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

Abstract

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.

Figure 1

Molecular characterization of ΦC31-generated recombinants of Ae. aegypti over-expressing FHV-B2 from the constitutive PUb promoter. (A) Diagram of the recombination event between the attP site of docking strain attP26 and the attB site of the donor pattB-DsRed2/PUb/_v5B2/svA. (B) Confirmation of site-directed donor integration in PUbB2 P61 mosquitoes by conventional PCR. Annealing sites of PCR primers are shown in the diagram.

Figure 2

FHV-B2 over-expression in midguts of transgenic mosquitoes of line PUbB2 P61. (A) Western blot analysis of PUbB2 P61 females to detect FHV-B2 expression. Total protein extracted from five midguts or carcasses of sugarfed PubB2 P61 and HWE (negative control) mosquitoes was electrophoretically separated in 10% SDS-PAGE. B2 over-expression was detected with an IgG antibody specific for the v5 epitope tag. Total protein of SINV-TE3’2J-B2 infected cell culture medium was used as positive control. Lane 1: HWE midguts, 5 days post-eclosion; lane 2: PUbB2 P61 midguts, 5 days post-eclosion; lane 3: PUbB2 P61 midguts, 7 days post-eclosion; lane 4: SINV-TE3’2J-B2 culture supernatant, 36 h post-infection; lane 5: molecular size marker; lane 6: PUbB2 P61 carcasses, 1 day post-eclosion; lane 7: PUbB2 P61 carcasses, 5 days post-eclosion; lane 8: PUbB2 P61 carcasses, 7 days post-eclosion. (B) Detection of FHV-B2 protein in midguts by Immunofluorescence assay using anti-v5 IgG as detection antibody. (1) sugarfed HWE (negative control); (2) sugarfed PUbB2 P61 female; (3) PUbB2 P61 female at 5 days pbm; (4) DENV2-QR94 infected female at 5 days pbm; (5) close-up image (400×) of midgut epithelium over-expressing FHV-B2 in a sugarfed PUbB2 P61 female at 7 days post-eclosion; (6) close-up image (400×) of midgut epithelium of a sugarfed HWE female at 7 days post-eclosion.

Figure 3

Functional assay to confirm suppression of induced silencing of the endogenous AeCPA-1 gene in midguts of PUbB2 P61 females. HWE and PUbB2 P61 females were intrathoracically injected with 1 µl of 1 µg/µl dsRNA targeting AeCPA-1 or with PBS (control). Twenty-four hours post-injection mosquitoes were given a bloodmeal. Following total RNA extraction from pools of 20 midguts at 7–48 h pbm, AeCPA-1 transcript copy number equivalents were quantified by qRT-PCR. NI = non-injected.

Figure 4

Intensity of SINV-TR339eGFP and DENV2-QR94 infections in PUbB2 P61 and HWE mosquitoes. (A) virus titers and infection rates at 7 days pbm; (B) virus titers and infection rates at 14 days pbm. One-week post-eclosion, females were orally challenged with SINV-TR339eGFP (titer in the bloodmeal: 2.0×10⁷ pfu/ml) or DENV2-QR94 (titer in the bloodmeal: 1.0×10⁶ pfu/ml). Each data point represents the virus titer (pfu/ml) in an individual midgut or carcass as analyzed by plaque assays. P-values (α=0.05) for infection rates and mean virus titers are shown in the table.