Response of the mosquito protein interaction network to dengue infection

<p>Abstract</p> <p>Background</p> <p>Two fifths of the world's population is at risk from dengue. The absence of effective drugs and vaccines leaves vector control as the primary intervention tool. Understanding dengue virus (DENV) host interactions is essential for the development of novel control strategies. The availability of genome sequences for both human and mosquito host greatly facilitates genome-wide studies of DENV-host interactions.</p> <p>Results</p> <p>We developed the first draft of the mosquito protein interaction network using a computational approach. The weighted network includes 4,214 <it>Aedes aegypti </it>proteins with 10,209 interactions, among which 3,500 proteins are connected into an interconnected scale-free network. We demonstrated the application of this network for the further annotation of mosquito proteins and dissection of pathway crosstalk. Using three datasets based on physical interaction assays, genome-wide RNA interference (RNAi) screens and microarray assays, we identified 714 putative DENV-associated mosquito proteins. An integrated analysis of these proteins in the network highlighted four regions consisting of highly interconnected proteins with closely related functions in each of replication/transcription/translation (RTT), immunity, transport and metabolism. Putative DENV-associated proteins were further selected for validation by RNAi-mediated gene silencing, and dengue viral titer in mosquito midguts was significantly reduced for five out of ten (50.0%) randomly selected genes.</p> <p>Conclusions</p> <p>Our results indicate the presence of common host requirements for DENV in mosquitoes and humans. We discuss the significance of our findings for pharmacological intervention and genetic modification of mosquitoes for blocking dengue transmission.</p

The Endosymbiotic Bacterium Wolbachia Induces Resistance to Dengue Virus in Aedes aegypti

Genetic strategies that reduce or block pathogen transmission by mosquitoes have been proposed as a means of augmenting current control measures to reduce the growing burden of vector-borne diseases. The endosymbiotic bacterium Wolbachia has long been promoted as a potential vehicle for introducing disease-resistance genes into mosquitoes, thereby making them refractory to the human pathogens they transmit. Given the large overlap in tissue distribution and intracellular localization between Wolbachia and dengue virus in mosquitoes, we conducted experiments to characterize their interactions. Our results show that Wolbachia inhibits viral replication and dissemination in the main dengue vector, Aedes aegypti. Moreover, the virus transmission potential of Wolbachia-infected Ae. aegypti was significantly diminished when compared to wild-type mosquitoes that did not harbor Wolbachia. At 14 days post-infection, Wolbachia completely blocked dengue transmission in at least 37.5% of Ae. aegypti mosquitoes. We also observed that this Wolbachia-mediated viral interference was associated with an elevated basal immunity and increased longevity in the mosquitoes. These results underscore the potential usefulness of Wolbachia-based control strategies for population replacement

Replacing a native Wolbachia with a novel strain results in an increase in endosymbiont load and resistance to dengue virus in a mosquito vector.

Wolbachia is a maternally transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce pathogen interference and spread into mosquito vector populations makes it possible to develop Wolbachia as a biological control agent for vector-borne disease control. Although Wolbachia induces resistance to dengue virus (DENV), filarial worms, and Plasmodium in mosquitoes, species like Aedes polynesiensis and Aedes albopictus, which carry native Wolbachia infections, are able to transmit dengue and filariasis. In a previous study, the native wPolA in Ae. polynesiensis was replaced with wAlbB from Ae. albopictus, and resulted in the generation of the transinfected "MTB" strain with low susceptibility for filarial worms. In this study, we compare the dynamics of DENV serotype 2 (DENV-2) within the wild type "APM" strain and the MTB strain of Ae. polynesiensis by measuring viral infection in the mosquito whole body, midgut, head, and saliva at different time points post infection. The results show that wAlbB can induce a strong resistance to DENV-2 in the MTB mosquito. Evidence also supports that this resistance is related to a dramatic increase in Wolbachia density in the MTB's somatic tissues, including the midgut and salivary gland. Our results suggests that replacement of a native Wolbachia with a novel infection could serve as a strategy for developing a Wolbachia-based approach to target naturally infected insects for vector-borne disease control

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells.

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections

<i>w</i>AlbB confers resistance to DENV-2 in both midguts and heads of MTB mosquitoes.

*<p>At 7 dpi through an infectious blood meal, the midguts of MTB and APM mosquitoes were collected and fixed, and the viral antigen was detected using a mouse anti-Dengue complex monoclonal antibody by indirect fluorescence assay (IFA).</p>#<p>At 14 dpi, the heads of both mosquitoes were collected to extract the total RNA. DENV-2 was diagnosed by RT-PCR using primers for the NS5 gene. Amplification of <i>Ae. polynesiensis</i> RPS6 was used to verify the quality of the RNA samples. Significance was shown in Fisher's exact test.</p

Localization of <i>Wolbachia</i> and DENV-2 in Aa23 cells.

<p>Double immunofluorescence staining of cells showing the localization of dengue virus (green) and <i>Wolbachia</i> (red). Cells were probed simultaneously with polyclonal anti-wsp antibody (<i>Wolbachia</i>) and monoclonal anti-DENV-2 antibody, followed by Alexa 594 (red) and Alexa 488 (green) conjugated antibodies, respectively. DNA (blue) is stained with DAPI. In panels (A, B, and C), the red, green and blue channels are merged. A and B show Aa23 cells with mock treatment and treatment with 5 µg/ml of rifampicin for 5 hr, respectively, followed by dengue infection. C is Aa23T cells without dengue infection (negative control). D to F or G to I is the same sample with different channel merged: D and G show only red and blue channel merged, E and H show only green and blue channel merged, F and I show all the red, green and blue merged. Aa23 cells treated with 5 µg/ml of rifampicin for 10 hr (D to F) and 40 hr (G to I), followed by dengue infection, are shown.</p

The native <i>Wolbachia</i> does not inhibit dissemination of DENV-2 to mosquito heads in <i>Ae. albopictus</i>.

<p>The HOU strain of <i>Ae. albopictus</i> and its aposymbiotic strain HT1 were infected with the blood containing DENV-2 at titers of 10⁶, 10⁵, and 10⁴ PFU/ml. At Day 14 post infection, heads of ten mosquitoes were collected and used for diagnosis of DENV-2 by RT-PCR. Data from two experiments were pooled together.</p

<i>w</i>AlbB induces a strong resistance to DENV-2 in mosquito cells.

<p><i>w</i>AlbB is a native infection in <i>Ae. albopictus</i> Aa23 cells (<b>A</b>) while Aa23T cells were initially generated by tetracycline treatment of Aa23 cell to remove <i>Wolbachia</i> infection. There is no <i>Wolbachia</i> in <i>Ae. aegypti</i> Aag2 cells (<b>B</b>). <i>w</i>-Aag2 was generated from Aag2 cells by introducing <i>w</i>AlbB from Aa23 using a shell-vial technique. Five days after inoculated with DENV-2, the cells were tested for dengue infection by plaque assay. Error bars are standard errors of the mean of at least three biological replicates. **, P<0.01; ***, P<0.001in Student's t-Test.</p