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Published Date: 2012-04-07 16:36:23
Subject: PRO/AH/EDR> Prion Disease update 2012 (04)
Archive Number: 20120407.1093352

PRION DISEASE UPDATE 2012 (04)
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A ProMED-mail post
http://www.promedmail.org
ProMED-mail is a program of the
International Society for Infectious Diseases
http://www.isid.org

In this update:

[1] UK CJD surveillance as of 2 Apr 2012
[2] Atypical BSE - Switzerland
[3] Mutation and Selection of prions

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[1] UK: National CJD Surveillance Unit -- monthly statistics
Date: Mon 2 Apr 2012
Source: UK National CJD Surveillance Unit, monthly statistics [edited]
http://www.cjd.ed.ac.uk/figures.htm

vCJD statistics
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The number of deaths due to definite or probable vCJD as of Mon 2 Apr 2012 remains 176. No definite/probable patient remains alive, so the total number of definite or probable vCJD cases (dead and alive) remains 176.

The overall picture remains consistent with the view that the vCJD outbreak in the UK is in decline, albeit now with a pronounced tail. The 1st cases were observed in 1995, and the peak number of deaths was 28 in the year 2000, followed by 20 in 2001, 17 in 2002, 18 in 2003, 9 in 2004, 5 in 2005, 5 in 2006, 5 in 2007, one in 2008, 3 in 2009, 3 in 2010, 5 in 2011, and none so far in 2012

Totals for all types of CJD cases in the UK in the year 2012
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During 2012 so far [as of 2 Apr 2012], there have been 27 referrals, 18 fatal cases of sporadic CJD, none of GSS, one cases of familial CJD, none cases of vCJD, and none cases of iatrogenic CJD.

Since records began in 1990, there have been 2912 referrals, 1322 fatal cases of sporadic CJD, 176 cases of vCJD, 95 cases of familial CJD, 68 cases of iatrogenic CJD, and 45 cases of GSS.

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[2] Atypical BSE -- Switzerland
Date: Mon 12 Mar 2012
Source: World Radio Switzerland [edited]
http://worldradio.ch/wrs/news/wrsnews/mad-cow-disease-strain-in-switzerland.shtml?29563

Mad cow strain found in Switzerland
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The Federal Veterinary Office has confirmed one case of mad cow disease in the canton of Bern. The cow was slaughtered last month. The case was discovered as part of a monitoring programme implemented by the Swiss authorities. According to vets the cow was not infected with a classical case of BSE, but rather an atypical strain. They say that means the disease was not triggered by a certain animal feed outlawed in 2005. The cow was imported to Switzerland in 2006. Until this case, no cows with BSE have been found in Switzerland since 2007. The Federal Veterinary Office confirms it may have found another one of these atypical cases but stresses this strain of disease is rare. BSE was first diagnosed in 1990, and since then 467 cows were diagnosed with it in Switzerland. No cases of the human strain have ever been detected here.

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Communicated by:
Terry S. Singeltary Sr.

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[3 Mutation and selection of prions
Date: Thu 29 Mar 2012
Source: Prions. PLoS Pathog 8(3): e1002582. doi:10.1371/journal.ppat.1002582 (2012)
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002582

Mutation and Selection of Prions
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(By Charles Weissmann, Department of Infectology, Scripps Florida, Jupiter, Florida, United States of America)

Introduction

Prion diseases, or transmissible spongiform encephalopathies (TSEs), occur naturally in several species, including humans, cattle, sheep, and deer, and can be transmitted experimentally to many others. Typically, incubation times are relatively long, extending to 40 years or more in humans; however, after appearance of clinical symptoms, death mostly ensues within less than a year, as a consequence of neurodegeneration accompanied by accumulation of abnormal conformers of the host protein PrP. Natural transmission usually occurs perorally, as exemplified by the kuru epidemic among the Fore people of Papua New Guinea, attributed to cannibalistic practices; the bovine spongiform encephalopathy (BSE) epizootic in the United Kingdom at the end of last century, caused by feeding of contaminated meat-and-bone meal to cattle; or the current epizootic of chronic wasting disease afflicting cervids in 19 states of the United States. Transmission of BSE prions to young humans gave rise to a limited outbreak of a novel illness, variant Creutzfeldt-Jakob disease (vCJD), almost exclusively in the UK. Sporadic cases of prion disease occur at very low frequency in human populations (sCJD) and in cattle herds (atypical BSE), and are attributed to spontaneous generation of prions in the affected individuals. Finally, familial forms of human prion disease are linked to a variety of different, dominant mutations in the PRNP gene, and while afflicted families are rare, penetrance is very high.

Replication of Prions

[To follow this section interested readers need to access the original text to view the diagram that accompany the original text. The text is included here because it is relevant to the reasoning underlying the arguments developed relating to design of antiprion drugs. - Mod.CP]

Prions consist mainly, if not solely, of PrPSc (scrapie prion protein), aggregated conformers of the GPI-linked host glycoprotein PrPC (cellular prion protein). PrPSc propagates by converting PrPC to a replica of itself (Figure 1A). PrPC may exist as an equilibrium mixture of conformers, some of which can accrete to PrPSc “seeds” at a critical rate [1], [2]. This seeding model is supported by the protein misfolding cyclic amplification (PMCA) reaction, in which brain homogenate, as a source of PrPC, is spiked with a seed of infected brain homogenate and subjected to multiple cycles of sonication and incubation, ultimately yielding a vast excess of infectious prions [3]. Infectious prions arose spontaneously in PMCA-mediated, cell-free reactions from defined components [4], in particular from recombinant PrP, a phospholipid, and poly(A) or poly(dT) [5], definitively laying to rest the perennial proposal that the infectious agent is a virus-like entity [6]. Prion-like, seeded conversion into an aggregated state has been proposed for several mammalian proteins such as Abeta, alpha-synuclein, or serum amyloid, which underlie protein misfolding diseases, and for several fungal, in particular yeast, proteins. [Figure 1 -- Propagation, mutation, and selection of prions in cultured cells, and associated text here]

Prion Strains

Prion populations may present as distinct strains: these differ in their phenotypic properties but are associated with PrPSc having the same amino acid sequence. Murine prion strains, originally characterized by the incubation time and the neuropathology they elicit, can be propagated indefinitely in mice homozygous for the PrP gene. Many “classical” strains currently propagated in mice and hamsters, such as 79A, 22L, and ME7, originated from scrapie-infected sheep or goats [7] and were cloned by endpoint dilution in mice.

Strain-specific properties of the prion are believed to be enciphered in the conformation of the cognate PrPSc [8], and indeed, distinct strains are often associated with PrPSc species differing in physicochemical properties. Experiments with yeast prion strains have shown that specific conformations can be propagated in vitro by pure, unglycosylated proteins [9]. Nonetheless, in view of the vast multiplicity of mammalian prion strains and their tropism for particular cell lines, it is conceivable that post translational modifications of PrP, such as glycosylation or association with some cellular components, might favor certain PrP conformations and hence account for cell-specific preferential propagation of particular strains.

The Species Barrier

In general, there is a considerable barrier to transmission of prions between animal species, in that even massive intracerebral trans-species inoculation causes disease at only low frequency (low “attack rate”) and/or only after very long incubation times, if at all. This barrier was abolished in some instances by replacing the PrP gene of the recipient by its counterpart from the donor, but clearly factors other than mismatch of PrP sequences contribute to the incompatibility. Importantly, when prions are serially transmitted from the initial trans-species recipients to further animals of the same species, attack rates increase and incubation times decrease, reflecting “adaptation” to the new host [10]. “Adaptation” implies as a first step accretion of PrPC from the recipient host to the incoming PrPSc seed, which may be a very inefficient process if the amino acid sequence of the host PrP entrains a spectrum of conformations that are poorly compatible with that of the seed. Efficient propagation may only be enabled when the conformation of the seed changes, perhaps initially at the “growing end” [11], resulting in a “mutation” at the conformational level. Subsequently, prions may evolve to replicate more rapidly in the new host, accounting for the striking reduction of their incubation period as they are sequentially transferred within the new species.

In some instances, transfer of a prion strain from one species to another, followed by several passages in the original host species, led to emergence of mutant strains. For example, when cloned murine 139A prions were passaged through hamster and subsequently passaged repeatedly in mouse a new strain, 139A-H2M, was recovered; however, ME7 subjected to the same procedure remained apparently unchanged [12].

Evolution of Prions

The finding that many murine prion strains replicated efficiently in selected murine cell lines created important new experimental opportunities. In particular, the slow, expensive, and imprecise mouse-based bioassay for murine prions could be replaced by a humane, rapid, and precise cell-based procedure, the standard scrapie cell assay (SSCA) [13]. The differential susceptibility of cell lines to various prion strains provided the basis of the cell panel assay (CPA), which rapidly differentiates between various prion strains on the basis of their cell tropism and their susceptibility to various drugs, such as swainsonine or kifunensine [14], [15].

The CPA [cell panel assay] revealed that serial propagation of brain-derived 22L prions in PK1 cells led to progressive change in their properties; while initially able to propagate in R33 cells (“R33 competent”) or in PK1 cells in the presence of swainsonine (“swainsonine resistant”), the prions gradually became completely R33 incompetent and swainsonine-sensitive [Figure 1B and related text here]. When these “cell-adapted” prions were returned to mouse brain, they gradually re-acquired their former properties and became indistinguishable from the original 22L strain [16]. Along similar lines, when swainsonine-sensitive prions were propagated in PK1 cells in the presence of the drug, a swainsonine-resistant prion population emerged after a few passages, documenting adaptation to the new environment. After withdrawal of the drug, further propagation for several splits again yielded drug-sensitive prions [16]. These findings suggested that prion populations constitute so-called quasispecies [17], that is, they are composed of a variety of conformational variants, each present at a low level; when the environment changes, the most efficiently replicating variant becomes the predominant component of the population, which then constitutes a distinct sub-strain [1], [16], [18]. Indeed, PK1 cell-adapted 22L populations were found to contain about 0.5% swainsonine-resistant variants before ever being exposed to the drug [16]. Because the 22L prions used in these experiments had been cloned by endpoint dilution years earlier, heterogeneity must have arisen by a mutation-like process in the interim. Mutations in the case of prions represent conformational changes and not modifications at the level of the protein sequence, because PrP is encoded by the host genome and the mutation is inherent to the proteinaceous particle. To verify whether heterogeneity of prion populations came about by mutation, swainsonine-sensitive prions were cloned by endpoint dilution into PK1 cells, and the infected cells were propagated serially for up to 100 doublings and challenged with swainsonine to determine at which stage the prion populations acquired the capacity for becoming resistant to the drug. Early after cloning the populations were incapable of doing so, but most clones developed this capability after 31–86 doublings [Figure 1C and related text here]. However, at least one of nine populations failed to do so even after 116 doublings, suggesting that the prions were heterogeneous in regard to their ability to develop swainsonine resistance [11], [16]. Acquisition of drug resistance by murine prions has also been reported by Ghaemmaghami et al. [19] and by yeast prions by Shorter [20]. Most if not all of the prion variants, or sub-strains, described above were reversible, suggesting that the underlying conformations were readily interconvertible. In contrast, strains are very stable, at least as long as they are propagated in the same species. As shown in Figure 2 [refer to original text], this suggests a low activation energy barrier between sub-strains, readily surmountable under physiological conditions, while high activation energy barriers prevent conversion between strains.

Sub-strains are depicted as distinguishable collectives of prions that can interconvert readily because they are separated by activation energy barriers that can be overcome in a particular environment under physiological conditions, while strains are separated by high energy barriers. When the environment changes, for example when prions are transferred between distinct tissues, different sub-strains may be favored.

Concluding thoughts

The finding that prions can acquire resistance to drugs has significant implications for drug design. Drugs targeted to PrPSc may have to be administered in combination, as in the case of viruses, in particular human immunodeficiency virus. Alternatively, drugs could be targeted to bind and stabilize PrPC or, in view of the finding that ablation of PrPC, at least in animals, is not detrimental to health [21], [22], to suppress its synthesis. At present no therapeutically useful drugs are available, but deepening insight into the molecular biology of prions may pave the way to novel approaches.

References

1. Collinge J, Clarke AR (2007) A general model of prion strains and their pathogenicity. Science 318: 930–936. Find this article online
2. Weissmann C (2009) Thoughts on mammalian prion strains. Folia Neuropathol 47: 104–113. Find this article online
3. Castilla J, Saa P, Hetz C, Soto C (2005) In vitro generation of infectious scrapie prions. Cell 121: 195–206. Find this article online
4. Deleault NR, Harris BT, Rees JR, Supattapone S (2007) Formation of native prions from minimal components in vitro. Proc Natl Acad Sci U S A 104: 9741–9746. Find this article online
5. Wang F, Zhang Z, Wang X, Li J, Zha L, et al. (2012) Genetic informational RNA is not required for recombinant prion infectivity. J Virol 86: 1874–1876. Find this article online
6. Manuelidis L (2010) Transmissible encephalopathy agents: virulence, geography and clockwork. Virulence 1: 101–104. Find this article online
7. Dickinson AG (1976) Scrapie in sheep and goats. In: Kimberlin RH, editor. Slow virus diseases of animals and man. Amsterdam: Elsevier/North Holland. pp. 209–241.
8. Prusiner SB (1991) Molecular biology of prion diseases. Science 252: 1515–1522. Find this article online
9. Tanaka M, Collins SR, Toyama BH, Weissman JS (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442: 585–589. Find this article online
10. Kimberlin RH, Walker C (1977) Characteristics of a short incubation model of scrapie in the golden hamster. J Gen Virol 34: 295–304. Find this article online
11. Li J, Mahal SP, Demczyk CA, Weissmann C (2011) Mutability of prions. EMBO Rep 14: 191. Find this article online
12. Kimberlin RH, Walker CA, Fraser H (1989) The genomic identity of different strains of mouse scrapie is expressed in hamsters and preserved on reisolation in mice. J Gen Virol 70: 2017–2025. Find this article online
13. Klohn PC, Stoltze L, Flechsig E, Enari M, Weissmann C (2003) A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proc Natl Acad Sci U S A 100: 11666–11671. Find this article online
14. Mahal SP, Baker CA, Demczyk CA, Smith EW, Julius C, et al. (2007) Prion strain discrimination in cell culture: the cell panel assay. Proc Natl Acad Sci U S A 104: 20908–20913. Find this article online
15. Browning S, Baker CA, Smith E, Mahal SP, Herva ME, et al. (2011) Abrogation of complex glycosylation by Swainsonine results in strain- and cell-specific inhibition of prion replication. J Biol Chem 19: 19. Find this article online
16. Li J, Browning S, Mahal SP, Oelschlegel AM, Weissmann C (2010) Darwinian evolution of prions in cell culture. Science 327: 869–872. Find this article online
17. Eigen M (1971) Selforganization of matter and the evolution of biological macromolecules. Naturwissenschaften 58: 465–523. Find this article online
18. Weissmann C, Li J, Mahal SP, Browning S (2011) Prions on the move. EMBO Rep 12: 1109–1117. Find this article online
19. Ghaemmaghami S, Ahn M, Lessard P, Giles K, Legname G, et al. (2009) Continuous quinacrine treatment results in the formation of drug-resistant prions. PLoS Pathog 5: e1000673. doi:10.1371/journal.ppat.1000673.
20. Shorter J (2010) Emergence and natural selection of drug-resistant prions. Mol Biosyst 6: 1115–1130. Find this article online
21. Büeler H, Aguzzi A, Sailer A, Greiner RA, Autenried P, et al. (1993) Mice devoid of PrP are resistant to scrapie. Cell 73: 1339–1347. Find this article online
22. Richt JA, Kasinathan P, Hamir AN, Castilla J, Sathiyaseelan T, et al. (2007) Production of cattle lacking prion protein. Nat Biotechnol 25: 132–138. Find this article online.

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[(1) In contrast to 2011 when there were 5 human cases of vCJD confirmed in the UK, there have been no cases during the first quarter of 2012.

(2) The significance of the typical BSE strain detected in Switzerland remains to be determined.

(3) The PLoS paper on mutation and selection of prions has been reproduced in full since the finding that prions can acquire resistance to drugs has significant implications for drug design. Drugs targeted to PrPSc may have to be administered in combination, or targeted to bind and stabilise PrPC. Or, in view of the finding that ablation of PrPC, at least in animals, is not detrimental to health, drugs designed to suppress its synthesis might be efficacious. At present no therapeutically useful drugs are available, but deepening insight into the molecular biology of prions may pave the way to novel approaches. - Mod.CP]

See Also