Highly Efficient Prion Transmission by Blood Transfusion

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 103ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed

Characterization of a prion distribution and infectivity level in sheep TSE models and evalution of leucodepletion / prion trap impact on endogenous infectivity

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Transfusion of VRQ/VRQ sheep with blood spiked with decreasing amounts of PG127 infected sheep brain homogenate.

<p>200 mL of whole blood was collected from each of 12 TSE Free VRQ/VRQ cheviot sheep. Whole blood pouches were spiked with decreasing amounts of sheep scrapie strain PG127 brain homogenate containing 10^6.6 ID₅₀/g as measured by IC inoculation in tg<i>338</i> mice. Each spiked blood was then transfused back into the sheep of origin. Two sheep were challenged at each dose (indicated as number of ID₅₀ IC in tg338). Sheep were observed until they developed clinical signs or reached 750 days post inoculation when they were euthanized. All recipients were tested for presence of abnormal PrP (PrP^Sc) deposition in brain and various lymphoid tissues by immunohistochemistry. Results confirmed the clinical diagnosis. Incubation periods in recipients are presented as days post inoculation (dpi).</p

Tg338 mice intracerebral inoculation with VRQ/VRQ sheep white blood cells homogenates.

<p>Blood was collected from three TSE free VRQ/VRQ donor sheep that had been orally challenged with PG127 scrapie (D1, D2, D3) and one TSE free VRQ/VRQ control sheep (C1). The date of collection from the infected animals was 210 days post inoculation. Donor sheep developed clinical signs within two to five weeks following blood collection. They were euthanized at 227 days, 256 days, 221 days respectively White blood cells (WBC) were prepared from whole blood and homogenised in 5% glucose solution. Successive 1/10 dilutions of WBC homogenates were inoculated intra-cerebrally to tg<i>338</i> mice (n = 6). The equivalent volume of whole blood inoculated in mice is indicated. Mice were euthanized when they showed clinical signs of infection or after 250 dpi. Mice were considered infected when abnormal PrP depositions were detected in brain. Infectious titer was estimated by the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Markus1" target="_blank">[19]</a>. Infectious titer is expressed as number of ID₅₀ per mL of whole blood. For each samples, the most likely value and (in parentheses) the lower and upper value of the 95% confidence interval are reported.</p

Intracerebral inoculation of tg338 mice with VRQ/VRQ sheep whole blood, plasma and red blood cell concentrate.

<p>Blood was collected from three TSE free VRQ/VRQ donor sheep that had been orally challenged with PG127 scrapie (D1, D2, D3) and one TSE free VRQ/VRQ control sheep (C1). The date of collection from the infected animals was 210 days post inoculation. Donors developed clinical scrapie two to five weeks following blood collection. They were euthanized at 227 days, 256 days, 221 days respectively. Whole blood, plasma, red blood cell concentrate (RBC) were each inoculated intracerebrally into 18 tg338 mice (20 µL per mouse). For each component, the volume of whole blood corresponding to the volume inoculated is given. Mice were euthanized when they showed clinical signs of infection or after 250 dpi. Mice were considered infected when abnormal PrP depositions were detected in brain. Infectious titers were estimated using limiting dilution titration method (application of Poisson model) described by Brown et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Brown3" target="_blank">[23]</a>. Infectious titers are given as the most likely infectious titer and, in parentheses, the values of the lower and upper limits of the 95% confidence interval.</p

End-point titration in tg338 mice of a 10% brain homogenate and white blood cells samples, collected in VRQ/VRQ sheep orally inoculated with PG127 scrapie.

<p>10% weight/volume homogenate (*) was prepared using posterior brainstem from VRQ/VRQ sheep inoculated with PG127 scrapie isolate and at the terminal stage of disease. Groups of 6 mice that over-express the VRQ ovine PrP (tg<i>338</i>) were intracerebrally (20 µL) inoculated with successive 1/10 dilutions of this homogenate. These data were already used in a previous publication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Andreoletti1" target="_blank">[16]</a>. In parallel, four susceptible VRQ/VRQ sheep (identified as D4, D5, D6 and D7) were orally challenged with PG127 classical scrapie isolate (total dose 10^6.7 ID₅₀ IC in tg338 mice). Scrapie incubation period in sheep were respectively 226 days, 238 days, 228 days and 242 days post inoculation (dpi). Aliquot of the same fresh and PFA 2% fixed WBC than those IV administrated sheep (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat-1002782-t005" target="_blank">Table 5</a>) were homogenised in 5% glucose before intracerebral inoculation in tg338 mice (n = 6 per sample, 20 µL per mice). Each mouse received a quantity of WBC that is equivalent to 2.5 mL of starting whole blood. Mice were observed till occurrence of clinical signs compatible with a transmissible spongiform encephalopathy and considered positive when abnormal PrP deposition was detected in brain. Incubation periods (days post inoculation: dpi) in mice are presented as mean +/−SD except for those dilutions with which less than 50% of mice were found positive. In that case individual incubation period are reported (†). Infectious titers were estimated by the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Markus1" target="_blank">[19]</a>. Infectious titer is expressed as number of ID₅₀ per mL of whole blood. For each samples, the most likely value and, in parentheses, the lower and upper value of the 95% confidence interval are reported.</p

Impact of leucocyte depletion and prion reduction filters on TSE blood borne transmission

The identification in the UK of 4 v-CJD infected patients thought to be due to the use of transfused Red Blood Cell units prepared from blood of donors incubating v-CJD raised major concerns in transfusion medicine. The demonstration of leucocyte associated infectivity using various animal models of TSE infection led to the implementation of systematic leuco-depletion (LD) of Red Blood cells concentrates (RBCs) in a number of countries. In the same models, plasma also demonstrated a significant level of infectivity which raised questions on the impact of LD on the v-CJD transmission risk. The recent development of filters combining LD and the capture of non-leucocyte associated prion infectivity meant a comparison of the benefits of LD alone versus LD/prion-reduction filters (LD/PR) on blood-borne TSE transmission could be made. Due to the similarity of blood/plasma volumes to human transfusion medicine an experimental TSE sheep model was used to characterize the abilities of whole blood, RBCs, plasma and buffy-coat to transmit the disease through the transfusion route. The impact of a standard RBCs LD filter and of two different RBCs LD/PR prototype filters on the disease transmission was then measured. Homologous recipients transfused with whole-blood, buffy-coat and RBCs developed the disease with 100% efficiency. Conversely, plasma, when intravenously administered resulted in an inconstant infection of the recipients and no disease transmission was observed in sheep that received cryo-precipitated fraction or supernatant obtained from infectious plasma. Despite their high efficacy, LD and LD/PR filtration of the Red Blood Cells concentrate did not provide absolute protection from infection. These results support the view that leuco-depletion strongly mitigates the v-CJD blood borne transmission risk and provide information about the relative benefits of prion reduction filters

Intravenous administration in sheep and intracerebral challenge in tg338 mice of blood derived products prepared from PG127 scrapie infected sheep.

<p>Four scrapie-susceptible VRQ/VRQ sheep (identified as D4, D5, D6 and D7) were orally challenged with 1 g of sheep scrapie strain PG127 infected brain homogenate containing 10^6.7 IC ID₅₀/g as measured by IC inoculation of tg338 mice Scrapie incubation periods were 226 dpi, 238 dpi, 228 dpi and 242 dpi, respectively. Blood was collected at 217 dpi, <i>i.e</i> few days (D4) to three weeks (D7) before clinical onset. Plasma and white blood cells (WBC) were prepared from 500 mL of whole blood. Half of the WBC preparation from each animal was fixed with paraformaldehyde (PFA 2% final concentration). Whole Blood, plasma and both fresh and fixed WBCs (re-suspended in 5% glucose), each corresponding to 200 mL of whole blood, were administered intravenously to VRQ/VRQ TSE free recipients. In addition, Plasma volume equivalent to 20 mL of whole blood was also intravenously administered to sheep. Recipients were euthanized when symptomatic with scrapie. 450 dpi (or 380 dpi for the 20 mL plasma) recipients that were still alive and apparently healthy were euthanized. Incubation periods in recipients are presented in days post inoculation (dpi). All recipient sheep were tested for the presence of abnormal PrP deposition in brain and various lymphoid tissues by immunohistochemistry.NA: not assessed.</p

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models

International audienceThe dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 106.5-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia