Description of the 2010 and 2013 ILPT panels.

<p>Description of the 2010 and 2013 ILPT panels.</p

Decisional algorithm for flavivirus serological diagnosis in case of negative results by molecular diagnosis.

<p>Decisional algorithm for flavivirus serological diagnosis in case of negative results by molecular diagnosis.</p

Box plot representing %S/N obtained with the ID screen WNV competition kit by each NRL during the 2010 and 2013 IPLTs.

<p>A single letter, A-0, refers to a unique NRL in 2010 and 2013.</p

NRLs having participated in the 2010 and 2013 WNV ILPTs and serological diagnostic assays used.

<p>Diagnostic assays implemented in 2010 and 2013 by European NRLs are shown in pale blue (IgG ELISA alone), medium blue (IgM ELISA alone) or dark blue (IgG and IgM ELISAs) and in yellow (WNV VNT). (Base layer: lemondedesetudes.fr/freebies-cartes-powerpoint).</p

Box plot representing the distribution of %S/N obtained with the ID screen WNV competition kit by NRLs during the 2010 and 2013 ILPTs according to the year and batch.

<p>Assays were performed and %S/N was calculated according to the manufacturer’s instructions (the threshold value for considering a serum as positive by the competitive ELISA was %S/N < 40%).</p

Percentage of NRLs having generated positive (red), doubtful (orange) and negative results (green) during the 2013 WNV ILPT.

<p>Doubtful are equivocal results; Striped results correspond to unsatisfactory results different from the accepted results. The below table summarises the accepted ILPT results taking into account the performance of the tests used by the participants with P (positive), D (doubtful) and N (negative).</p

Number of participants and serological methods used by participating laboratories during the 2010 and 2013 WNV ILPTs.

<p>Number of participants and serological methods used by participating laboratories during the 2010 and 2013 WNV ILPTs.</p

Individual production of total IgG after immunization with rVHSV-SP_GE_VWN and rVHSV-SP_GE_VWNΔTMTM_G.

<p>5-week-old BALB/c mice in groups of 12 individuals were immunized three times with different antigens as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091766#pone-0091766-g002" target="_blank">fig.2</a> for schedule details). Antibody production by each mouse was assessed by ELISA with individual sera collected on day 56 just before the WNV challenge. Each serum was diluted to 1∶100. OD values for each individual are represented directly after removal of the white (OD measured in the absence of serum).</p

Detection of DIII_WNV at the virus surface of rVHSV-SP_GE_WNV and rVHSV-SP_GDIII_WNVTM_G by immunogold.

<p>Sucrose purified-recombinant viral particles were adsorbed on electron microscopy nickel grids. After fixation, DIII_WNV and the glycoprotein G of VHSV (G_VHSV) were detected using specific mouse primary monoclonal antibodies. These mouse primary antibodies were detected by an anti-mouse secondary antibody coupled with a gold particle (black dots of 5 nm in diameter). After negative staining, recombinant viral particles were observed by transmission electron microscopy.</p

Expression of E_WNV antigens in rVHSV-infected cells.

<p>The expression of E_WNV antigens was assessed by indirect-immunofluorescence in BF-2 cells. The cells were infected or not infected (Mock) either with the empty vector (rVHSV) or the six recombinant viruses expressing E_WNV domains (as indicated). Cells were incubated for 48 h at 14°C. (A) At 48 h post-infection, cells were fixed and permeabilized with a mixture of alcohol/acetone, and protein expression was detected using a monoclonal antibody against E_WNV DIII (E24). (B) Detection of membrane expression of E_WNV antigens was performed on live cells using the E24 antibody, except for rVHSV-SP_GDIIDIII_WNVTM_G infected cells where E_WNV expression was achieved with mAb8150 (magnification X63).</p