Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers

Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers

Kristi M. Green1¤a, Joaquín Castilla2¤b, Tanya S. Seward3, Dana L. Napier3, Jean E. Jewell4, Claudio Soto2, Glenn C. Telling1,3,5*

1 Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America, 2 Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America, 3 Sanders Brown Center on Aging, University of Kentucky, Lexington, Kentucky, United States of America, 4 Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming, United States of America, 5 Department of Neurology, University of Kentucky, Lexington, Kentucky, United States of America

Abstract 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.

Author Summary Prions are unique pathogens that result from conversion of a normal host-encoded prion protein, PrPC, into a self-propagating, disease-associated conformation, referred to as PrPSc. An important aspect of prion diseases is their transmissibility, frequently as epidemics. The contagious transmission of chronic wasting disease (CWD) of deer and elk is of particular concern. The elements governing prion transmission between species, including the influence of agent strain properties, remain enigmatic, in large part because of considerable difficulties associated with experimental manipulation of prions. The aim of this study was to evaluate the feasibility of expediting studies of intra- and inter-species prion transmission. We made use of transgenic mice as a source of deer prion protein for the production of CWD prions by protein misfolding cyclic amplification (PMCA). Characterization of infectivity in the same transgenic mice demonstrated that PMCA results in the efficient amplification of CWD prions with unaltered strain characteristics. Also, whereas adaptation of mouse prions to form novel cervid prions required several hundred days and subsequent stabilization in transgenic mice, we show that PMCA rapidly abrogated this inter-species transmission barrier. Our results indicate that PMCA can be used to replace the process of prion strain adaptation and selection occurring in vivo.

Citation: Green KM, Castilla J, Seward TS, Napier DL, Jewell JE, et al. (2008) Accelerated High Fidelity Prion Amplification Within and Across Prion Species Barriers. PLoS Pathog 4(8): e1000139. doi:10.1371/journal.ppat.1000139

Editor: Neil Mabbott, University of Edinburgh, United Kingdom

Received: February 19, 2008; Accepted: August 1, 2008; Published: August 29, 2008

Copyright: © 2008 Green et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by grants from the US Public Health Service, namely 2RO1 NS040334-04 from the National Institute of Neurological Disorders and Stroke, and N01-AI-25491 from the National Institute of Allergy and Infectious Diseases. K.M.G. was supported in part by funds from the T32 AI49795 Training Program in Microbial Pathogenesis. None of the sponsors or funders played any role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, or approval of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: gtell2@uky.edu

¤a Current address: Department of Neurology, University of Texas Medical Branch, Galveston, Texas, United States of America

¤b Current address: Department of Infectology, Scripps Research Institute, Jupiter, Florida, United States of America

Introduction

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Our studies convincingly show that PMCA of murine RML prions using Tg(CerPrP)1536+/- brain homogenate generates a novel strain of cervid-adapted prions with properties distinct from either naturally occurring or PMCA-generated CWD prions. It currently is less clear whether the PrPSc structures and strain properties of amplified and in vivo derived prions are equivalent. Direct comparisons of the strain properties of PMCA Cer/RML cervid prions and in vivo-adapted strains are complicated by our observations that in vivo adaptation gives rise to individual isolates with different strain-related properties, at least as judged by histoblot and immunohistochemical profiles of PrPSc. While certain strain-related attributes, including comparably rapid prion incubation times, and similar denaturation profiles of CerPrPSc after infection, suggest shared biological properties between Cer/RML-4827 and PMCA Cer/RML prions, other differences, including targeting of cerebral PrPSc deposition of Tg(CerPrP)1536+/- mice infected with Cer/RML-4827 and PMCA Cer/RML prions, point to divergent strain properties and thus would rather argue for different strains. Whether inter-species PMCA-mediated prion adaptation also results in the generation of multiple and distinct prion strains remains to be determined, but our limited analyses consisting of uniform histoblot profiles, reproducible onsets of disease, and similar conformation stabilities of CerPrPSc in individual infected mice may indicate that PMCA selectively and stably propagates distinct strains following abrogation of a species barrier.

Finally, we note that under certain conditions, PMCA may result in the spontaneous formation of PK-resistant PrP species [40] and de novo generated infectivity under conditions that do not involve seeding with infectious prions [50]. While it would be of considerable interest to determine the biological properties of spontaneously-produced cervid prions by PMCA of CerPrPC, we feel that the possibility of spontaneous generation of infectivity in the context of the current studies is remote. The experiments of Deleault and co-workers using purified PrPC plus poly(A) RNA, indicated that spontaneous generation of PrPSc was a stochastic and relatively infrequent event, estimated at <1>400 d (Soto and co-workers, unpublished observations). For these reasons we feel that the generation of PrPSc and cervid prions reported in the present study, when normal brain homogenate from Tg(CerPrP)1536+/- mice was mixed with prion seeds, is unlikely to be influenced by spontaneous, de novo generated infectivity.

Materials and Methods

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http://www.plospathogens.org/article/info:doi%2F10.1371%2Fjournal.ppat.1000139;jsessionid=392A2E2CC0411D8DC0382922D7D4A163




Journal of Virology, April 2007, p. 4305-4314, Vol. 81, No. 8 0022-538X/07/$08.00+0 doi:10.1128/JVI.02474-06 Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transmission and Adaptation of Chronic Wasting Disease to Hamsters and Transgenic Mice: Evidence for Strains

Gregory J. Raymond,1 Lynne D. Raymond,1 Kimberly D. Meade-White,1 Andrew G. Hughson,1 Cynthia Favara,1 Donald Gardner,2 Elizabeth S. Williams,3, Michael W. Miller,4 Richard E. Race,1* and Byron Caughey1* Laboratory of Persistent Viral Diseases,1 Rocky Mountain Veterinary Branch, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, Montana 59840,2 Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming 82070,3 Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, Colorado 80526-20974

Received 9 November 2006/ Accepted 23 January 2007

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

-------------------------------------------------------------------------------- * Corresponding author. Mailing address: Rocky Mountain Labs, 903 S. 4th St., Hamilton, MT 59840. Phone for Byron Caughey: (406) 363-9264. Fax: (406) 363-9286. E-mail: bcaughey@nih.gov. Phone for Richard Race: (406) 363-9358. Fax: (406) 363-9286. E-mail: rrace@nih.gov

Published ahead of print on 7 February 2007.


http://jvi.asm.org/cgi/content/abstract/81/8/4305



Chronic Wasting Disease CWD


http://chronic-wasting-disease.blogspot.com/




Infectious, test tube-produced prions can jump the 'species barrier'

Published: Thursday, September 4, 2008 - 11:28 in Biology & Nature Learn more about: bovine spongiform encephalopathy infectious microorganisms infectious proteins prion strains species barrier test tube Researchers have shown that they can create entirely new strains of infectious proteins known as prions in the laboratory by simply mixing infectious prions from one species with the normal prion proteins of another species. The findings are reported in the September 5th issue of the journal Cell, a Cell Press publication. Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are infectious neurodegenerative diseases affecting the brain of several species of mammals including humans. Creutzfeldt-Jakob disease (CJD) is the most common prion disease in humans, along with scrapie in sheep, bovine spongiform encephalopathy (BSE, aka mad cow) in cattle, and chronic wasting disease (CWD) in deer and other cervids.

Unlike conventional infectious microorganisms, the infectious agent in the case of prion diseases consists exclusively of a misfolded form of the prion protein, earlier studies showed.

The researchers now find that prion strains produced by combining normal hamster proteins with infectious mouse proteins can infect hamsters and vice versa. Although they are both rodents, prions from one of the two species normally don't readily infect the other, a common phenomenon amongst prions known as a species barrier, the researchers explained.

The novel prions they produced not only look different, but they also produce symptoms in the animals that differ from any known strain found in nature, they report.

" We are forcing the system by putting everything together, but this suggests that the variety of possible prions is really very large," said Claudio Soto of the University of Texas Medical Branch. "We shouldn't be surprised if new barriers are crossed and new prions arise. There is the potential for a large variety of new infectious prions—some of which may have dramatic effects."

"The infectous agent is nothing like what we're used to," Soto said. "It's just a protein with a different shape from the normal protein we all have." Those misfolded and misshapen proteins can spread by causing normal protein to change their shape. Those aberrant forms band together, forming fibrils.

Soto's team recently reported the generation of infectious prions by amplification of prion misfolding in the test tube. In those experiments, they used a technology called protein misfolding cyclic amplification (PMCA) that mimics some of the fundamental steps involved in the replication of infectious prions in living animals, but at an accelerated rate. The method involves placing small quantities of infectious prions with large quantities of the normal protein from the same species together, allowing the infectious form to imprint on the normal form and thereby replicate itself.

Now, they show that the same method can generate new strains when infectious prions from one species are mixed with normal prion proteins from another species. The finding provides conclusive evidence that the imprinting of disease-causing prions on normal forms can overcome species barriers, and doesn't require any other infectious agent.

This new insight has profound implications for public health, according to the researchers.

" One of the scariest medical problems of the last decades has been the emergence of a new and fatal human prion disease--variant CJD--originated by cross-species transmission of BSE from cattle," the researchers said. BSE has also spread to other animals, including exotic cats, other primates and domestic cats, after they ate feed derived from diseased cows.

The new method might provide insight into the risk that other prion diseases could spread from one species to another, Soto said. For instance, scientists don't know whether chronic wasting disease, a condition now on the rise amongst deer in some parts of the U.S., can be transmitted to humans or not.

Test tube studies like this one might help answer that question, and-- in the case that the deer prions can make the leap—such studies may inform scientists about what those prions might look like, he said. By studying any new prion strains created in mice with the human prion protein, scientists might also gain insight into the potential symptoms associated with those diseases.

" The data demonstrate that PMCA is a valuable tool for the investigation of the strength of the barrier between diverse species, its molecular determinants, and the expected features of the new infectious material produced," the researchers concluded. "Finally, our findings suggest that the universe of possible prions is not restricted to those currently known but that likely many unique infectious foldings of the prion protein may be produced and that one of the sources for this is cross-species transmission."

Source: Cell Press



http://esciencenews.com/articles/2008/09/04/infectious.test.tube.produced.prions.can.jump.species.barrier




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