Efficient In Vitro Amplification of Chronic Wasting Disease PrPRES (original) (raw)
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Species barriers for chronic wasting disease by in vitro conversion of prion protein
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP CWD of elk can readily induce the conversion of normal cervid PrP (PrP C ) molecules to a protease-resistant form, but is less efficient in converting the PrP C of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP CWD -induced conversion can be greatly enhanced in all species. Our results demonstrate that PrP C from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP C can apparently overcome the structural barriers between more distant species.
Journal of Veterinary Diagnostic Investigation, 2012
Cerebrospinal fluid (CSF) has been examined as a possible source for preclinical diagnosis of prion diseases in hamsters and sheep. The present report describes the detection of chronic wasting disease (CWD) in the CSF of elk and evaluates its usefulness as an antemortem test for CWD. The CSF from 6 captive and 31 free-ranging adult elk was collected at necropsy and evaluated for the presence of the abnormal isoform of the prion protein that has been associated with CWD (PrP CWD) via protein misfolding cyclic amplification. Additionally, the obex from each animal was examined by immunohistochemistry (IHC). Four out of 6 captive animals were CWD-positive and euthanized due to signs of terminal CWD. The remaining 2 were CWD negative. None of the 31 free-range animals showed overt signs of CWD, but 12 out of 31 tested positive for CWD by IHC. Protein misfolding cyclic amplification detected PrP CWD from 3 of the 4 captive animals showing clinical signs of CWD and none of the nonclinical animals that were CWD positive by IHC. The data suggests that CWD prions can be detected in the CSF of elk, but only relatively late in the course of the disease.
Journal of Virology, 2006
North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP CWD ) was used as an indicator of CWD infection. Although no PrP CWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP CWD -positive clone out of 51. This clone, designated MDB CWD , has maintained stable PrP CWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP CWD -positive subclones out of 30, one of which was designated MDB CWD2 . The MDB CWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP CWD accumulation in MDB CWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP CWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.
Scientific Reports
Rocky mountain elk (Cervus elaphus nelsoni). There are 2 allelic variants in the elk prion protein gene: L132 (leucine) and M132 (methionine). Following experimental oral challenge with the CWD agent incubation periods are longest in LL132 elk, intermediate in ML132 elk, and shortest in MM132 elk. In order to ascertain whether such cWD-infected elk carry distinct prion strains, groups of Tg12 mice that express M132 elk prion protein were inoculated intracranially with brain homogenate from individual CWD-infected elk of various genotypes (LL132, LM132, or MM132). Brain samples were examined for microscopic changes and assessment of the biochemical properties of disease-associated prion protein (prp Sc ). On first passage, mice challenged with LL132 elk inoculum had prolonged incubation periods and greater prp Sc fibril stability compared to mice challenged with MM132 or LM132 inoculum. On second passage, relative incubation periods, western blot profiles, and neuropathology were maintained. These results suggest that the CWD prion isolated from LL132 elk is a novel CWD strain and that M132 PrP c is able to propagate some biophysical properties of the L132 PrP Sc conformation.
Generation of human chronic wasting disease in transgenic mice
Acta Neuropathologica Communications
Chronic wasting disease (CWD) is a cervid prion disease caused by the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC). It has been spreading rapidly in North America and also found in Asia and Europe. Although bovine spongiform encephalopathy (i.e. mad cow disease) is the only animal prion disease known to be zoonotic, the transmissibility of CWD to humans remains uncertain. Here we report the generation of the first CWD-derived infectious human PrPSc by elk CWD PrPSc-seeded conversion of PrPC in normal human brain homogenates using in vitro protein misfolding cyclic amplification (PMCA). Western blotting with human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPSc was derived from the human PrPC substrate. Two lines of humanized transgenic mice expressing human PrP with either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated ...
Scientific Reports
chronic wasting disease (cWD) is a rapidly spreading prion disease of cervids, yet antemortem diagnosis, treatment, and control remain elusive. We recently developed an organotypic slice culture assay for sensitive detection of scrapie prions using ultrasensitive prion seeding. However, this model was not established for cWD prions due to their strong transmission barrier from deer (Odocoileus spp) to standard laboratory mice (Mus musculus). therefore, we developed and characterized the ex vivo brain slice culture model for CWD, using a transgenic mouse model (Tg12) that expresses the elk (Cervus canadensis) prion protein gene (PRNP). We tested for cWD infectivity in cultured slices using sensitive seeding assays such as real-time quaking-induced conversion (Rt-Quic) and protein misfolding cyclic amplification (PMCA). Slice cultures from Tg12, but not from prnp −/− mice, tested positive for cWD. Slicegenerated CWD prions transmitted efficiently to Tg12 mice. Furthermore, we determined the activity of anti-prion compounds and optimized a screening protocol for the infectivity of biological samples in this cWD slice culture model. our results demonstrate that this integrated brain slice model of cWD enables the study of pathogenic mechanisms with translational implications for controlling cWD.
Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease
PLoS ONE, 2013
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP d ) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP d detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP d identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.
De Novo Generation of a Unique Cervid Prion Strain Using Protein Misfolding Cyclic Amplification
mSphere, 2017
Substantial evidence supports the hypothesis that prions are misfolded, infectious, insoluble, and protease-resistant proteins (PrP RES) devoid of instructional nucleic acid that cause transmissible spongiform encephalopathies (TSEs). Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRes acts as a template that can convert the normal cellular prion protein (PrP C) present in uninfected normal brain homogenate (NBH) into the infectious misfolded PrP RES isoform. Human PrP C has been shown to spontaneously convert to a misfolded pathological state causing sporadic Creutzfeldt-Jakob disease (sCJD). Several investigators have reported spontaneous generation of prions by in vitro assays, including PMCA. Here we tested the rate of de novo generation of cervid prions in our laboratory using our standard PMCA protocol and NBH from transgenic mice expressing cervid PrP C (TgCerPrP mice). We generated de novo prions in rounds 4, 5, and 7 at low cumulative rates of 1.6, 5.0, and 6.7%, respectively. The prions caused infectious chronic wasting disease (CWD) upon inoculation into normal uninfected TgCerPrP mice and displayed unique biochemical characteristics compared to other cervid prion strains. We conclude that PMCA of cervid PrP C from normal brain homogenate spontaneously generated a new cervid prion strain. These data support the potential for cervids to develop sporadic CWD. IMPORTANCE CWD is the only known TSE that affects free-ranging wildlife, specifically cervids such as elk, deer, moose, caribou, and reindeer. CWD has become endemic in both free-ranging and captive herds in North America, South Korea, and, most recently, northern Europe. The prion research community continues to debate the origins of CWD. Original foci of CWD emergence in Colorado and Wyoming coincident with the sheep TSE scrapie suggest that scrapie prions may have adapted to cervids to cause CWD. However, emerging evidence supports the idea that cervid PrP C may be more prone to misfolding to the pathological isoform. Here we test the hypothesis that cervid PrP C can spontaneously misfold to create de novo prions. Whether CWD can arise spontaneously as a sporadic TSE or represents a new TSE caused by cervid-adapted scrapie prions profoundly impacts surveillance and mitigation strategies.
Scientific Reports, 2020
Chronic wasting disease (CWD) is a prionopathy affecting wild and farmed cervids. This disease is endemic in North America and has been recently identified in Europe. Ante-mortem CWD tests of pre-clinical cervids may be an important tool in helping control the spread of this disease. Unfortunately, current CWD diagnostic methods are not suitable for non-tissue type samples. We reported that CWD prions can be detected in blood of pre-clinical CWD-infected white-tailed deer (WTD) with high sensitivity and specificity using the Protein Misfolding Cyclic Amplification (PMCA) assay. However, that report only included animals homozygous for codon 96G, the most common polymorphic version of the prion protein within this animal species. Here, we report CWD prion detection using blood of naturally infected WTD coding one or two copies of the PrP-96S polymorphic variant. Our results, from a blinded screening, show 100% specificity and ~ 58% sensitivity for animals harboring one 96S codon, reg...