°C. The marker indicates DNA sizes in base pairs.
Fig. 2.
Fig. 2.
Northern analysis of IR RNA expression in mosquito midguts and detection of IR RNA-derived siRNAs by ribonuclease protection assay. (A) Detection of the transcribed IR construct. Ten to 15 μg of total midgut RNA extracted at different times after a bloodmeal or sugarmeal (−) were loaded in each lane. Membranes were hybridized with probe “Mnp+” transcribed in vitro as an RNA probe with [α-32P] UTP. Ribosomal RNAs are shown (Lower) to indicate amounts of RNA loaded per lane. (B) Detection of siRNAs among total midgut RNA extracted from Carb77 and HWE 1 and 2 days after receiving a sugarmeal (−) or bloodmeal. A 30-nt sense or antisense RNA fragment containing 22 nt of sequence complementary to a DENV-2 (Jamaica 1409) siRNA was end-labeled with [γ-32P] ATP as a probe. Hybridizations were performed at 42°C.
Fig. 3.
Fig. 3.
Characterization of the Carb77 DENV-2 resistance phenotype. Detection of DENV-2 antigen in midguts (scale bars, 0.5 mm) (A and B) and in salivary glands (three salivary glands/image; scale bars, 0.5 mm) (C and D) of Carb77 and HWE by IFA by using mAb 3H5 recognizing an epitope of DENV-2 E. Images represent typical infection patterns. (E) DENV-2 plaque titrations of single whole body Carb77 and HWE mosquitoes 7, 10, and 14 days pbm (bars indicate mean values of titers). Plaque assays were performed in LLC-MK2 monkey kidney cells at 10-fold dilutions. (F and G) Detection of DENV-2 viral RNA in midguts of HWE (F) and Carb77 (G) 1–14 days pbm by Northern analysis by using “Mnp+” probe. Ribosomal RNAs are shown (Lower) to indicate amounts of RNA loaded per lane. (H) Transient silencing of A. aegypti ago2 in Carb77 and HWE followed by challenge with a DENV-2-containing bloodmeal. Four days before bloodfeeding, 1 μg dsRNA was injected into 15 3-day-old females. β-gal dsRNA was used as a control. Virus titers of single mosquitoes were assessed 7 days pbm by plaque titration in LLC-MK2 cells (bars indicate mean values of titers).
Fig. 4.
Fig. 4.
In vitro transmission assay of DENV-2 by Carb77 and HWE 14 days pbm. Three batches of 15 mosquitoes each (Carb77 or HWE) were allowed to feed for 1–2 h on a solution that was placed between two parafilm membranes stretched over a glass feeder. The solution and fed mosquitoes were collected after feeding and separately analyzed for infectious DENV-2 by plaque assays in LLC-MK2 cells (bars indicate mean values of titers). (A) Virus titers in feeding solutions after exposure to mosquitoes. (B) Virus titers of the three groups of mosquitoes after feeding on the solutions analyzed in A.

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