Biological functions that are predicted to be activated by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.

*<p>the expression of the underlined molecules is down-modulated.</p

Validation of gene expression induced by CAV2 vector with qRT-PCR.

<p>A selection of genes was checked for CAV2-induced upregulation using qRT-PCR on cDNA from the CD103⁺ and CD11b⁺ -type DCs (4 different sheep were used in total, 3 per validated gene). Each gene detection was normalized with GAPDH expression and the relative gene expressions (log2(CTgene-CTGAPDH)) are reported. The gene names printed with regular font were selected as being up-regulated in the CD11b⁺ type DC microarrays (p < 0.05 ANOVA followed by Benjamini-Hochberg FDR and > 2 fold), the gene names printed in italic are not represented by any probe on the ovine microarray, the underlined gene names were close to be selected by the variance analysis in the CD11b⁺ -type DCs.</p

Top canonical pathways induced by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.

<p>Top canonical pathways induced by CAV2 vector in CD103⁺ and CD11b⁺ -type DCs.</p

Transcriptional response to CAV2 vector in the CD103⁺ and CD11b⁺ -type DC subsets.

<p>(A)The mean fold induction of the selected genes up-regulated by Cav-null R⁰ are represented in graded yellow to red color (from 1 to >3) for both CD103⁺ and CD11b⁺ -type DCs from 4 sheep. The represented genes were selected from the significantly induced genes in the CD11b⁺ -type DC subset (222 genes, p<0.05, fold > 2) and they were ranked based on their fold induction in the CD11b⁺ -type DC subset. The significantly activated genes in the CD103⁺ subset (21 genes, p<0.05, fold > 2) are indicated by a star. (B) The mean fold reduction levels of the selected down-modulated genes by Cav-null R⁰ are represented in graded yellow to blue color (from 1 to <0.2) for both CD103⁺ and CD11b⁺ -type DCs from 4 sheep. The represented genes were selected from the significantly reduced genes in the CD11b⁺ -type DC subset (29 genes, p<0.05) and they were ranked based on their fold induction in the CD11b⁺ -type DC subset. No gene expression was significantly reduced in the CD103⁺ -type DC subset.</p

Antimicrobial gene network induced by CAV2 vector in CD11b⁺ -type DCs.

<p>An antimicrobial gene network centered on IFN was generated by the Ingenuity Pathways Analysis on the selected genes dys-regulated by CAV2 vector in CD11b⁺ -type DCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052513#pone.0052513.s003" target="_blank">Table S1</a>, p < 0.05 and fold > 2, or p < 0.05 and folds confirmed by qRT-PCR). Molecule types are represented by symbols: diamonds (enzymes), triangles (kinase), square (cytokine), double circle (complex), oval (transcription regulator), circle (others).</p

Measurement of viraemia in needle and <i>C. nubeculosous</i>- infected sheep.

<p>BTV viral RNA was detected in blood by qRT-PCR at 0–9, 11, 15, 21 dpi. The number of BTV copies per ml of blood is reported for each sheep with a specific color and symbol shape. The 3 infected group are represented by a distinct symbol, i.e. a circle for the intradermal needle- inoculation group (ID, <b>A</b>), a square for the control <i>C.</i> bite + intradermal needle- inoculation group (CB + ID, <b>B</b>), and triangle for the infected C. bite group (IB, <b>C</b>). In <b>D</b>, the viral load over the 21- day monitoring period was integrated for each sheep using a calculation of area under the curve (color and shape symbol as in A, B, C). Statistical comparisons between 2 groups were done with <i>a Mann-Whitney U</i> test and the p values are reported to show significance and major tendencies.</p

Body temperature and clinical scoring in needle and <i>C. nubeculosous-</i> infected sheep. A.

<p>Body temperature was monitored for each sheep over 17 days. <b>B.</b> The increase in temperature over time was calculated relatively to day 0 and the peak area under the curve was calculated for each sheep. <b>C.</b> A clinical score was evaluated for each sheep and reported from 6 to 17 dpi in B. <b>D.</b> The integrated area under the curve of the clinical scores from 6 to 17 dpi was calculated for each sheep. The same color and shape are used for each sheep as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083683#pone-0083683-g001" target="_blank">Fig. 1</a>. Statistical comparisons between 2 groups were done with <i>a Mann-Whitney U</i> test and the p values are reported to show significance and major tendencies.</p

<i>Culicoides</i> Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

<div><p>Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of <i>Culicoides</i> midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected <i>C. nubeculosus</i> feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected <i>C. nubeculosus</i> bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected <i>Culicoides</i> in order to possibly develop new control strategies against BTV and other <i>Culicoides</i> transmitted viruses.</p></div

IFN- induced gene expression in blood cells is delayed in <i>C. nubeculosous-</i> infected sheep.

<p>Total RNA was extracted from blood cells of each sheep from 0–5 dpi. qPCR was performed on reverse transcribed RNA using the primers for CXCL10 and MX1 genes detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083683#pone.0083683.s006" target="_blank">Table S3</a>. The 2^{−<b>ΔΔ</b>CT} method was used to calculate the cytokine gene expression relatively the T0 time point level using the ribosomal RPS24 RNA as an internal control. The blood cells from control and control CB group did not show any induction of CXCL10 and MX1 gene expression during the observation period (not shown). The same color and shape symbol are used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083683#pone-0083683-g001" target="_blank">Fig. 1</a>. Statistical comparisons between 2 groups were done with <i>a Mann-Whitney U</i> test and the p values are reported to show significance.</p

Anti-VP7 and neutralizing antibody response in serum is delayed in <i>C. nubeculosous-</i> infected sheep.

<p><b>A.</b> Anti-VP7 antibodies were detected by competitive ELISA at 0, 5, 8, 15 and 21 dpi. <b>B.</b> Neutralizing antibodies were assessed at 0, 5, 8, 15 and 21 dpi in BHK21 cells by serum neutralization with 50 TCID₅₀ BTV8 (triplicates per sheep and per day). Statistical differences between groups were calculated by <i>a Mann-Whitney U</i> test on each day and the p values are reported for day 8.</p