Jean E. Jewell

Prion Strain Mutation Determined by Prion Protein Conformational Compatibility and Primary Structure

CWD Strain Variation So-called prion diseases are fatal neurogenerative disorders that include chronic wasting disease (CWD) found in deer and other cervids. Prion diseases are thought to be caused by infectious proteins (prions) in the absence of associated infectious DNA. Nevertheless, prion strains have been isolated that can mutate in the absence of nucleic acids, and these strain properties control the ability of prions to cross species barriers. Angers et al. (p. 1154, published online 13 May; see the Perspective by Collinge) address the issue of strain variation in the context of CWD. Whereas the host range of this contagious disease continues to expand, the prevalence of CWD strains has not been determined. Understanding CWD strain variation may be important in predicting and preventing any future risks to human health. The stability of two related strains is influenced by a species-specific amino acid difference in deer and elk prions. Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

A Natural Case of Chronic Wasting Disease in a Free-ranging Moose (Alces alces shirasi)

Chronic wasting disease (CWD) was diagnosed in a free-ranging moose (Alces alces shirasi) killed by a hunter in Jackson County, Colorado, USA, in September 2005. The diagnosis was based upon immunohistochemistry (IHC) demonstrating the presence of accumulations of CWD-associated prion protein (PrPCWD) in tissue sections of medulla oblongata at the level of the obex (dorsal motor nucleus of the vagus) and in retropharyngeal lymph node (RPLN); additional testing by IHC revealed deposits of PrPCWD in multiple sections of medulla oblongata and cervical spinal cord as well as palatine tonsil and submandibular lymph node tissues. Western blot confirmed the presence of PrPCWD in RPLN and tonsil tissue. The PrPCWD also was detected via enzyme-linked immunosorbent assay of RPLN tissue. Spongiform encephalopathy was observed in sections of the brainstem and cervical spinal cord, although no clinical signs were noted by the hunter who killed the animal. The affected moose was homozygous for methionine at codon 209 of the prion protein coding region. In October 2006, two additional free-ranging moose were diagnosed with CWD. Epidemiology and implications of CWD in moose remain to be determined.

Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers

Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.

Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10-7 dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 107 protease resistant cervid prion protein (PrPCWD) monomers. We also detected PrPCWD in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrPCWD detected was below infectious levels. These data demonstrate detection of very low levels of PrPCWD in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.

The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation.

The elk prion protein gene (PRNP) encodes either methionine (M) or leucine (L) at codon 132, the L132 allele apparently affording protection against chronic wasting disease (CWD). The corresponding human codon 129 polymorphism influences the host range of bovine spongiform encephalopathy (BSE) prions. To fully address the influence of this cervid polymorphism on CWD pathogenesis, we created transgenic (Tg) mice expressing cervid PrPC with L at residue 132, referred to as CerPrPC-L132, and compared the transmissibility of CWD prions from elk of defined PRNP genotypes, namely homozygous M/M or L/L or heterozygous M/L, in these Tg mice with previously described Tg mice expressing CerPrPC-M132, referred to as Tg(CerPrP) mice. While Tg(CerPrP) mice were consistently susceptible to CWD prions from elk of all three genotypes, Tg(CerPrP-L132) mice uniformly failed to develop disease following challenge with CWD prions. In contrast, SSBP/1 sheep scrapie prions transmitted efficiently to both Tg(CerPrP) and Tg(CerPrP-L132) mice. Our findings suggest that the elk 132 polymorphism controls prion susceptibility at the level of prion strain selection and that cervid PrP L132 severely restricts propagation of CWD prions. We speculate that the L132 polymorphism results in less efficient conversion of CerPrPC-L132 by CWD prions, an effect that is overcome by the SSBP/1 strain. Our studies show the accumulation of subclinical levels of CerPrPSc in aged asymptomatic CWD-inoculated Tg(CerPrP-L132) mice and also suggests the establishment of a latent infection state in apparently healthy elk expressing this seemingly protective allele.

Chronic Wasting Disease

Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed ungulates (deer, elk, and moose) in North America and South Korea. First described by the late E.S. Williams and colleagues in northern Colorado and southern Wyoming in the 1970s, CWD has increased tremendously both in numerical and geographical distribution, reaching prevalence rates as high as 50% in free-ranging and >90% in captive deer herds in certain areas of USA and Canada. CWD is certainly the most contagious prion infection, with significant horizontal transmission of infectious prions by, e.g., urine, feces, and saliva. Dissemination and persistence of infectivity in the environment combined with the appearance in wild-living and migrating animals make CWD presently uncontrollable, and pose extreme challenges to wild-life disease management. Whereas CWD is extremely transmissible among cervids, its trans-species transmission seems to be restricted, although the possible involvement of rodent and carnivore species in environmental transmission has not been fully evaluated. Whether or not CWD has zoonotic potential as had Bovine spongiform encephalopathy (BSE) has yet to be answered. Of note, variant Creutzfeldt-Jakob disease (vCJD) was only detected because clinical presentation and age of patients were significantly different from classical CJD. Along with further understanding of the molecular biology and pathology of CWD, its transmissibility and species restrictions and development of methods for preclinical diagnosis and intervention will be crucial for effective containment of this highly contagious prion disease.

Oral Transmission of Chronic Wasting Disease in Captive Shira's Moose

Three captive Shiras moose (Alces alces shirasi) were orally inoculated with a single dose (5 g) of whole-brain homogenate prepared from chronic wasting disease (CWD)–affected mule deer (Odocoileus hemionus). All moose died of causes thought to be other than CWD. Histologic examination of one female moose dying 465 days postinoculation revealed spongiform change in the neuropil, typical of transmissible spongiform encephalopathy. Immunohistochemistry staining for the proteinase-resistant isoform of the prion protein was observed in multiple lymphoid and nervous tissues. Western blot and enzyme-linked immunosorbent assays provided additional confirmation of CWD. These results represent the first report of experimental CWD in moose.

DNA sequence homology relationships among six lepidopteran nuclear polyhedrosis viruses.

Summary The DNA sequence homology relationships among six lepidopteran nuclear polyhedrosis viruses (NPVs) have been explored by hybridization of 32P-labelled NPV DNAs to Southern blots of restriction endonuclease-digested NPV DNA. The Autographa californica NPV (AcMNPV) shows extensive DNA sequence homology throughout the entire genome with the Rachiplusia ou NPV (RoMNPV). Both the Orgyia pseudotsugata NPV (OpMNPV) and the Porthetria dispar NPV (PdMNPV) share homologous regions, equivalent to 1% of the DNA genome, with AcMNPV and RoMNPV. This homology is localized in two regions on the AcMNPV physical map although other regions are also weakly homologous. Approx. 1% of the DNA of OpMNPV and PdMNPV show sequence homology with each other; the homology is primarily localized in two to four regions of the genomes. Heliothis zea NPV (HzSNPV) and Trichoplusia ni NPV (TnSNPV) share less than 0.2% sequence homology with the MNPVs and share less than 0.2% sequence homology with each other.

Levels of abnormal prion protein in deer and elk with chronic wasting disease.

Infected deer may pose a higher risk than elk for disease transmission.

Efficient Insertion of Odd-numbered Transmembrane Segments of the Tetracycline Resistance Protein Requires Even-numbered Segments

Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly “helical hairpins,” are necessary for efficient membrane insertion of the tetracycline resistance protein.