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 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

<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

Resistance of MTB mosquitoes to DENV-2 is associated with a high density of <i>Wolbachia</i> in mosquito somatic tissues.

<p>The fold change in genome copy of the <i>Wolbachia</i> surface protein (WSP) gene in MTB mosquitoes is compared to APM mosquitoes. The copy number of the <i>Wolbachia</i> wsp was normalized by <i>Ae. polynesiensis</i> RPS6. In all the assays, the midguts, salivary glands, fat bodies, and ovaries of 7-day-old non-blood-fed females were dissected and used for extraction of total genomic DNA. Error bars are standard errors of the mean of twelve biological replicates.</p

Generation of Aa23 cells with different <i>Wolbachia</i> density.

<p>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.</p

<i>Wolbachia</i> density in somatic tissues of <i>Ae. albopictus</i> is too low to induce resistance to DENV.

<p>(A). The density of <i>w</i>AlbA is significantly lower than <i>w</i>AlbB in midgut, fatbody, salivary gland and ovary of <i>Ae. albopictus</i>. The copy number of the <i>Wolbachia</i> wsp was normalized by <i>Ae. albopictus</i> actin; (B). <i>Wolbachia</i> density in somatic tissues is significantly lower in the <i>Ae. albopictus</i> HOU strain than in the transinfected <i>Ae. aegypti</i> WB1 strain. The copy number of the <i>Wolbachia</i> wsp was normalized by one conserved RPS6 in both <i>Ae. albopictus</i> and <i>Ae. aegypti</i>. 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.</p

Inhibition of dengue infection in the midgut of MTB mosquitoes.

<p>(<b>A</b>) At 4, 7 and 10 dpi with a blood meal containing DENV-2, mosquito midguts were collected, and the number of genome copies of the DENV-2 genome was determined by qRT-PCR using primers for the NS5 gene; the results were normalized to the <i>Ae. polynesiensis</i> RPS6. Lines indicate the median of the ten biological replicates. Significance was determined using a Mann-Whitney U test. (<b>B</b>) At 7 dpi through a blood meal, mosquito midguts were collected and fixed, and the viral antigen was detected using a mouse anti-Dengue complex monoclonal antibody by indirect fluorescence assay (IFA). One representative midgut from each category is shown. APM mosquitoes fed with dengue-infected or -uninfected blood meal was used as positive (POS.) and negative (NEG.) control, respectively. +, dengue-positive; −, dengue-negative.</p

<i>Wolbachia</i> induces density-dependent inhibition to DENV-2 in Aa23 cell lines.

<p>Cell cultures derived from twelve different treatments in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001754#pntd-0001754-g002" target="_blank">Fig. 2</a> 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 <i>Wolbachia</i> density and DENV copy. Each point is the mean of at least three biological replicates.</p

Inhibition of dengue infection in the whole bodies of MTB mosquitoes.

<p>At 14 dpi (days post infection) through a blood meal or intrathoracic injection, the whole bodies of MTB and APM mosquitoes were collected, and the number of genome copies of the DENV genome was determined by qRT-PCR using primers for the NS5 gene; the results were normalized to the <i>Ae. polynesiensis</i> ribosomal protein S6 (RPS6). Lines indicate the median of the ten biological replicates. Significance was determined using a Mann-Whitney U test.</p