Influence of N and NSs on translation.

<p>BHK-21 cells were infected with <i>Vaccinia virus</i> and co-transfected with 100 ng of expression vectors encoding REN luciferase, FF luciferase and MBP, N, NSs, combination of N and NSs, or pUC19 at the amount of 450 ng (A) and 700 ng (B). pUC19 was added as negative control. Luciferase expression was measured 23 h post transfection. The relative luciferase expression is shown, corrected for the internal FF control (REN/FF). (C) Cells were analysed for expression of MBP, N, or NSs by Western blotting and using antisera specific for MBP, N or NSs respectively. Abbreviation: MBP, Maltose binding protein; N, nucleoprotein; NSs, non-structural protein; H, hairpin; ½H, half hairpin; pA, polyA; (-), negative control.</p

Analysis of the A- and U-rich part of the predicted hairpin structure sequence in translation.

<p>(A) Localization of the A- and U-rich part within the predicted hairpin structure sequence. (B) BHK-21 cells were infected with vv-T7 and co-transfected with 100 ng of pREN sensor constructs (pREN-H^A/U-rich, pREN-halfH^A-rich, pREN-halfH^A*-rich, pREN-halfH^U-rich, or pREN-polyA) and 0.5 ng of pIRES-FF as internal control. Relative luciferase expression was measured after 23 h post transfection. Error bars show the standard deviations from the means of three replicate experiments.</p

Comparison of the predicted hairpin structure sequence from TSWV (N gene transcript) with the analogous one from TYRV.

<p>(A) Alignment of the TSWV and TYRV N-based hairpin structure sequence. (B) Predicted hairpin structure at the 3′ end of the N gene of TSWV and TYRV respectively. (C) BHK-21 cells were infected with <i>Vaccinia virus</i> and transfected with 100 ng of either pREN-H^A/U-rich, pREN-TYRV H, or pREN-polyA. In addition to the REN construct, 0.5 ng of pIRES-FF was added as internal control. After 23 h, the cells were lysed and assayed for relative luciferase activity. Error bars show the standard deviations from the means of three replicate experiments.</p

Structural features within the S RNA segment.

<p>Structural features within the S RNA segment.</p

Requirement of the 5′ UTR sequence in translation.

<p>BHK-21 cells were infected with <i>Vaccinia virus</i>, and subsequently co-transfected with 100 ng of the indicated REN constructs and 0.5 ng of pIRES-FF as internal control. The relative luciferase expression (REN/FF) was measured after 23 h post transfection. Error bars show the standard deviations from the means of three replicate experiments.</p

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

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

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

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