Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells - PubMed (original) (raw)
Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells
Peng Lu et al. PLoS Negl Trop Dis. 2012.
Abstract
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.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. _w_AlbB induces a strong resistance to DENV-2 in mosquito cells.
_w_AlbB is a native infection in Ae. albopictus Aa23 cells (A) while Aa23T cells were initially generated by tetracycline treatment of Aa23 cell to remove Wolbachia infection. There is no Wolbachia in Ae. aegypti Aag2 cells (B). _w_-Aag2 was generated from Aag2 cells by introducing _w_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.
Figure 2. Generation of Aa23 cells with different Wolbachia density.
Cells were treated using sub-lethal doses of rifampicin for a different time periods. Three dosages (0.05 µg/ml, 0.5 µg/ml and 5 µg/ml) and four time periods (4 h, 10 h, 40 h and 70 h) were used. The genome copies of wsp were measure by q-PCR, normalized by host gene actin. Error bars are standard errors of the mean of at least three biological replicates. Statistical significance is represented by letters above each column, with different letters signifying distinct statistical groups. Student's t test: a vs. b, P<0.001; b vs. c, P<0.001; d vs. a, P<0.05.
Figure 3. Wolbachia induces density-dependent inhibition to DENV-2 in Aa23 cell lines.
Cell cultures derived from twelve different treatments in Fig. 2 were used in the assay. Five days after these cells were infected with DENV-2, viral genome copies were measured by qRT-PCR. Actin was used as a host gene to normalize the data. There is a negative linear correlation between Wolbachia density and DENV copy. Each point is the mean of at least three biological replicates.
Figure 4. Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to DENV.
(A). The density of _w_AlbA is significantly lower than _w_AlbB in midgut, fatbody, salivary gland and ovary of Ae. albopictus. The copy number of the Wolbachia wsp was normalized by Ae. albopictus actin; (B). Wolbachia density in somatic tissues is significantly lower in the Ae. albopictus HOU strain than in the transinfected Ae. aegypti WB1 strain. The copy number of the Wolbachia wsp was normalized by one conserved RPS6 in both Ae. albopictus and Ae. aegypti. In all the assays, midguts, salivary glands, fatbodies and ovaries of 7-day-old non blood fed females were dissected and used for extraction of total genomic DNA. ***, P<0.001; **, P<0.01 in Student's t-Test. Error bars are standard errors of the mean of ten biological replicates.
Figure 5. The expression of DEFD was induced by Wolbachia in density-dependent manner in Aa23 cell lines.
Seven days after rifampicin treatment, cells were collected to measure Wolbachia densities and DEFD expression. Because treatment with 5 µg/ml of rifampicin for 70 h resulted in both Wolbachia infection and DEFD expression at lowest level, it serves as a reference for all the other treatments to calculate a fold increase in both Wolbachia density and DEFD expression. Each point is the mean of at least three biological replicates.
Figure 6. Localization of Wolbachia and DENV-2 in Aa23 cells.
Double immunofluorescence staining of cells showing the localization of dengue virus (green) and Wolbachia (red). Cells were probed simultaneously with polyclonal anti-wsp antibody (Wolbachia) 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.
References
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