Infection and transmission rates of <i>Ae</i>. <i>aegypti</i> intrathoracically injected with ZIKV, CHIKV, or both viruses.

<p>Mosquitoes were infected through intrathoracic injections with a dose of 2.8 × 10³ TCID₅₀ units of ZIKV^SUR, CHIKV³⁷⁹⁹⁷, or both. (A) Infection and (B) transmission rates of <i>Ae</i>. <i>aegypti</i> mosquitoes at 7 dpi presented as percentage of the total number of injected mosquitoes. The percentage of co-infected mosquito bodies and saliva samples is indicated by the dashed line. Shown are the mean percentages from three independent replicates. Error bars show the standard error of the mean. Sample size ranged between 48–49 female mosquitoes per treatment. Results were evaluated with Chi-squared tests, and corrected for multiple comparisons with the Bonferroni correction. Significant differences between treatments (<i>P</i> < 0.05) are indicated by different letters.(C-D). Virus titers of CHIKV and ZIKV mosquito (C) bodies and (D) saliva. Each dot represents one mosquito body, and the horizontal bars indicate median titers. The detection limit of the EPDA is indicated by a dashed line. Results were evaluated with a Kruskal-Wallis test, and Dunn’s test for multiple comparisons, corrected with the Bonferroni correction. Significant differences between treatments (<i>P</i> < 0.05) are indicated by different letters.</p

Infection rates, transmission rates, and median titers of <i>Ae</i>. <i>aegypti</i> mosquitoes orally exposed to different doses of ZIKV and CHIKV.

<p>Infection and transmission rates determined at 14 dpi are presented as percentages (number of virus positive mosquito bodies or saliva samples / total number of engorged mosquitoes). Titers were determined for mosquitoes with a fully disseminated infection of ZIKV or CHIKV. The results represent the cumulative data from three independent biological replicates.</p

Infection rates, transmission rates and median titers of <i>Ae</i>. <i>aegypti</i> mosquitoes intrathoracically injected with ZIKV, CHIKV, or both ZIKV and CHIKV.

<p>Infection and transmission rates of mosquitoes were determined as the percentage of mosquitoes with either ZIKV, CHIKV, or both viruses in their body or saliva, respectively, out of the total number of injected mosquitoes within the respective treatment. Infection and transmission rates are presented as percentages (number of virus positive mosquito bodies or saliva samples / total number of engorged mosquitoes). Titers were determined for mosquitoes with a fully disseminated infection of ZIKV, CHIKV, or both. The results represent the cumulative data from three independent biological replicates.</p

Infection rates, transmission rates and median titers of <i>Ae</i>. <i>aegypti</i> mosquitoes orally exposed to ZIKV, CHIKV, or both ZIKV and CHIKV.

<p>Infection and transmission rates of mosquitoes in the co-infection treatment were determined as the percentage of mosquitoes with either ZIKV, CHIKV, or both viruses in their body or saliva, respectively, out of the total number of orally exposed mosquitoes within the respective treatment. Infection and transmission rates are presented as percentages (number of virus positive mosquito bodies or saliva samples / total number of engorged mosquitoes). Titers were determined for mosquitoes with a fully disseminated infection of ZIKV, CHIKV, or both. The results represent the cumulative data from three independent biological replicates.</p

Infection and transmission rates of <i>Ae</i>. <i>aegypti</i> orally infected with different doses of ZIKV or CHIKV.

<p>Mosquitoes were inoculated with an infectious blood meal containing a dose of 2.0 × 10⁵, 2.0 × 10⁶, or 2.0 × 10⁷ TCID₅₀/ml ZIKV^SUR or CHIKV³⁷⁹⁹⁷. (A) Ingested virus titers of <i>Ae</i>. <i>aegypti</i> immediately after blood feeding. Each dot represents one mosquito body, and horizontal bars indicate median titers. The detection limit of the EPDA is indicated by the dashed line. Results were evaluated with a Kruskal-Wallis test. Significant differences between viral doses (<i>P</i> < 0.05) are indicated by different letters. (B) Infection and (C) transmission rates of <i>Ae</i>. <i>aegypti</i> mosquitoes at 14 dpi presented as the percentage of the total number of engorged mosquitoes. Shown are the mean percentages from three independent replicates. Error bars show the standard error of the mean. Sample size ranged between 48–101 female mosquitoes per treatment. Results were evaluated with Chi-squared test. Significant differences between viral doses (<i>P</i> < 0.05) are indicated by different letters. (D-E) Virus titers of CHIKV and ZIKV mosquito (D) bodies and (E) saliva. Each dot represents one mosquito body or saliva sample, and the horizontal bars indicate median titers. The detection limit of the EPDA is indicated by a dashed line. Results were evaluated with a Kruskal-Wallis test. Significant differences between viral doses (<i>P</i> < 0.05) are indicated by different letters.</p

Immunofluorescence of ZIKV and CHIKV single- and co-infections in mammalian and mosquito cells.

<p>(A) Vero and (B) C6/36 cells were infected with ZIKV^SUR, CHIKV³⁷⁹⁹⁷ or both. At 48 hpi the monolayers were fixed, permeabilized, stained with antibodies for ZIKV-E (pan-Flavivirus α-E (4G2)), CHIKV-E2 and Hoechst33258 (details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005654#sec005" target="_blank">Materials and methods</a>) and visualized by immunofluorescence. Magnifications are indicated in each picture. Bottom panels indicate zoomed and merged images of co-infected cells.</p

Cell viability of Vero cells during ZIKV, CHIKV single- or co-infections.

<p>(A-B) Vero cells were infected with ZIKV^SUR, CHIKV³⁷⁹⁹⁷, or co-infected at an MOI of 0.1. At 0, 24, 48, 72 and 96 hpi cells were visualized by (A) bright field microscopy and (B) lysed before measuring the cell viability by CellTiter-Glo assay. Cell viability is presented as percentage normalized to the mock.</p

Latitudinal Diversity of <i>Culex pipiens</i> Biotypes and Hybrids in Farm, Peri-Urban, and Wetland Habitats in Europe - Fig 1

<p><b>Main effects of (A) trap type, (B) country, and (C) habitat on the ratio of <i>Culex pipiens</i> biotypes and their hybrids.</b> The total sample size (n) is indicated for each bar. Significance is displayed for each pairwise comparison, with ns = not significant, ** = p < 0.01, *** = p < 0.001. BGS = Biogents Sentinel trap, MMLP = Mosquito Magnet Liberty Plus, SW = Sweden, NL = The Netherlands, and IT = Italy.</p

Total number of collected <i>Cx</i>. <i>pipiens</i> females per trap type, habitat, and country.

<p>BGS = Biogents Sentinel trap, MMLP = Mosquito Magnet Liberty Plus trap, SW = Sweden, NL = The Netherlands, and IT = Italy.</p

Latitudinal Diversity of <i>Culex pipiens</i> Biotypes and Hybrids in Farm, Peri-Urban, and Wetland Habitats in Europe

<div><p>Despite the presence of <i>Culex (Cx</i>.<i>) pipiens</i> mosquitoes and circulation of West Nile virus (WNV), WNV outbreaks have so far not occurred in northern Europe. The species <i>Cx</i>. <i>pipiens</i> consists of two morphologically identical biotypes, <i>pipiens</i> and <i>molestus</i>, which can form hybrids. Until now, population dynamic studies of <i>Cx</i>. <i>pipiens</i> have not differentiated between biotypes and hybrids at the European scale, nor have they used comparative surveillance approaches. We therefore aimed to elucidate the relative abundance of <i>Cx</i>. <i>pipiens</i> biotypes and hybrids in three habitat types at different latitudes across Europe, using two different surveillance traps. BG-Sentinel and Mosquito-Magnet Liberty Plus traps were placed in three habitat types (farms, peri-urban, wetlands), in three European countries (Sweden, The Netherlands, Italy). Collected <i>Cx</i>. <i>pipiens</i> mosquitoes were identified to biotype with real-time PCR. Both trap types collected equal ratios of the biotypes and their hybrids. From northern to southern latitudes there was a significant decrease of <i>pipiens</i> and an increase of <i>molestus</i>. Habitat types influenced the relative ratios of biotypes and hybrids, but results were not consistent across latitudes. Our results emphasize the need to differentiate <i>Cx</i>. <i>pipiens</i> to the biotype level, especially for proper future WNV risk assessments for Europe.</p></div