Estimating relative CWD susceptibility and disease progression in farmed whitetail deer with rare PRNP alleles

Chronic wasting disease is a prion disease affecting both free-ranging and farmed cervids in North America and Scandinavia. A range of cervid species have been found to be susceptible, each with variations in the gene for the normal prion protein, PRNP, reportedly influencing both disease susceptibility and progression in the respective hosts. Despite the finding of several different PRNP alleles in whitetail deer, the majority of past research has focused on two of the more common alleles identified – the 96G and 96S alleles. In the present study, we evaluate both infection status and disease stage in nearly 2100 farmed deer depopulated in the United States and Canada, including 714 CWD-positive deer and correlate our findings with PRNP genotype, including the more rare 95H, 116G, and 226K alleles. We found significant differences in either likelihood of being found infected or disease stage (and in many cases both) at the time of depopulation in all genotypes present, relative to the most common 96GG genotype. Despite high prevalence in many of the herds examined, infection was not found in several of the reported genotypes. These findings suggest that additional research is necessary to more properly define the role that these genotypes may play in managing CWD in both farmed and free-ranging whitetail deer, with consideration for factors including relative fitness levels, incubation periods, and the kinetics of shedding in animals with these rare genotypes.

Human stem cell–derived astrocytes replicate human prions in a PRNP genotype–dependent manner

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Deletion of the Prion Protein Does not Affect Erythropoiesis in a Mouse Model of Beta-Thalassemia

Abstract 3213 The cellular prion protein (PrPc) is a highly conserved GPI-linked cell surface sialoglycoprotein which plays a key role in the pathogenesis of neurodegenerative prion diseases. Although PrPc is conserved across species, its normal function is not clearly understood. PrPc tissue distribution varies by cell type or by level of expression in different species. Circulating red blood cells (RBCs) in humans and mice have similar levels of PrPc and reports in the literature suggest that it has a role in survival of cells under stress conditions. We previously reported that an absence of PrPc in PrPc knock out (Prnp-/-) mice negatively influenced their response to acute anemia induced with phenylhydrazine (PHZ) (Zivny et al. 2008. Blood Cells, Molecules and Diseases. 40: 302–307). Prpn-/- mice displayed a reduction of erythroid cells and erythropoietin production suggesting the importance of PrPc expression for stress erythropoiesis. In order to further explore this observation, we mated chronically anemic β-thalassemic (th3/+) male mice (generously donated by Dr. M Sadelein, NY) with female Prnp-/- mice. Th3/+ mice have a defect in the β chain of hemoglobin and have lower hematocrits (HCT). Their offspring, heterozygous for PrPc (Prnp+/−), were genotyped and anemic th3/+ Prnp+/− males were mated with non-anemic (WT) Prnp+/− females to produce mice for the study. The PrPc genotype of two cohorts of offspring combined (n=127 total), Prnp+/+ (18%), Prpn+/− (56%) and Prnp-/- (26%), did not show significant deviation from a Mendelian distribution. Similarly, the proportion of Prnp-/- genotype among anemic th3/+ offspring (29.5%) was slightly higher than the proportion of Prnp+/+ genotype (18.1%), suggesting that PrPc deletion does not lead to higher in utero mortality of th3/+ mice. Evaluation of the microhematocrit at 12 weeks of age demonstrated decreased HCT levels of th3/+ mice (37.4±5.0% and 35.1±2.9%) in comparison to their WT siblings (51.6±2.5% and 49.1±3.5%). No significant differences were detected among different Prnp genotypes in both th3/+ and WT mice. In order to test if PrPc expression in these mice is important for the recovery from acute anemia, the th3/+ and the WT siblings of all PrPc genotypes (n=6 per group) were injected with PHZ (80 mg/kg). The following parameters were measured over a 7 day period using standard methods: HCT levels, plasma erythropoietin (EPO) levels, peripheral reticulocyte count, percentage of cells in the spleen expressing CD71, and the weight of animals and their spleens at the end of the study. On day 7 after induction of acute anemic stress, plasma EPO levels were higher in th3/+ animals compared to WT animals, 1125±48.4 pg/mL and 338±104.5 pg/mL respectively. The percent of erythroid precursor cells (CD71+) were lower in th3/+ (10.0±0.7%) than in WT (21.8±2.8%) splenocytes but the spleen sizes were larger in the th3/+ mice. Hematocrits were not different with statistical significance between th3/+ (28.8±0.6%) and WT (31.5±0.75%) animals on day 7, but circulating reticulocyte counts were higher in th3/+ (54.2±4.9% vs. 40.9±3.8%) animals. The PrPc genotype (Prnp+/+, Prnp+/− and Prnp-/-) did not affect the results in either the th3/+ or WT animals. Th3/+ animals were chronically anemic and had inefficient erythropoiesis which produced a lower percentage of CD71+ cells, in spite of higher levels of plasma EPO in response to similar levels of hypoxic stress as in the WT mice. However, the overall RBC production of th3/+ mice in response to acute hypoxia was maintained by a large spleen size. The prion protein deletion did not affect the percent of RBC precursors or the final RBC output. Thus, prion protein expression does not appear to influence RBC production in a chronically anemic mouse model. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. (GAUK86408, GACR 310/08/0878) Disclosures: No relevant conflicts of interest to declare.

Blood Chimerism Confounds Genetic Relative Susceptibility Testing for Classical Scrapie in Sheep

Classical scrapie disease is a transmissible spongiform encephalopathy of sheep that is enzootic in the United States. Susceptibility of sheep to classical scrapie is linked to single nucleotide polymorphisms in the prion protein gene ( PRNP), forming the basis for genetic testing strategies used by national efforts to eradicate scrapie. Such efforts are occasionally hampered by inconclusive results stemming from the detection of “complex” genotypes. Naturally occurring cases of ovine chimerism are thought to account for some of these instances. In the current report, 4 naturally occurring ovine chimeras are documented through cytogenetic and molecular analyses. All 4 of these sheep had chimeric cells circulating in their blood. Blood and alternate tissue samples of ear punch and hair bulbs from one of these chimeras was submitted in batch with similar samples from control sheep for routine scrapie genetic relative susceptibility testing. A complex PRNP genotype was detected in the blood of the chimeric female but not in the alternate tissue samples or in the control sheep samples. The results demonstrate that naturally occurring blood chimerism can confound current testing efforts. The potential impacts of undetected chimeras on current scrapie eradication efforts are discussed.