Transgene-mediated suppression of the RNA interference pathway in Aedes aegypti interferes with gene silencing and enhances Sindbis virus and dengue virus type 2 replication.

RNA interference (RNAi) is the major innate antiviral pathway in Aedes aegypti that responds to replicating arboviruses such as dengue virus (DENV) and Sindbis virus (SINV). On the one hand, the mosquito's RNAi machinery is capable of completely eliminating DENV2 from Ae. aegypti. On the other, transient silencing of key genes of the RNAi pathway increases replication of SINV and DENV2, allowing the viruses to temporally overcome dose-dependent midgut infection and midgut escape barriers (MEB) more efficiently. Here we expressed Flock house virus B2 (FHV-B2) from the poly-ubiquitin (PUb) promoter in Ae. aegypti using the ΦC31 site-directed recombination system to investigate the impact of transgene-mediated RNAi pathway suppression on infections with SINV-TR339eGFP and DENV2-QR94, the latter of which has been shown to be confronted with a strong MEB in Ae. aegypti. FHV-B2 was constitutively expressed in midguts of sugar- and blood-fed mosquitoes of transgenic line PUbB2 P61. B2 over-expression suppressed RNA silencing of carboxypeptidase A-1 (AeCPA-1) in midgut tissue of PUbB2 P61 mosquitoes. Following oral challenge with SINV-TR339eGFP or DENV2-QR94, mean titres in midguts of PUbB2 P61 females were significantly higher at 7 days post-bloodmeal (pbm) than in those of nontransgenic control mosquitoes. At 14 days pbm, infection rates of carcasses were significantly increased in PubB2 P61 mosquitoes infected with SINV-TR339eGFP. Following infection with DENV2-QR94, midgut infection rates were significantly increased in the B2-expressing mosquitoes at 14 days pbm. However, B2 expression in PUbB2 P61 did not increase the DENV2-QR94 dissemination rate, indicating that the infection phenotype was not primarily controlled by RNAi.

Infection, growth and maintenance of Wolbachia pipientis in clonal and non-clonal Aedes albopictus cell cultures.

Insect cell lines provide useful in vitro models for studying biological systems, including interactions between mosquitoes and obligate intracellular endosymbionts such as Wolbachia pipientis. The Aedes albopictus Aa23 cell line was the first cell line developed to allow examination of Wolbachia infections. However, Wolbachia studies using Aa23 can be complicated by the presence of different cell types in the cell line and the substantial temporal variation in infection level. Two approaches were examined to ameliorate infection variability. In the first approach, multiple Aa23 passaging regimes were tested for an effect on infection variability. Fluorescence in situ hybridization (FISH) staining was used to characterize Wolbachia infection level over time. The results demonstrate an impact of passaging method on Wolbachia infection level, with some methods resulting in loss of infection. None of the passaging methods succeeded in effectively mitigating infection level variation. In a second approach, the clonal C7-10 A. albopictus cell line was infected with Wolbachia from Aa23 cells and Drosophila simulans (Riverside), resulting in cell lines designated C7-10B and C7-10R, respectively. Characterization via FISH staining showed greater stability and uniformity of Wolbachia infection in C7-10R relative to the infection in C7-10B. Characterization of the Aa23, C7-10B and C7-10R lines is discussed as a tool for the study of Wolbachia-host cell interactions.

Comparison of transgene expression in Aedes aegypti generated by mariner Mos1 transposition and ΦC31 site-directed recombination.

Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.

Stability and loss of a virus resistance phenotype over time in transgenic mosquitoes harbouring an antiviral effector gene.

Transgenic Aedes aegypti were engineered to express a virus-derived, inverted repeat (IR) RNA in the mosquito midgut to trigger RNA interference (RNAi) and generate resistance to dengue virus type 2 (DENV2) in the vector. Here we characterize genotypic and phenotypic stabilities of one line, Carb77, between generations G(9) and G(17). The anti-DENV2 transgene was integrated at a single site within a noncoding region of the mosquito genome. The virus resistance phenotype was strong until G(13) and suppressed replication of different DENV2 genotypes. From G(14)-G(17) the resistance phenotype to DENV2 became weaker and eventually was lost. Although the sequence of the transgene was not mutated, expression of the IR effector RNA was not detected and the Carb77 G(17) mosquitoes lost their ability to silence the DENV2 genome.