Rodrigo Morales

Karim Abid

Claudio Soto
email: clsoto@utmb.edu

The prion strain phenomenon: Molecular basis and unprecedented features

Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathies. Compelling evidences indicate that prions are composed exclusively by a misfolded form of the prion protein (PrP Sc ) that replicates in the absence of nucleic acids. One of the most challenging problems for the prion hypothesis is the existence of different strains of the infectious agent. Prion strains have been characterized in most of the species. Biochemical characteristics of PrP Sc used to identify each strain include glycosylation profile, electrophoretic mobility, protease resistance, and sedimentation. In vivo, prion strains can be differentiated by the clinical signs, incubation period after inoculation and the vacuolation lesion profiles in the brain of affected animals. Sources of prion strain diversity are the inherent conformational flexibility of the prion protein, the presence of PrP polymorphisms and inter-species transmissibility. The existence of the strain phenomenon is not only a scientific challenge, but it also represents a serious risk for public health. The dynamic nature and inter-relations between strains and the potential for the generation of a very large number of new prion strains is the perfect recipe for the emergence of extremely dangerous new infectious agents.

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I. Introduction

Transmissible Spongiform Encephalopathies (TSEs), also known as prion disorders, are infectious and fatal neurodegenerative diseases affecting humans and other mammals. In humans, TSEs include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), Gertsmann-Straussler-Scheinker Syndrome (GSS) and Kuru [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. In other mammals, bovine spongiform encephalopathy (BSE) is found in cattle, scrapie in sheep and goats and chronic wasting disease (CWD) in elk and deer [1,[START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF]. Although the clinical symptoms vary in distinct diseases, they usually include dementia and/or ataxia with progressive loss of brain function, irreversibly resulting in death [START_REF] Budka | Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)[END_REF]. The hallmark of prion diseases is the misfolding of the prion protein observed in the brain of affected individuals [1]. Misfolded proteins have the intrinsic tendency to form large aggregates and fibrillar structures, that may form amyloid deposits in a similar fashion as observed in Alzheimer's, Parkinson's diseases and many other protein misfolding disorders [START_REF] Soto | Unfolding the role of protein misfolding in neurodegenerative diseases[END_REF].

Although of rare occurrence, prion diseases have drawn considerable attention from the public and led to severe economic and political consequences in Europe and in the United

States. The two main reasons of this impact include the unique nature of the infectious agent and the appearance of a new human disease (vCJD) linked to consumption of cattle meat infected by BSE. At present, it is impossible to estimate accurately the number of upcoming cases of vCJD due to the very long incubation time of the disease in humans [START_REF] Cohen | When did bovine spongiform encephalopathy (BSE) start? Implications on the prediction of a new variant of Creutzfeldt-Jakob disease (nvCJD) epidemic[END_REF][START_REF] Ghani | Estimation of the number of people incubating variant CJD[END_REF][START_REF] Will | Variant Creutzfeldt-Jakob disease[END_REF]. Prion research has been plagued with the discovery of new and heretic scientific findings that have confronted the most solid paradigms in modern biology. The current evidence suggest that an abnormal form of the prion protein (termed PrP Sc ) is the main, and possibly the only, constituent of prion infectious agent [1]. This so called protein-

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only hypothesis [1,[START_REF] Griffith | Self-replication and scrapie[END_REF] proposes that amplification of PrP Sc occurs at expenses of the normal host's version of the prion protein (termed PrP C ). The new generated PrP Sc have new biochemical characteristics compared to PrP C , for example its insolubility, resistance to denaturation, and its partial resistance to protease degradation [START_REF] Cohen | Pathologic conformations of prion proteins[END_REF]. PrP Sc treatment with proteases reveal the protease resistant core of the infectious agent (termed PrP 27-30 according to its molecular weight) [START_REF] Prusiner | Purification and structural studies of a major scrapie prion protein[END_REF].

II. Molecular basis of Prion strains

Among the unique features that have contributed to place the prion field in the spotlight, one of the most interesting is the prion strain phenomenon. It has been observed that animals affected by prion diseases may develop different pathologies and the clinical and biochemical outcomes could be maintained through several passages in rodents models of prion diseases. In analogy to other infectious agents, these variants have been termed strains. A classical definition of strain makes mention to a genetic variant or subtype of the infectious agent responsible for the disease, but this concept, valid in virology, can not be extended to prions. In early days, the strain phenomenon was claimed as one of the strongest evidences against the protein-only hypothesis [START_REF] Chesebro | BSE and prions: uncertainties about the agent[END_REF][START_REF] Soto | The controversial protein-only hypothesis of prion propagation[END_REF]. It was assumed that the different phenotypes found in animals were due to differences in the genetic information contained within the TSE causing agent. However, currently it is widely accepted that the main differences between prion strains arise from alternative conformations of PrP Sc that can be stably and faithfully propagated [START_REF] Bartz | Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy[END_REF][START_REF] Peretz | Strain-specified relative conformational stability of the scrapie prion protein[END_REF].

The first evidence about the existence of prion strains was described in goats affected by scrapie by Pattison and Millson in 1961 [15]. In this report, goats infected by

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the same batch of infectious scrapie agent developed two different clinical phenotypes, termed by the authors "scratching" and "nervous", according to disease's manifestation.

The differences between these infectious agents were alleged to be the consequence of differences in the genetic background of the host. The current evidence supports this hypothesis. Clinical signs could be very useful to differentiate between prion strains [START_REF] Pattison | Scrapie produced experimentally in goats with special reference to the clinical syndrome[END_REF][START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF][START_REF] Dickinson | Scrapie in sheep and goats[END_REF]. Each prion strain has the capability to affect specific brain areas producing differences in clinical signs. In the case of scrapie in sheep and goats, after identification and isolation of the prion protein gene (prnp) several polymorphic differences were recognized when numerous sheep flocks were compared [START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF].

Prion strains can be classified by different parameters. Incubation periods, profile of histological damage and clinical signs are the main in vivo characteristics which can be used to differentiate between prion strains [START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF][START_REF] Bruce | Scrapie strain variation and mutation[END_REF][START_REF] Fraser | Diversity in the neuropathology of scrapie-like diseases in animals[END_REF]. The most commonly used is incubation period which correspond to the time elapsed between experimental inoculation of the infectious agent and clinical onset of the disease. Intra-species inoculation of prions is usually very reproducible [START_REF] Bruce | Scrapie strain variation and mutation[END_REF]. Inoculation of different prion strains preparations usually results in different and reproducible incubation times [START_REF] Bruce | Scrapie strain variation and mutation[END_REF][START_REF] Westaway | Distinct prion proteins in short and long scrapie incubation period mice[END_REF].

Histological studies have also shown substantial differences when animals were inoculated with distinct strains. The differences are mainly on the distribution and characteristics of PrP Sc deposition and the degree of vacuolation in specific brain regions [START_REF] Bruce | Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie[END_REF][START_REF] Bruce | Strain characterization of natural sheep scrapie and comparison with BSE[END_REF][START_REF] Lasmezas | Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy[END_REF][START_REF] Fraser | Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation[END_REF]. In order to quantify this aspect, a well standardized procedure for vacuolization scoring (lesion profile) in mice [START_REF] Fraser | Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation[END_REF] has been described; nine gray matter and three white matter brain areas are analyzed and scored according to the magnitude of the damage.

Using this approach, prion strains having similar incubation times were recognized, such

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as ME7 and 79A [START_REF] Fraser | Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation[END_REF]. In a similar way, PrP Sc accumulation profile has been useful to track the origin of the infectious material, such as in the case of the transmission of BSE into humans, originating vCJD, since both strains produce similar neuropathological signatures [START_REF] Scott | Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans[END_REF][START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF]. The clinical signs are also a characteristic that can be very useful to differentiate strains. For example, in human prion diseases, motor incoordination, dementia, ataxia, depression, and insomnia are just few from a much larger list of clinical symptoms that can appear depending on the strain of the agent [START_REF] Budka | Neuropathological diagnostic criteria for Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)[END_REF]. In other animals, such as the case of hamsters, the clinical features can be diametrically opposed. That is the case of the Drowsy (DY) and Hyper (HY) prion strains [START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF]. Unfortunately, clinical signs can not always be applied to differentiate and classify prion strains. In mouse for example, several prion strains have the same rough signs, which include ataxia, rough coat, and hunch [START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF][START_REF] Dickinson | Identification of a gene which controls the incubation period of some strains of scrapie agent in mice[END_REF]. However, recent publications using more detailed tests have identified dissimilar behavioral deficits when different prion strains are administrated to mice [START_REF] Dell'omo | Early behavioural changes in mice infected with BSE and scrapie: automated home cage monitoring reveals prion strain differences[END_REF]. Since different brain lesion patterns appear to be responsible for the variation in clinical signs, behavioral studies could give us more specific information about the type of brain damage produced by different prion isolates [START_REF] Dell'omo | Early behavioural changes in mice infected with BSE and scrapie: automated home cage monitoring reveals prion strain differences[END_REF][START_REF] Cunningham | Neuropathologically distinct prion strains give rise to similar temporal profiles of behavioral deficits[END_REF].

In addition to the in vivo differences, each prion strain has a particular group of biochemical characteristics in the infectious protein that could be specifically associated to them. Among them the most important are electrophoretical mobility after PK digestion [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF][START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF], glycosylation pattern [START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Khalili-Shirazi | PrP glycoforms are associated in a strain-specific ratio in native PrPSc[END_REF], extent of PK resistance [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF],

sedimentation [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF] and resistance to denaturation by chaotropic agents [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF][START_REF] Safar | Eight prion strains have PrP(Sc) molecules with different conformations[END_REF]. Recently, differences in the binding affinity for copper among strains have been described [START_REF] Wadsworth | Strain-specific prion-protein conformation determined by metal ions[END_REF]. As illustrated in figure 1, the biochemical features of PrP Sc in various forms of CJD are

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The western blot profile of different sources of human PrP Sc shows diversity in terms of glycosylation pattern and electrophoretical mobility after proteinase K (PK) digestion [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF][START_REF] Collinge | Molecular analysis of prion strain variation and the aetiology of 'new variant[END_REF][START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF]. Fourier Transform Infrared Spectroscopy (FTIR) studies involving different prion strains [START_REF] Safar | Eight prion strains have PrP(Sc) molecules with different conformations[END_REF][START_REF] Caughey | Strain-dependent differences in betasheet conformations of abnormal prion protein[END_REF][START_REF] Aucouturier | Biochemical and conformational variability of human prion strains in sporadic Creutzfeldt-Jakob disease[END_REF], conformation dependent immunoassays [START_REF] Safar | Eight prion strains have PrP(Sc) molecules with different conformations[END_REF][START_REF] Bellon | Improved conformation-dependent immunoassay: suitability for human prion detection with enhanced sensitivity[END_REF] and atomic force microscopy of synthetic prion protein polymers [START_REF] Jones | Fibril conformation as the basis of species-and strain-dependent seeding specificity of mammalian prion amyloids[END_REF] confirm the hypothesis that differences between prion strains lies in the diversity of structures that PrP Sc can acquire. However, the definitive proof for the structural nature of the differences between prion strains is still missing.

III. Species barrier and generation of new prion strains

The principal source of strain diversity arise from interspecies infection [START_REF] Bruce | Strain characterization of natural sheep scrapie and comparison with BSE[END_REF][START_REF] Bruce | Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier[END_REF][START_REF] Fraser | Transmission of bovine spongiform encephalopathy and scrapie to mice[END_REF][START_REF] Muramoto | Successful transmission of Creutzfeldt-Jakob disease from human to mouse verified by prion protein accumulation in mouse brains[END_REF][START_REF] Hill | Prion strains and species barriers[END_REF]. One of the characteristics of the agent responsible for prion diseases is its ability to infect some species and not others. This phenomenon is known as "species barrier" and is manifested as the prolongation in the incubation periods when prions from one species are used to infect a different one [START_REF] Peretz | A change in the conformation of prions accompanies the emergence of a new prion strain[END_REF][START_REF] Moore | Species barriers in prion diseases--brief review[END_REF]. Differences in the sequence of prion protein could lead to different conformations, explaining both, species barrier and diversity of PrP Sc conformations [START_REF] Jones | Fibril conformation as the basis of species-and strain-dependent seeding specificity of mammalian prion amyloids[END_REF][START_REF] Vanik | Molecular basis of barriers for interspecies transmissibility of mammalian prions[END_REF][START_REF] Prusiner | Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication[END_REF][START_REF] Chen | A journey through the species barrier[END_REF]. In some species, PrP C conformation does not permit conversion by prions coming from other species. A clear example of this is found in rabbit, an animal that has been unable to be infected by various sources of prions. In these cases, it is considered that the species barrier is absolute.

Interspecies prion transmission from cattle to human is probably the most relevant problem in terms of public health [START_REF] Smith | Bovine spongiform encephalopathy (BSE) and its epidemiology[END_REF][START_REF] Collee | BSE: a decade on--Part I[END_REF][START_REF] Collee | BSE: a decade on--Part 2[END_REF]. It is widely accepted that consumption of BSE infected material is the cause of vCJD in humans [START_REF] Bruce | Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent[END_REF][START_REF] Hill | The same prion strain causes vCJD and BSE[END_REF]. Strikingly, vCJD is a very

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different strain of prions than the previously known human strains, arisen sporadically.

Differences between vCJD and sCJD include the clinical manifestation of the disease, the profile of brain damage and the biochemical features of PrP Sc [START_REF] Collinge | Variant Creutzfeldt-Jakob disease[END_REF]. The BSE epidemic in the United Kingdom demonstrated how dangerous prions could be. So far, BSE is the only non-human prion described to be transmissible to humans. Despite the fact that people have consumed for centuries sheep potentially affected by scrapie, no correlation has been found between patients suffering by CJD and sheep consumption. Scrapie transmissibility experiments using transgenic animal models expressing chimeric human/mouse PrP support this assumption [START_REF] Gombojav | Susceptibility of transgenic mice expressing chimeric sheep, bovine and human PrP genes to sheep scrapie[END_REF].

BSE has not only been transmitted to humans. The extensive use of cow-derived material for feeding other animals led to the generation of new diseases in exotic felines such as tiger and cheetah, non human primates, and domestic cats [START_REF] Collee | BSE: a decade on--Part I[END_REF][START_REF] Kirkwood | Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles[END_REF][START_REF] Pearson | Feline spongiform encephalopathy: fibril and PrP studies[END_REF][START_REF] Taylor | Bovine spongiform encephalopathy: the causal role of ruminant-derived protein in cattle diets[END_REF][START_REF] Bons | Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents[END_REF]. As it was mentioned before, the transmission of BSE into these different species could create many new prion strains, each one of them with particular biological and biochemical characteristics and thus a potentially new hazard for human health. Successful transmission of BSE in pigs has been described [START_REF] Ryder | The neuropathology of experimental bovine spongiform encephalopathy in the pig[END_REF][START_REF] Wells | Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs[END_REF] and also in transgenic mice expressing pig PrP (PoPrP) [START_REF] Castilla | Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein[END_REF]. Porcine derivates are widely consumed and the hypothetic case of "mad pigs" could increase the events of zoonotic transmission of prions to humans. Fortunately, transmission of BSE to pigs is possible only in very drastic conditions, not likely to be occurring naturally [START_REF] Wells | Studies of the transmissibility of the agent of bovine spongiform encephalopathy to pigs[END_REF][START_REF] Castilla | Subclinical bovine spongiform encephalopathy infection in transgenic mice expressing porcine prion protein[END_REF]. More frightening is perhaps the possibility that BSE has been passed into sheep and goats. Studies have already shown that this transmission is possible and actually relatively easy and worrisomely produces a disease clinically similar to scrapie [START_REF] Foster | Transmission of bovine spongiform encephalopathy to sheep and goats[END_REF]. The cattle origin of this

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new scrapie makes possible that the new strain may be transmissible to humans.

Transmission experiments of BSE infected sheep brain homogenate into human transgenic animal models are currently ongoing in several laboratories. It is very important to note that all materials generated by transmission of BSE in experimental and natural cases show similar biochemical behavior compared to the original inoculum [START_REF] Stack | Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies[END_REF],

suggesting that all these new generated infectious agents could potentially be hazardous for humans. The origin of BSE is still a mystery. Abundant evidence supports the hypothesis that BSE was produced by cattle feeding with scrapie derivated material [START_REF] Wilesmith | Bovine spongiform encephalopathy: epidemiological studies[END_REF][START_REF] Wilesmith | Bovine spongiform encephalopathy: epidemiological studies on the origin[END_REF], indicating that bovine PrP Sc might be a "conformational intermediary" between ovine PrP Sc and human PrP C .

There is currently no mean to predict which will be the conformation of a newly generated strain and how this new PrP Sc conformation could affect other species. One interesting new prion disease is CWD, a disease affecting farm and wild species of cervids [START_REF] Sigurdson | Other animal prion diseases[END_REF][START_REF] Williams | Chronic wasting disease[END_REF]. The origin of CWD and its potential to transmit to humans are currently unknown. This is worrisome, considering that CWD has became endemic in some parts of USA and the number of cases continues to increase [START_REF] Williams | Chronic wasting disease[END_REF]. It is presumed that a large number of hunters in the US have been in contact or consumed CWD-infected meat [START_REF] Belay | Creutzfeldt-Jakob disease in unusually young patients who consumed venison[END_REF].

CWD transmissibility studies have been performed in many species in order to predict how this disease could be spread by consumption of CWD meat [START_REF] Marsh | Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus)[END_REF][START_REF] Hamir | Preliminary findings on the experimental transmission of chronic wasting disease agent of mule deer to cattle[END_REF][START_REF] Hamir | Experimental transmission of chronic wasting disease agent from mule deer to cattle by the intracerebral route[END_REF]. In these studies, a special attention has been done to scavenging animals [START_REF] Hamir | Experimental inoculation of scrapie and chronic wasting disease agents in raccoons (Procyon lotor)[END_REF], which are presumed to be exposed to high concentration of cervid prions, resulting in the putative generation of many new forms of TSEs. Fortunately negative results were obtained in one experiment done in raccoons infected with CWD [START_REF] Hamir | Experimental inoculation of scrapie and chronic wasting disease agents in raccoons (Procyon lotor)[END_REF]. Transmission of CWD to humans cannot be

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ruled out at present and a similar infective episode to BSE involving CWD could result in catastrophic events, spreading the disease in a very dangerous way through the human population. No clinical evidence linking CWD exposed humans and CJD patients have been found [START_REF] Belay | Creutzfeldt-Jakob disease in unusually young patients who consumed venison[END_REF], but experimental inoculation of CWD prions into squirrel monkeys propagated the disease [START_REF] Marsh | Interspecies Transmission of Chronic Wasting Disease Prions to Squirrel Monkeys (Saimiri sciureus)[END_REF]. It is important to mention that the species barrier between humans and cervids appears to be greater than with cattle, as judged by experiments with transgenic mice models [START_REF] Kong | Chronic wasting disease of elk: transmissibility to humans examined by transgenic mouse models[END_REF]. Finally, it is important to be aware about CWD transmissibility to other species in which a "conformational intermediary" could be formed, facilitating human infection.

IV. Use of experimental animals to study prion strains

Probably the best way to study the strain phenomenon is to use experimental animals for the generation of diverse strains through inoculation with prion infectious material coming from different species. Among the experimental models, perhaps mouse is the most useful one, in which more than 20 phenotypically distinct strains have been isolated [START_REF] Bruce | Scrapie strain variation and mutation[END_REF]. Many of these strains have their origin in the transmission of different sources of scrapie from goat and sheep, BSE derived material from cattle [START_REF] Bruce | Strain characterization of natural sheep scrapie and comparison with BSE[END_REF][START_REF] Lasmezas | Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy[END_REF][START_REF] Lasmezas | Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein[END_REF] and human sources as sCJD and GSS [START_REF] Muramoto | Successful transmission of Creutzfeldt-Jakob disease from human to mouse verified by prion protein accumulation in mouse brains[END_REF][START_REF] Manuelidis | Transmission of Creutzfeldt-Jakob disease with scrapie-like syndromes to mice[END_REF][START_REF] Tateishi | Experimental transmission of human subacute spongiform encephalopathy to small rodents. IV. Positive transmission from a typical case of Gerstmann-Straussler-Scheinker's disease[END_REF]. Serial passage of infectious prions in one species, with constant biological background is necessary to stabilize and define a prion strain.

PrP Sc obtained from mouse adapted scrapie prion strains such as RML, ME7, 139A and 79A show similar electrophoretical characteristics after PK digestion. All PrP Sc coming from these strains show an electrophoretical mobility of ~21 KDa for the

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unglycosylated band and a similar glycosylation pattern, with the monoglycosilated form as the most abundant [START_REF] Kascsak | Immunological comparison of scrapie-associated fibrils isolated from animals infected with four different scrapie strains[END_REF][START_REF] Rubenstein | Scrapie-infected spleens: analysis of infectivity, scrapieassociated fibrils, and protease-resistant proteins[END_REF][START_REF] Kascsak | Biochemical differences among scrapie-associated fibrils support the biological diversity of scrapie agents[END_REF][START_REF] Legname | Strain-specified characteristics of mouse synthetic prions[END_REF][START_REF] Baron | Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice[END_REF]. Despite the lack of biochemical differences, these strains can be differentiated when inoculated in mice by measuring the incubation time or the profile of brain lesions [START_REF] Fraser | Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation[END_REF][START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF][START_REF] Legname | Strain-specified characteristics of mouse synthetic prions[END_REF][START_REF] Thackray | Subclinical prion disease induced by oral inoculation[END_REF]. Other mouse strains have been generated by inoculation of animals with BSE and sCJD prions, leading to strains termed 301C and Fukuoka, respectively [START_REF] Bruce | Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier[END_REF][START_REF] Fraser | Transmission of bovine spongiform encephalopathy and scrapie to mice[END_REF][START_REF] Tateishi | Transmission of chronic spongiform encephalopathy with kuru plaques from humans to small rodents[END_REF]. The transmission of BSE into mice generated two different phenotypes: one presenting PK resistant isoform of PrP Sc , and another lacking this characteristic [START_REF] Lasmezas | Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein[END_REF]. This phenotype is maintained after two serial passages in mice, but finally only the PK resistance phenotype remains. The presence of a PK sensitive infectious material (termed sPrP Sc ) has been also described in some cases of human prion diseases [START_REF] Collinge | Prion dementia without characteristic pathology[END_REF][START_REF] Medori | Fatal familial insomnia: a second kindred with mutation of prion protein gene at codon 178[END_REF]. At this time, the isolation of a protease sensitive form of infectious prions has not been successful.

The characteristics of mouse strains generated from scrapie or from BSE are quite different. For example, intracerebral inoculation of RML strain into mice present an onset of ~150 days post inoculation (dpi), while 301C preparations cause the disease at ~200 dpi [START_REF] Bruce | Scrapie strain variation and mutation[END_REF][START_REF] Lloyd | Identification and characterization of a novel mouse prion gene allele[END_REF]. Intraperitoneal inoculation of RML and 301C material in the same animals shows a larger difference in the incubation periods: 200 and 300 dpi, respectively [START_REF] Lasmezas | Strain specific and common pathogenic events in murine models of scrapie and bovine spongiform encephalopathy[END_REF][START_REF] Thackray | Subclinical prion disease induced by oral inoculation[END_REF].

There are also differences in the brain affected areas by both mouse adapted prions (Castilla J., Morales R., Saá P., and Soto C.; unpublished data). In addition, it is also possible to find biochemical differences between RML and 301C strains. As mentioned above, PK digestion pattern of RML shows an electrophoretical mobility of ~21 KDa for the unglycosylated band and is rich in the monoglycosilated isoform of prion protein. In contrast, 301C prion strain show a different electrophoretical pattern compared to RML.

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Unglycosilated isoform of BSE adapted mouse strain shows an electrophoretical mobility of ~19 KDa and its glycoform distribution favors the diglycosylated isoform [START_REF] Baron | Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice[END_REF].

How different PrP Sc conformations could induce stable conformational changes in the same host protein is still unknown. Even more interesting is the isolation of different prion strains into the same host after inoculation of PrP Sc from a single species. Probably the most representative experience is the isolation of DY and HY strains after inoculation of the agent associated to transmissible mink encephalopathy (TME) in Syrian hamsters (Fig. 2) [START_REF] Bartz | Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy[END_REF][START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF]. This interspecies transmission of prions presented the expected behavior of the species barrier phenomenon: a long incubation period in the first passage, but shorter incubation periods after inoculation of serial passages from the resulting infectious material into Syrian hamsters. Incubation periods became stable in two different groups with different clinical signs: the first one with an incubation period of ~150 dpi presented lethargy, while a shorter incubation period strain (~60 dpi) presented hyperactivity. These strains were called Drowsy (DY) and Hyper (HY) respectively according to their clinical signs [START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF]. Histhopathological analysis of animal groups infected with both TME hamster adapted agents show differences in the vacuolation distribution among different brain regions [START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF] and also in the PrP Sc deposition areas [START_REF] Bessen | Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy[END_REF] (Fig. 2).

As 301C and RML in mouse, DY and HY present differences in their electrophoretical mobility after PK treatment. The unglycosylated band of DY has a molecular weight of 19 KDa, while HY show the same band at 21 KDa [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF][START_REF] Bessen | Non-genetic propagation of strain-specific properties of scrapie prion protein[END_REF]. This is the most direct evidence that suggested conformational differences between both PrP Sc species. Supporting this assumption, structural differences using Fourier transform

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infrared spectroscopy (FTIR) between both Syrian hamster adapted TME strains were found [START_REF] Caughey | Strain-dependent differences in betasheet conformations of abnormal prion protein[END_REF]. Another biochemical difference found between DY and HY lies in their differential resistance to PK digestion, where DY is the most sensitive to digestion compared to HY [START_REF] Bessen | Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent[END_REF] (Fig. 2). All these biological and biochemical characteristics make DY and HY one of the most intriguing examples of prion strain variation.

V. Polymorphisms and prion strains

Polymorphisms in the prion protein and their effects in the prion strain phenomenon were indirectly described a long time before the prion hypothesis was developed [START_REF] Pattison | Scrapie produced experimentally in goats with special reference to the clinical syndrome[END_REF]. Differences in prion pathology were found and extensively described in sheep and mice [START_REF] Dickinson | Scrapie in sheep and goats[END_REF][START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF][START_REF] Bruce | Scrapie strain variation and mutation[END_REF][START_REF] Fraser | Scrapie in mice. Agent-strain differences in the distribution and intensity of grey matter vacuolation[END_REF]. The drowsy and scratchy phenotypes found in sheep were attributed to polymorphic differences in the infectious agent inoculated in experimental animals [START_REF] Dickinson | Scrapie in sheep and goats[END_REF]. The identification of "scrapie incubation period gene" ( sinc) and its polymorphic differences was a very big hit in the study of prion strains [START_REF] Dickinson | Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent[END_REF]. In mouse, two polymorphic animal groups were originally described: sinc s7 and sinc p7 . Later, it was discovered that the sinc gene was indeed the gene encoding PrP and the polymorphisms resulted in differences in the prion protein at positions 108 and 189 [START_REF] Carp | Genetic control of scrapie: incubation period and plaque formation in I mice[END_REF]. The transmission of infectious agents from sheep, goats and cattle to both mice groups resulted in the emergence of a wide diversity of prion strains [START_REF] Bruce | Scrapie strain variation and mutation[END_REF][START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF]. When incubation periods of mouse adapted prions were stabilized in each group, new generated infectious agent could be assayed in the other animal group. It was found that the presence of the polymorphism produced a prolongation in the incubation period in a similar way as observed in the species barrier phenomenon [START_REF] Bruce | Scrapie strain variation and mutation[END_REF]. It was postulated that prion strains in

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mouse could be differentiated inoculating infectious material in both animals' types and identifying the short and long incubation period animal cluster [START_REF] Bruce | Scrapie strain variation and mutation[END_REF]. Interestingly, when a mouse prion strain is inoculated in sinc heterozygous animals, either intermediate or longer incubation periods are observed [START_REF] Bruce | The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis[END_REF]. Inter-polymorphic transmissions can lead to the generation of new prion strains [START_REF] Bruce | Scrapie strain variation and mutation[END_REF], which implies new vacuolation, infectivity and/or dominance characteristics, among others. All this information suggests that polymorphisms in the prion protein are able to favor strain diversity. Table 1 shows mice and prion strains corresponding to each polymorphic group.

After the prion hypothesis development and isolation of prion protein gene [START_REF] Carlson | Linkage of prion protein and scrapie incubation time genes[END_REF], it was described that sinc and prnp genes were congruent [START_REF] Hunter | Are Sinc and the PrP gene congruent? Evidence from PrP gene analysis in Sinc congenic mice[END_REF]. Analysis of long and short incubation period animal groups revealed the expected polymorphic differences in the prion protein gene [START_REF] Westaway | Distinct prion proteins in short and long scrapie incubation period mice[END_REF]. These findings strongly supports the prion hypothesis, because as observed in the species barrier phenomenon, differences in the sequence of the prion protein affect extensively the transmission and strain characteristics of the infectious agent. According to a new nomenclature generated, sinc s7 animals are re-baptized as prnp a , while sinc p7 as prnp b [START_REF] Westaway | Distinct prion proteins in short and long scrapie incubation period mice[END_REF]. Recently a new group of mice have been identified and named prnp c (Table 1) [START_REF] Lloyd | Identification and characterization of a novel mouse prion gene allele[END_REF].

PrP polymorphisms are not unique of mouse. Indeed, polymorphisms in the prion protein have been described in most of the species. In sheep, several inter-bred crosses have been performed in order to optimize the quality and productivity of these animals, producing a wide range of polymorphic variants for prnp [START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF]. However, only five alleles of the PrP gene are significantly present giving a total of 15 possible PrP genotypes, each likely to favor or disfavor the selection of different scrapie strains [START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF]. These five age of death and five polymorphic groups was observed. Many prion strains have been described for scrapie. Each strain is associated to a particular allelic group, and allelic groups are associated to a particular breed of animals [START_REF] Bruce | Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier[END_REF][START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type, Nor98[END_REF]. However, as previously described, prion protein diversity could exist with the same sequence in the prion protein and sheep is not the exception. CH1641, a prion strain with clear biochemical differences compared to other scrapie strains was isolated from a natural case of scrapie in Cheviot sheep [START_REF] Foster | Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats[END_REF]. Recently a new scrapie strain designated Nor98 has been described [START_REF] Benestad | Cases of scrapie with unusual features in Norway and designation of a new type, Nor98[END_REF],

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mostly in animals having AHQ/AHQ and AHQ/ARQ genotypes (a variation relatively resistant to scrapie). In this case, "classically susceptible" alleles seem to be resistant for this class of prions [START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF][START_REF] Tranulis | Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway[END_REF]. All this information arise questions about how natural cases of scrapie are developed.

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In humans there is a polymorphism at codon 129, where an ATG or GTG results in either a methionine (Met) or a valine (Val) at that position. A large body of evidence indicates that this polymorphism alone or in conjunction with mutations in the prion gene modulates disease susceptibility and phenotypic expression of human TSE [START_REF] Collinge | Prion diseases of humans and animals: their causes and molecular basis[END_REF][START_REF] Collinge | Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease[END_REF][START_REF] Collinge | Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease[END_REF][START_REF] Palmer | Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Lee | Increased susceptibility to Kuru of carriers of the PRNP 129 methionine/methionine genotype[END_REF][START_REF] Mead | Balancing selection at the prion protein gene consistent with prehistoric kurulike epidemics[END_REF][START_REF] Hauw | Neuropathologic variants of sporadic Creutzfeldt-Jakob disease and codon 129 of PrP gene[END_REF][START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects[END_REF][START_REF] Goldfarb | Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism[END_REF].

Both Met and Val homozygous are over-represented, while heterozygous cases are underrepresented in sCJD [START_REF] Collinge | Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease[END_REF][START_REF] Palmer | Homozygous prion protein genotype predisposes to sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF]. About 40% of the normal population is Methomozygous, however 78%, 50% and 100% of patients affected by the sporadic, iatrogenic and variant forms of CJD are Met-homozygous, respectively [START_REF] Hill | Molecular classification of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Brandel | Distribution of codon 129 genotype in human growth hormonetreated CJD patients in France and the UK[END_REF][START_REF] Bishop | Predicting susceptibility and incubation time of human-to-human transmission of vCJD[END_REF][START_REF] Aguzzi | Prion diseases of humans and farm animals: epidemiology, genetics, and pathogenesis[END_REF][START_REF] Collinge | Prion protein gene analysis in new variant cases of Creutzfeldt-Jakob disease[END_REF]. These data suggest that the presence of Met at position 129 confers a higher susceptibility for the protein to be converted into the pathogenic isoform. The polymorphism has also been shown to alter the neuropathological pattern of lesions in sporadic CJD, the glycoform profile of protease-resistant PrP Sc and the duration and severity of the disease [START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Hauw | Neuropathologic variants of sporadic Creutzfeldt-Jakob disease and codon 129 of PrP gene[END_REF][START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects[END_REF][START_REF] Glatzel | Human prion diseases: molecular and clinical aspects[END_REF][START_REF] Gambetti | Sporadic and familial CJD: classification and characterisation[END_REF][112][113]. A study involving 300 patients showed that Met-homozygous develop a more aggressive phenotype characterized by a short duration of disease (4.5 months), while heterozygous and Val-homozygous have a much longer disease duration (14.3 and 16.9 months, respectively) [START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects[END_REF]. Val-homozygous seems to cause damage preferentially in the deep gray matter, while Met-homozygous seems to target mainly cortical structures [START_REF] Parchi | Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects[END_REF]. Codon 129 polymorphism also influences the phenotypic expression of mutations elsewhere in the prion gene [START_REF] Goldfarb | Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism[END_REF][START_REF] Puoti | Polymorphism at codon 129 of PRNP affects the phenotypic expression of Creutzfeldt-Jakob disease linked to E200K mutation[END_REF][START_REF] Bianca | Gerstmann-Straussler-Scheinker disease with P102L-V129 mutation: a case with psychiatric manifestations at onset[END_REF][START_REF] Montagna | Clinical features of fatal familial insomnia: phenotypic variability in relation to a polymorphism at codon 129 of the prion protein gene[END_REF][START_REF] Petersen | A novel mechanism of phenotypic heterogeneity demonstrated by the effect of a polymorphism on a pathogenic mutation in the PRNP (prion protein gene)[END_REF][START_REF] Cortelli | Fatal familial insomnia: clinical features and molecular genetics[END_REF][START_REF] Hainfellner | A novel phenotype in familial Creutzfeldt-Jakob disease: prion protein gene E200K mutation coupled with valine at codon 129 and type 2 protease-resistant prion protein[END_REF]. For example, people with a mutation at codon 178 resulting in a change of aspartic acid to asparagine develop either familial CJD or FFI depending on whether the amino acid at codon 129 is Val or Met, respectively [START_REF] Goldfarb | Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism[END_REF].

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Despite the clear importance of PrP polymorphism at position 129 in the disease propensity and pathogenesis, the molecular mechanism of this effect is unknown.

Experimental and computational modeling studies of the tridimensional structure of PrP have been unable to identify any significant difference between the two isoforms [START_REF] Liemann | Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein[END_REF].

In addition, no difference was reported on the in vitro thermodynamic stability of recombinant PrP bearing either Met or Val at position 129 [START_REF] Liemann | Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein[END_REF][START_REF] Zahn | NMR solution structure of the human prion protein[END_REF]. Structural studies show evidence for hydrogen bonding between Asp178 and Tyr128, which might provide a structural basis for the influence of the polymorphism on the disease phenotype that segregates with the mutation Asp178Asn [START_REF] Zahn | NMR solution structure of the human prion protein[END_REF]. In addition, it has been reported that a slightly different conformation of recombinant Met-or Val-containing PrP isoforms was induced upon copper binding [START_REF] Wong | Copper has differential effect on prion protein with polymorphism of position 129[END_REF]. Using short model peptides, we found that M at position 129 increases the propensity of this region to aggregate into β-sheet rich fibrillar structures [START_REF] Petchanikow | Biochemical and structural studies of the prion protein polymorphism[END_REF]. These findings were interpreted to suggest that Met induces a higher local propensity to extend the short β-sheet present in the normal protein into a larger sheet, which results in an increase in the rate of PrP conversion to the pathological isoform [START_REF] Petchanikow | Biochemical and structural studies of the prion protein polymorphism[END_REF].

VI. Unique features of prion strains

The biological and infectious characteristics of prions are dramatically different to the conventional infectious agents. These differences are manifested in the prion strains phenomenon in unique and unprecedented features, such as for example strain adaptation and memory, the coexistence and competition of prion strains, among others. In this section, some of these interesting phenomena will be briefly described.

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Adaptation of Prion strains. Interspecies transmission of prions could result in the emergence of more than one variety of infectious material. All new collected infectious agents could present particular strain characteristics. That is the case of DY and HY prion strains generation [START_REF] Bartz | Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy[END_REF][START_REF] Bessen | Identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters[END_REF]. When interspecies transmission of prions occurs, serial passages in the new host are needed in order to stabilize the characteristics of new generated infectious material. In the case of TME transmission in hamsters, at least four serial passages in the new species were required for stabilization [START_REF] Bartz | Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy[END_REF]. The first passage was characterized by long incubation periods and a dominance of a 19 KDa fragment when newly obtained PrP Sc was analyzed after PK digestion. In the three first passages, clinical symptoms were not characteristic of the hamster-adapted HY or DY TME strains.

This phenotype was attributed to the combination effects of both strains replicating simultaneously. Thereafter, each of the strains was stabilized in some of the animals and once they are adapted and stabilized, they can be serially propagated in vivo and the characteristics are maintained. It is accepted that both strains present differential conversion kinetics in vitro, with DY being the slowest and HY the fastest [START_REF] Mulcahy | Strain-specific Kinetics of Prion Protein Formation in Vitro and in Vivo[END_REF]. For this reason, in order to select efficiently this prion strain, limit dilutions must be performed [START_REF] Bartz | Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy[END_REF]. In that way, the most abundant and less convertible DY is favored against the less abundant but fastest HY strain.

Co-existence of prion strains: Related to the above, it has been shown that two or more prion strains can co-exist in natural cases of TSE. Co-existence of prion strains has been found in sporadic cases of CJD [113,[START_REF] Puoti | Sporadic Creutzfeldt-Jakob disease: co-occurrence of different types of PrP(Sc) in the same brain[END_REF]. Analyses of several sCJD tissue showed that

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different biochemical profiles of PrP Sc could be found in different brain areas from the same patient [113]. Co-existence of prion strains was mainly observed in patient heterozygous for codon 129 [113]. As many as 50% of these patients present different types of PrP Sc in their brains, whereas 9% of MM patients were positive for co-existence of strains. On the other hand, more than one PrP Sc type was not observed in VV patients [113].

The biochemical and structural properties of the protein seem to be the major cause of this differential distribution. This observation may explain why sCJD is so heterogeneous in terms of clinical manifestation [START_REF] Parchi | Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Pocchiari | Predictors of survival in sporadic Creutzfeldt-Jakob disease and other human transmissible spongiform encephalopathies[END_REF][START_REF] Brown | Creutzfeldt-Jakob disease of long duration: clinicopathological characteristics, transmissibility, and differential diagnosis[END_REF]. In a recent publication by Bishop et al.

[107], vCJD infected transgenic mice expressing human PrP C , present changes in their PrP Sc and vacuolation patterns in the brain according to their polymorphic classification for codon 129.

Competition of prion strains:

In particular experimental conditions, some prion strains can extend their specific incubation period when co-infected with another strain. Long incubation period prions increase the incubation period of "faster" prions. This phenomenon of "competition of prion strains" has been observed in mice and hamster. In mice, competition between 22A and 22C strains was reported in 1975 by Dickinson et al. [START_REF] Dickinson | Scrapie incubation time can exceed natural lifespan[END_REF]. In this study, RIII mice (homozygous for sinc s7 allele) were used. These authors treated mice using 22A and 22C prion strain. Before inoculation, 22A was treated with different chemical and physical agents in order to see if the "competitor" or "blocking" characteristics of 22A were maintained. From all treatments, 12M urea was shown to almost abolish the blocking properties of 22A agent. This information suggests that infective properties of long incubation period agent are strictly necessary in order to increase the incubation period of faster prions.

In hamster, similar observations were reported using DY and HY [START_REF] Bartz | Delay in onset of prion disease for the HY strain of transmissible mink encephalopathy as a result of prior peripheral inoculation with the replication-deficient DY strain[END_REF]. DY prion strain was inoculated 30 and 60 days prior intraperitoneal inoculation of HY at three different doses. When incubation periods of HY inoculated control group were compared with the animals inoculated at 60 days with DY, significant differences in the incubation periods were found, especially when HY prions were administrated in a higher dose [START_REF] Bartz | Delay in onset of prion disease for the HY strain of transmissible mink encephalopathy as a result of prior peripheral inoculation with the replication-deficient DY strain[END_REF]. On the other hand no differences were observed in the case of intranerve inoculation, revealing that competition phenomenon occurs only when peripheral inoculation is performed. These results are surprising considering the fact that DY was reported not to be infectious when intraperitoneally inoculated in hamsters [START_REF] Bartz | Delay in onset of prion disease for the HY strain of transmissible mink encephalopathy as a result of prior peripheral inoculation with the replication-deficient DY strain[END_REF]. This data suggest that replication of DY is occurring in peripheral tissues but is not able to reach the central nervous system.

In general, the principal variables that need to be observed for a successful competition are the route of infection, the interval between injections and the particular strains and doses of agent used. Prolongation of incubation periods in TSE are therapeutically beneficial and several strategies are under development to reach this aim,

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including antibodies, beta-sheet breakers, and other chemical agents [START_REF] Weissmann | Approaches to therapy of prion diseases[END_REF][START_REF] Soto | Reversion of prion protein conformational changes by synthetic betasheet breaker peptides[END_REF][START_REF] Peretz | Antibodies inhibit prion propagation and clear cell cultures of prion infectivity[END_REF]. The experimental evidence described above suggests that prions could be potentially useful for this purpose. In order to prevent spread of prion disease in cattle or humans, prion strains with incubation periods longer than species' lifespan could be used to slowdown the replication of BSE or vCJD prions.

VII. Concluding Remarks

The existence of different strains of an infectious agent composed exclusively of a protein has been one of the most puzzling issues in the prion field.

  22A and 22C showed long and short incubation period (550 and 230 days), respectively. When 22C strain was intraperitoneally inoculated 100, 200 and 300 days after intraperitoneal administration of the 22A agent, all three experimental groups resulted in incubation periods and lesion patterns matching 22A prions, suggesting that 22C prions were A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT degraded or excreted, in animals previously infected by 22A. Similar results were obtained by Kimberlin and Walker in 1985 [129] using a different strain of sinc s7 mice.

Figure legends Figure 1 .

 legends1 Figure legendsFigure 1. PrP Sc western blot profiles associated to different strains of human prions. Schematic representation of human PrP Sc types after PK digestion. Particular polymorphic groups in position 129 are associated with specific PrP Sc patterns. Types 1

Figure 2 .

 2 Figure 2. Origin and properties of the HY and DY prion strains in hamsters. The

  R I P TACCEPTED MANUSCRIPTcommon polymorphic alleles are ARQ, ARR, AHQ, ARH and VRQ. Polymorphic changes are present principally in codons 136, 154 and 171, but in order to simplify the nomenclature they are designated by the amino acid present in each position. In a recent revision by Baylis and Goldman[START_REF] Baylis | The genetics of scrapie in sheep and goats[END_REF] it is documented that VRQ/VRQ, ARH/VRQ and ARQ/VRQ alleles are most susceptible for sheep to develop scrapie, whereas the less vulnerable are animals having ARR/ARR, ARR/ARH and AHQ/ARH alleles. Therefore, it is generally agree that the VRQ allele promotes susceptibility to scrapie, whereas ARR diminishes the manifestation of the disease. Interestingly, other alleles such as ARH alone appear to favor the development of the disease while in combination with other alleles appear to confer resistance. In the same study, a correlation was established between incubation periods and the type of polymorphism. A linear relationship between

Acknowledgements

This research was supported in part by NIH grant NS049173.

species can a given prion strain be transmissible?; what other cellular factors control the origin and properties of prion strains?.

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