Viral infection, dissemination, transmission (A-C) and saliva viral loads (D) at 3 and 7 days after <i>Haemagogus leucocelaenus</i> (LEU) and <i>Aedes terrens</i> (TER) from Rio de Janeiro, Brazil, challenge with two CHIKV isolates of the two genotypes currently circulating in the Americas, CHIKV-115 (ESCA genotype) and CHIKV_SM (Asian genotype) when provided at a titer of 10^7.5 PFU/mL and 10^6.5 PFU/mL, respectively.

<p>Infection rate (IR) refers to the proportion of mosquitoes with infected bodies among tested females. Disseminated infection rate (DIR) corresponds to the proportion of mosquitoes with infected heads among the previously detected infected mosquitoes (i.e, abdomen/thorax positive). Transmission rate (TR) represents the proportion of mosquitoes with infectious saliva among mosquitoes with disseminated infection. Transmission efficiency (TE) represents the proportion of mosquitoes with infectious saliva among the initial number of females tested [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005698#pntd.0005698.ref029" target="_blank">29</a>]. The number of mosquitoes analyzed for each vector competence rate is given on top of bars. The horizontal black bars (in panel D) represent the mean viral loads.</p

Dicer 2 activity in <i>Ae. albopictus</i> C6/36, C7-10 and U4.4 cells.

<p>Cells were transfected with BUNV-specific long dsRNA, <i>Renilla</i>-specific long dsRNA or left untransfected (untreated) as indicated. At 24 h post-transfection, cells were infected with wtBUNV or rBUNdelNSs2 at an MOI of 5 PFU/cell. Supernatants were collected at the indicated times post infection for determination of the presence of infectious virus by plaque assay.</p

Expression of NSs protein in wtBUNV infected mosquito cells.

<p><i>Ae. albopictus</i> C6/36 cells were infected at an MOI of 10 PFU/cell and samples were harvested at the indicated time points. Cell lysates were electrophoresed on a 4–12% NuPAGE gel (Invitrogen). After transfer the membrane was cut into three and strips were incubated with anti-BUNV NSs protein (NSs), anti-BUNV N protein (N) or anti-tubulin (T) antibodies as indicated.</p

Virus replication in mosquito midguts and salivary glands.

<p>Following infection via blood-meal, 9 engorged mosquitoes were collected at days 1 to 13, and 6 engorged mosquitoes were collected at days 15, 18 and 21. Midguts and salivary glands were dissected, pooled into groups of 3 organs, and infectious virus determined by plaque assay. <b>A.</b> Midgut infection rates. The percentage of virus positive midguts over total number of tested mosquitoes was calculated. <b>B.</b> Average virus titre per mosquito midgut. Error bars show the standard error between the different pools. <b>C.</b> Disseminated infection rates. The percentage of virus positive salivary glands over total number of positive mosquito midguts was calculated. <b>D.</b> Average virus titres per mosquito salivary gland. Error bars show the standard error between the different pools.</p

Establishment of persistent infection in <i>Ae. albopictus</i> cell lines.

<p><b>A.</b> Titres of virus released into the supernatant from each passage of cells. Cells were initially infected at MOI of 0.1 PFU/cell, and passaged regularly at a 1∶5 split ratio. The supernatant medium was collected at the time of each passage and the presence of virus measured by plaque assay. <b>B.</b> Detection of viral N protein. An aliquot of cells was collected at each passage, lysed and 10 µg of total protein was analyzed by Western blotting with anti-BUNV N protein antibodies. Numbers at the top of the blots indicate the passage number.</p

Immunofluorescence analysis of morphological changes in <i>Ae. albopictus</i> cell lines.

<p>Mosquito cells were infected with rBUN-GcGFP or rBUNdelNSs-GcGFP at an MOI of 3 PFU/cell. The results present cells stained with anti-BUNN/anti-rabbit TexasRed (red signal), and with anti-tubulin/anti-mouse CY5 (light blue signal). The green signal shows autofluorescence of GFP-tagged Gc and the blue signal is DAPI staining of nuclei. <b>A.</b> C6/36 cell infected with rBUN-GcGFP virus during the acute stage of infection. <b>B.</b> C6/36, C7-10 and U4.4 cells were infected with fluorescent viruses. Three morphological stages of infection are presented: early, acute, and late. Only the merged images are shown.</p

BUNV replication in <i>Ae aegypti</i> Ae cells.

<p><b>A.</b> Growth curves. Ae cells were infected with wtBUNV or rBUNdelNSs2 at an MOI of 1 PFU/cell. At the indicated times post infection supernatants were collected and assayed for the presence of infectious virus by plaque titration. <b>B.</b> Viral N protein expression. Cell extracts were prepared at the indicated times (h P.I.), separated by 12% SDS-PAGE, and proteins transferred to a membrane. The membrane was incubated with anti-BUNV N protein and tubulin (T) antibodies. <b>C.</b> Establishment of persistent infection. The titres of infectious virus in supernatants collected at each passage of infected cells were determined by plaque assay.</p

Infection of female <i>Ae. aegypti</i> mosquitoes with wtBUNV or rBUNdelNSs2.

<p>Following infection via blood-meal, 8 to 10 engorged mosquitoes were collected at the indicated days after feeding, individually homogenized and the presence of infectious virus determined by plaque assay. Mosquito infection rates were calculated as the percentage of virus positive females over total number of engorged mosquitoes tested.</p

Growth of wtBUNV and rBUNdelNSs2 in <i>Aedes albopictus</i> cell lines.

<p><b>A.</b> Growth curves. C6/36, C7-10 and U4.4 cells were infected at an MOI of 1 PFU/cell. At the indicated time points, supernatants were collected and viral titres were determined by plaque assay. <b>B.</b> Viral N protein expression. Cells were infected at an MOI of 1 and at the indicated time points (hpi), cells were lysed, and proteins separated by 12% SDS-PAGE. The proteins were transferred to a membrane and incubated with anti-BUNV N protein (N) or anti-tubulin (T) antibodies.</p

Mean numbers ± standard deviations of infectious viral particles in saliva of <i>Aedes albopictus</i> Providence co-infected with CHIKV and DENV-1.

<p>The saliva of females detected positive for both viruses by IFA on head squashes, 14 days after an infectious blood-meal were titrated. Titers were expressed in FFU (foci forming units). For CHIKV, both trials used a blood-meal at a titer of 10⁶ FFU/mL. For DENV-1, trial 1 corresponded to a blood-meal at a titer of 10^4.5 FFU/mL and trial 2 to a titer of 10^5.9 FFU/mL. N refers to the total number of females tested.</p