Evaluation of the infectivity present in platelets prepared from scrapie infected sheep by two different methods: PMCA and inoculation into tg338 mice (bioassay).

<p>Five susceptible VRQ/VRQ sheep were orally challenged with 2 g of brain homogenate (10^6.6 ID₅₀/g IC in tg<i>338</i> mice) between 6 and 10 months of age. The five VRQ/VRQ sheep respectively died at 198, 193, 198, 194 and 191days post inoculation (dpi). Classical scrapie was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposit in central nervous system and lymphoid tissues. At different time points, whole blood was collected from each donor and aliquots of platelets corresponding to 15 mL of plasma were prepared. Platelet homogenates (in 200μL) were inoculated in groups of six tg338 mice. Mice were monitored up to occurrence of TSE compatible clinical sign onset or killed at 250 days post inoculation. All mice were tested for presence of abnormal PrP deposition in brain. Incubation period in mice are presented in days (+/−SD). When less than 3 mice were positive, individual incubation period are given. In parallel the same homogenates were tested by PMCA (using tg338 mice brain homogenate as substrate). Each sample was run in 5 replicates for 2 successive rounds and the number of positive reactions is presented.</p

Cell-based assay of white blood cells infectivity from asymptomatic scrapie sheep.

<p>White blood cells from 5 infected sheep (D1 to D5) were isolated 80 days and 130 days post inoculation (dpi) when sheep were still asymptomatic. White blood cell homogenates (4×10⁷ cells) were inoculated to recipient ovRK13 cells. After 2 successive rounds of cell assay, the cultures were assayed for PrP^res by immunoblotting. PrP^res level is higher in cells infected with D3 130 dpi sample but its banding pattern is similar to that in cells infected with the other samples. M are molecular mass marker proteins (20, 30 and 40 kDa).</p

Evaluation of the infectivity present in white blood cells prepared from scrapie infected sheep by Cerebellar Organotypic Slice Culture Assay.

<p>Immunoblots of PK-treated slice culture homogenates probed with anti-PrP antibody Sha31, showing PrP^res accumulation in slice culture. (A) Cerebellar organotypic slices were prepared from tg338 pups and maintained in culture during 42 days <i>in vitro</i> after exposure to white blood cells prepared from blood collected from five scrapie infected sheep (D1, D2, D3, D4 and D5) at different times: 50 days post inoculation (dpi), 80 dpi, 130 dpi and at the terminal stage (180 dpi). For quantification purposes, slice cultures were also exposed to serial dilutions of PG127 scrapie-infected brain stock prepared from terminally ill tg338 mice, previously used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104287#pone.0104287-ArellanoAnaya1" target="_blank">[31]</a>. To visualize low levels of PrP^res, membranes were exposed over-night (B).</p

PMCA analysis of white blood cells and platelets samples.

<p>Platelets (A) and white blood cells (WBC) (B) from sheep D2 collected at the indicated time points (dpi) were subjected to two successive rounds of PMCA. Unseeded reactions were run in parallel. Samples were processed for PrP^res isolation and analyzed by immunoblotting. A western-blotting positive control (cont) is included in each gel.</p

Overlapping PK-resistant PrP^Sc and infectivity density profiles of prion strains.

<p>Brain homogenates from <i>tg338</i> mice infected with LA21K <i>fast</i> (<b>A</b>), LA19K (<b>B</b>), sheep BSE (<b>C</b>) and Nor98 (<b>D</b>) were solubilized and fractionated by sedimentation at the equilibrium. The fractions collected from the gradient were analyzed for PK-resistant PrP^Sc content (black line) and for infectivity (red line). The mean levels of PK-resistant PrP^Sc per fraction shown are the combined and fit replicates (left axis) obtained from the immunoblot analysis of n≥3 independent fractionations. For each fraction analyzed, infectivity was determined by applying the mean survival times values in <i>tg338</i> mice (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003702#ppat-1003702-t001" target="_blank">Table 1</a>) to standard dose response curves established for each strain <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003702#ppat.1003702-Tixador1" target="_blank">[20]</a>. The right, logarithmic scale provides the reciprocal relative infectious dose found. A relative infectious dose of 0 corresponds to animal inoculated with the equivalent of 2 mg of infectious brain tissue.</p

Overlapping PK-resistant PrP^Sc and infectivity density profiles of ‘fast’ prions upon additional solubilization with digitonin.

<p>Brain homogenates from <i>tg338</i> mice infected with LA21K <i>fast</i> (<b>A</b>) or 127S (<b>B</b>, <b>C</b>) were solubilized in the standard conditions (<b>A</b>, <b>B</b>) or by adding digitonin first (<b>C</b>) before fractionation by sedimentation at the equilibrium. The fractions collected from the gradient were analyzed for PK-resistant PrP^Sc content (black line; left axis) and for infectivity (red line; right axis). The mean levels of PK-resistant PrP^Sc per fraction shown are the combined and fit replicates obtained from the immunoblot analysis of n≥3 independent fractionations. Fraction infectivity was determined by a Rov cell assay as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003702#ppat-1003702-g001" target="_blank">Figure 1</a> (mean ± SEM of n = 3 independent titrations).</p

Immunoblot analysis of PrP material from <i>tg338</i> mouse brain sedimented at the equilibrium.

<p>Solubilized brain material from uninfected (<b>A, D</b>) or 127S (<b>B</b>, <b>C, E</b>) infected <i>tg338</i> mice was fractionated by sedimentation at the equilibrium. The collected fractions (numbered from top to bottom of the gradient) were analyzed for PrP^C (<b>A</b>; <b>B</b>), Thy1 (<b>A</b>), flotillin and caveolin (<b>C</b>) content by immunoblot without (<b>A</b>; <b>B</b>, right axis; <b>C</b>) or after PK digestion (<b>B</b>, left axis). The relative amounts of those proteins per fraction were reported on the graphs, as indicated on left or right axis. The data presented are the mean ± SEM of n≥3 independent fractionations. The density values estimated by refractometry are indicated on the top of the graph. (<b>D</b>, <b>E</b>) Representative immunoblots of PrP^C (<b>D</b>) and PK-resistant PrP^Sc (<b>E</b>) distribution at the equilibrium are shown.</p

Mean survival time of <i>tg338</i> mice intracerebrally inoculated with prion strains fractionated by sedimentation at the equilibrium.

1<p>n/n₀: Number of diseased, brain PrP^res-positive/inoculated mice.</p>2<p>Mean survival time of the infected mice in days ± SEM.</p

Effect of solubilization temperature and increase in ultracentrifugation time on the sedimentation velocity profile of PK-resistant PrP^Sc and infectivity.

<p>Brain homogenates from <i>tg338</i> mice infected with LA21K <i>fast</i> prions were solubilized at 4°C (<b>A</b>; <b>C</b>) or 37°C (<b>B</b>), loaded on top of a linear optiprep gradient and ultracentrifuged for 45 min (<b>A</b>, <b>B</b>) or 90 min (<b>C</b>). Fractions collected from the gradient (numbered from top to bottom) were analyzed for PK-resistant PrP^Sc content (black line; left axis) and for infectivity (red line; right axis). The mean levels of PK-resistant PrP^Sc per fraction have been obtained from the immunoblot analysis of n≥3 independent fractionations. As the replicates gave consistent results, these data were combined and fit. Fraction infectivity was determined by a Rov cell assay. It is based on the quantification of PrP^Sc-containing Rov cells by immunofluorescence using PrP^Sc-specific antibodies. The cells were exposed in parallel to fraction aliquots and to serial tenfold dilutions of a LA21K <i>fast</i>-infected brain (expressed as relative infectious doses) prepared in the same conditions. The data presented are the mean ± SEM of n = 3 independent titrations. Data presented in A are from ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003702#ppat.1003702-Tixador1" target="_blank">[20]</a> and this study.</p

Templating activity of sedimentation velocity fractions of ‘fast’ prions.

<p>Brain homogenates from <i>tg338</i> mice infected with LA21K <i>fast</i> prions were solubilized in the standard conditions before fractionation by sedimentation velocity. The fractions collected from the gradient were pooled as indicated and serially diluted 10³ to 10¹⁰-fold before being used as templates in PMCA reaction (one round). The resulting product was PK-digested, denatured and quantified by immunoblot analysis. The data shown are the mean ± SEM levels of PK-resistant PrP^Sc from n = 4 independent experiments. The last positive dilution is indicated in red. The white bars are considered as nonspecific background.</p