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Prion2006 Abstracts Welcome Address Dear colleagues: on behalf of the European Network of Excellence NeuroPrion and the Italian prion researchers, we are happy to welcome you in Torino to the international conference “Prion 2006: strategies, advances and trends towards protection of society”. The aims of the European Network of TSE researchers are to integrate and coordinate the research efforts of their members in prevention, treatment, control and management of prion diseases and to avoid future crises related to prions. On the heels of Paris 2004 and Düsseldorf 2005, Prion 2006 is a further step in the better coordination and reinforcement of international research activities. It was your contribution that enabled us to put together a programme of exceptional scientific excellence which will be equally attractive in the different areas of prion research. The scientific programme of “Prion 2006” includes 5 plenary lectures, more than 50 oral presentations selected from almost 400 abstracts, and poster sessions on the classical themes of the NeuroPrion network (i.e., prevention, control, treatment, management and risk analysis of prion diseases). This event provides a great opportunity for scientists from all over the world to share their thoughts, findings and progress in this attractive and interesting setting. The meeting is also an opportunity to know one of the nicer town in Italy. Torino was the first Italian capital and a lot of beautiful memories passed on through buildings, royal residences and gardens in the heart of the city. Torino is a treasure of historic cafes, located throughout the twelve kilometers of arcades that wind through the center’s streets and squares. We would like to thank the Italian Ministry of Health, the Regione Piemonte, the City of Torino, the CRT Foundation, the European Commission and a number of industrial sponsors for their generous funding of this great event. We wish you all a pleasurable and inspiring time in Torino! Maria Caramelli, Torino Gianluigi Forloni and Fabrizio Tagliavini, Milano Jean-Phillippe Deslys and Jens Schell, Paris Content WELCOME ADDRESS 1 ORAL PRESENTATIONS 21 POSTER SESSION Epidemiology 51 Risk Assessment 65 Genetics 74 Structural Determinants of Infectivity and Strains 90 Prion Strains in Humans and Animals 106 Pathogenesis 126 Cell Biology of PrPC and PrPSc 158 Diagnosis 186 Therapy 218 Prion Safety 231 WORKSHOP OF THE NEUROPRION CERVID GROUP 240 Final program 241 Abstracts 243 snip... ORAL-05 EPIDEMIOLOGY OF HUMAN TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES M. Pocchiari, M. Puopolo, for the EUROCJD surveillance Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy; pocchia@iss.it An international study of the epidemiologic characteristics of human transmissible spongiform encephalopathy (TSE) diseases was established in 1993 and included national registries in France, Germany, Italy, the Netherlands, Slovakia, and the United Kingdom. In 1997, the study was extended to Australia, Austria, Canada, Spain, and Switzerland. Data were pooled from all participating countries for the years 1993 to 2004 and included deaths from definite or probable TSE diseases of all etiologic subtypes. Five thousand six hundred fifteen cases were available for analysis and included 4,727 cases of sporadic Creutzfeldt-Jakob disease (CJD), 560 genetic cases, 170 iatrogenic cases, and 158 variant cases. The overall annual mortality rate between 1999 and 2004 was 1.68 per million for all cases and 1.42 per million for sporadic CJD. There was heterogeneity in the distribution of TSE cases by etiologic subtype. Data for the analyses of predictors of survival were available in sporadic (n = 4618), iatrogenic (n = 163) and variant Creutzfeldt–Jakob disease (n = 158), and in cases associated with mutations of the prion protein gene (n=530). Overall, survival for each disease type was assessed by the Kaplan– Meier method and the multivariate analyses by the Cox proportional hazards model. snip... ORAL-07 RISK LINKED TO BLOOD M L Turner, Scottish National Blood Transfusion Service Although Blood Services have taken a precautionary approach over the past 10 years in relation to the possibility that variant CVD may be transmissible by blood and tissues, emerging evidence that there may be a significant cohort of individuals with sub-clinical disease, along with three cases of transmission of variant CJD prions by blood components, has increased concern that blood transfusion and tissue transplantation may provide a route to extension of the outbreak of this disease. The exact level of risk remains difficult to assess because of continuing uncertainties around the prevalence of sub-clinical disease, the concentration and distribution of infectivity and the overall transmissibility of variant CJD by these routes. Similarly, the extent to which current risk reduction measures including donor selection and universal leucodepletion are effective in reducing the risk of transmission is unclear. Newer technologies including prion reduction filters and peripheral blood prion assays may provide a significant improvement in management of this risk, but there are problems in terms of validation, countervailing risks and ethical and social considerations. snip... ORAL-09 THE OIE SYSTEM FOR THE EVALUATION OF COUNTRY STATUS FOR BOVINE SPONGIFORM ENCEPHALOPATHY Elisabeth Erlacher-Vindel and Gideon Brückner; World Organisation for Animal Health (OIE), 12 rue de Prony, 75017, Paris, France The Terrestrial Animal Health Code of the World Organisation for Animal Health (OIE) (Chapter 2.3.13) makes provision for the BSE risk status of the cattle population in a country, zone or compartment to be determined on the basis of a risk assessment and other evaluation criteria described in the Terrestrial Code. Pending the outcome of the risk assessment the cattle population in a country, zone or compartment can be classified as a negligible BSE risk, a controlled BSE risk or an undetermined BSE risk. Applications of Member Countries are assessed using the information in the completed questionnaire for BSE status recognition, the requirements of Chapter 2.3.13 and the guideline for Surveillance for BSE (Appendix 3.8.4) of the Terrestrial Code. The applications of Member Countries are evaluated by an ad hoc Group of experts and recommendations made to the OIE Scientific Commission for Animal Diseases. Every year, during the General Session in May, the International Committee of the OIE composed of the Official delegates of the OIE Member Countries adopts a list by Resolution of countries recognised as a negligible BSE risk or a controlled BSE risk The adoption of Resolution XXVII at the 74th General Session of the OIE in May 2006, giving recognition by the International Committee of the OIE to the bovine spongiform encephalopathy (BSE) categorisation status of several Member Countries, also signifies the acceptance by the European Commission, the EU Council and the EU Parliament of a process that would now be the sole responsibility and mandate of the OIE replacing the evaluation and classification process for the allocation of a GBR index in respect of BSE to Member States and third countries by the European Commission. The Member Countries of the OIE that were listed following the adoption of Resolution XXVII, were evaluated in accordance with the 2004 edition of the Terrestrial Animal Health Code with the provision that any re-evaluations or new applications from Member Countries, will be done in accordance with the 2006 edition of the Terrestrial Code or the Terrestrial Code current at the date of evaluation and also using the questionnaire for BSE risk assessment that was adopted at the 74th General Session. In the Resolution four countries were listed as BSE free and four countries as provisionally free from BSE in accordance with the 2004 Terrestrial Code classification. The status allocated to these countries will be published by the OIE until May 2008. snip... ORAL-11 BIOLOGICAL PROPERTIES OF THE PRION STRAIN LINKED TO THE AMYLOIDOTIC FORM OF BOVINE SPONGIFORM ENCEPHALOPATHY (BASE) F. Tagliavini1, R. Capobianco1, C. Casalone2, S. Suardi1, M. Mangieri1, C. Miccolo1, L. Limido1, M. Catania1, G. Rossi1, G. Giaccone1, C. Corona2, D. Gelmetti3, G. Lombardi3, M.H. Groschup4, A. Buschmann4, G. Zanusso5, S. Monaco5, M. Caramelli2 1Istituto Nazionale Neurologico Carlo Besta, Milan, Italy, 2Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy, 3Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy, 4Friedrich- Loeffler-Institut, Greifswald-Insel Riems, Germany, 5Department of Neurological and Visual Science, University of Verona, Italy In the past few years, two atypical forms of bovine spongiform encephalopathy (BSE) have been recognized in different European countries, Japan and USA through active surveillance systems. One of these phenotypes has been identified in Italy and is distinguishable from classical BSE for remarkable differences in pattern of deposition and brain regional distribution of PrPSc, with presence of PrP-immunoreactive amyloid plaques and severe involvement of the olfactory system with relative sparing of the brainstem. The molecular signature of this “amyloidotic” form of bovine spongiform encephalopathy, named BASE, is a PrPSc type having a protease-resistant core of lower molecular mass than BSE-PrPSc with predominance of the monoglycosylated species. We carried out strain typing studies using transgenic mice expressing bovine PrP (Tg Bov mice) and inbred lines of nontransgenic mice, including SJL, C57Bl/6, RIII and VM mice. Both BSE and BASE transmitted readily to Tg Bov mice, and produced different clinical, neuropathological and molecular disease phenotypes indicating the propagation of two distinct prion strains. Conversely, all inbred mouse lines showed a substantial barrier to primary transmission of BASE. Unexpectedly, second-passage transmission of the BASE strain to non-transgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species including humans. snip... ORAL-24 STRAIN VARIABILITY IN BOVINE ATYPICAL TSES T. Baron1, A.G. Biacabe1, A. Bencsik1, J. Jacobs2, J.P.M. Langeveld2, P.L. Acutis3, M. Polak4, D. Gavier- Widèn5, A. Buschmann6, M.H. Groschup6, J. A. Richt7 1 AFSSA, Lyon, France t.baron@lyon.afssa.fr; 2 CIDC-Lelystad, Dept. Bacteriology & TSEs, Lelystad, The Netherlands, 3 CEA – Istituto Zooprofilattico del Piemonte, Turin, Italy, Turino, Italy; 4 NVRI, Dept. Virology, Pulawy, Poland; 5 SVA, Upssala, Sweden; 6FLI-INEID, Riems, Greifswald, Germany; 7 NADC/USDA, Ames, Iowa, U.S.A. Since 2003, some cases of prion diseases in cattle have shown unusual features as assessed by molecular characterization of the protease-resistant prion protein (PrPres) and/or histopathology, when compared to the unique features of BSE described previously. Similar cases have now been recognized in a number of countries, and an overview of the current situation will be presented. Such studies have allowed to refine the molecular definition of such cases using Western blotting, referred as H-type (Biacabe et al., 2004) or L-type (BASE)(Casalone et al., 2004). While a single strain of infectious agent had previously been recognized when BSE was transmitted to a panel of genetically defined inbred wild-type mice, the recent unusual findings raised the question of transmission of prion disease from such unusual isolates. We could show transmission of prion disease from unusual BSE isolates in murine experimental models (including wild-type and transgenic mouse lines). Most importantly, data obtained during the characterization of experimentally infected mice showed different features when compared to those previously described in mice infected with typical BSE isolates. The unusual PrPres molecular features initially described in the brain of cattle by Western blot were maintained following transmission of the agent into mice. In this presentation, the potential origin of such cases, including the possible existence of “sporadic” forms of prion diseases in cattle, will be discussed. snip... ORAL-17 NOVEL PRION PROTEIN CONFORMATION AND GLYCOTYPE IN ATYPICAL CREUTZFELDTJAKOB DISEASE G. Zanusso1, A. Polo2, A. Farinazzo1, R. Nonno6, F. Cardone5, M. Di Bari6, S. Ferrari1, S. Principe5, M. Gelati1, E. Fasoli1, M. Fiorini1, F. Prelli8, B. Frangione8, G. Tridente3, M. Bentivoglio4, A. Giorgi7, M. E. Schininà7, B. Maras7, U. Agrimi6, M. Pocchiari5, N. Rizzuto1 and S. Monaco1 1 Department of Neurologic and Visual Sciences, University of Verona, Verona; 2 Ospedale Piove di Sacco; 3 Department of Pathology, University of Verona, Verona; 4 Department of Morphologic and Biomedical Sciences, University of Verona, Verona; 5 Department of Cell Biology and Neurosciences and 6 Department of Food Safety and Veterinary Public Health, Istituto Superiore di Sanità, Rome; 7 Department of Biochemistry and Centro di Eccellenza BEMM, University of Rome "La Sapienza", Rome; 8 Department of Pathology, New York University Medical Center, New York, New York, U.S.A. E-mail address: gianluigi.zanusso@univr.it Here we report a 69-year-old woman who presented with behavioral and personality changes followed by rapidly evolving dementia. Post-mortem examination of the brain showed an atypical Creutzfeldt-Jakob disease (CJD) phenotype characterized by intracellular prion protein deposition and the presence of axonal swellings filled with amyloid fibrils. Biochemical analysis of the pathological prion protein (PrPSc) disclosed a previously unrecognized PrPSc tertiary structure lacking diglycosylated PrPSc species. Genetic analysis revealed a wild-type prion protein gene with methionine/valine heterozygosity at the polymorphic codon 129. The present results define a new prion disorder with disease phenotype and PrPSc glycotype similar to familial CJD with frontotemporal presentation. This work was supported by Neuroprion (FOOD-CT-2004-506579) snip... ORAL-25 PRION STRAINS IN HUMAN PRION DISEASES P. Gambetti National Prion Disease Pathology Surveillance Center Department of Pathology Case Western Reserve University Cleveland, USA pxg13@case.edu The prion protein (PrP) genotype, as determined by the polymorphic codon 129, is thought to have an effect on the conformational characteristics of the scrapie PrP (PrPSc) revealed by the size of the protease-resistant PrPSc fragment or PrPSc type. Based on PrP genotype and PrPSc type we have proposed a classification of sporadic Creutzfeldt-Jakob disease (sCJD) in six subtypes. Each of these subtypes appears to be associated with a distinct PrPSc or prion strain. The purpose of this presentation is to review the current state of human prion strains, of the disease phenotypes with which the prion strains associate, and of current classifications of human prion diseases. Alternative classifications proposed by others or based on our own experience in examining over thousand cases of prion disease will be discussed. Of special interest is the recent identification of a novel phenotype of human prion disease associated with a distinct PrPP Sc strain that is largely protease sensitive. The contribution of transgenic mice expressing human PrPC to the study of human prion strains and disease phenotypes will also be examined. (Supported by NIH AG-14359 and CDC UR8/CCU515004 awards and the Charles S. Britton Fund). snip... ORAL-26 GERSTMANN-STRÄUSSLER-SCHEINKER DISEASE (GSS) AND PRION PROTEIN CEREBRAL AMYLOID ANGIOPATHY (PRP-CAA) B. Ghetti Department of Pathology and Laboratory Medicine, Indiana University, Indianapolis, IN. GSS and PrP-CAA are degenerative dementias inherited in an autosomal dominant pattern and associated with the deposition of PrP in the cerebral parenchyma and cerebral blood vessels, respectively. GSS has been found to be associated with ten missense mutations in PRNP. PrP-CAA has been found to be associated with three nonsense mutations in PRNP. We report clinical, and genetic data collected from 34 affected and 19 unaffected gene-carriers from 7 families in which the following mutations in the PRNP gene were found: P102L-129M, A117V-129V, H187R-129V, F198S- 129V. We report pathologic, and genetic data collected from 52 affected and 3 unaffected genecarriers from 15 families in which the following mutations in the PRNP gene were found: P102L- 129M, P102L-129V, A117V-129V, G131V-129M, Y145STOP-129M, H187R-129V, F198S-129V, D202N-129V, Q212P-129M, Q217R-129V. Clinically, GSS is characterized by a movement disorder and dementia. PrP-CAA is characterized by a dementing illness reminiscent of Alzheimer disease. The age at onset varies between the fourth and eighth decades; however, in GSS associated with the F198S mutation, we have observed a statistically significant difference (p<0.001) in the mean age at onset of clinical signs between individuals homozygous for valine at residue 129 (mean: 43.7 years) and individuals heterozygous methionine/valine at residue 129 (mean: 56.5 years). Pathologically, deposition of intraneuronal insoluble tau occurs in association with the Y145STOP, F198S, D202N and Q217R mutations while it is sporadically present in association with the A117V and H187R. Spongiform degeneration is only seen in GSS associated with the P102L mutation, but not in all cases. Transmission of GSS into experimental animals has been shown to occur only following the inoculation of brain tissue from individuals with the P102L mutation. snip... ORAL-18 BSE AGENT SHOWS AN ENHANCED VIRULENCE AFTER PASSAGE IN SHEEP J.M. Torres1, J.C. Espinosa1, O. Andréoletti2, M.E. Herva1, E. Alamillo1, C. Lacroux2 1 Centro de Investigación en Sanidad Animal. INIA, 28130 Valdeolmos, Madrid (Spain). jmtorres@inia.es 2 UMR INRA-ENVT 1225, Interactions Hôte Agent Pathogène, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France Sheep are fully susceptible to BSE infection and the ensuing disease is un-differentiable from scrapie in terms of pathogenesis and clinical signs. The biological properties of BSE adapted in sheep, and more particularly its capacity to infect other species is an animal and human health concern. We investigated, using transmission in various mice models, biological properties of BSE passaged in sheep by comparison to the original cattle BSE and various sheep scrapie prions. Our results indicate, in all mice models we used (bovine, porcine, ovine or murine), that sheep BSE have an increased attack rate and/or a reduced incubation time when compared to the effects of the original cattle BSE isolate. The reduced incubation duration was conserved in subsequent passage in BoPrPTg mice indicating that alterations of biological properties in BSE in sheep are not due to differences in infectious titre. In the meanwhile, pathological and biochemical features in mice inoculated with sheep BSE prions were very similar to those exhibited by cattle BSE prions but differed from those observed with sheep scrapie isolates. The enhanced virulence of BSE after passage in sheep raises new concerns about the infectivity of this new prion to infect other species including human. 30 snip... ORAL-19 IINTERSPECIES PRION TRANSMISSION IS CONTROLLED BY CONFORMATIONAL COMPATIBILITY BETWEEN PRPSC AND HETEROTYPIC PRPC K.M. Green*1, S.R. Browning*1,6, T.S. Seward2, M. Green4, E.A. Hoover5, G.C. Telling1,2,3,7 1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders Brown Center on Aging,3Department of Neurology, 4UK Transgenic Facility University of Kentucky, Lexington, Ky USA. 5Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Co, USA 6Present address: Department of Infectology, Scripps Research Institute, Jupiter, Florida, USA 7To whom correspondence should be addressed:gtell2@uky.edu * These authors contributed equally The threats to humans and livestock from interspecies prion transmission are difficult to assess because the factors controlling this process remain uncertain. To address this we have used transgenic mouse models to understand the roles played by PrP primary structure, prion strains and the species specificity of protein X in controlling interspecies prion infection in the context of cervid transmission barriers. Cervid prions are of particular concern because chronic wasting disease (CWD) of North American and South Korean cervids is the only recognized prion disease of wild animals and its increasing geographic range, contagious nature, and environmental persistence have raised concerns about prion dissemination and the potential for further interspecies transmission. We show that conformational compatibility of PrPSc in a prion strain and PrP primary structure in a new host is the most important determinant of interspecies prion transmission barriers. Although prion strains can acquire totally new host range properties following heterologous conversion of PrPP C in a new host, the strain-related biochemical properties of PrPSc may remain relatively stable. We also show that the cervid PrP polymorphism at residue 132, which is equivalent to the human PrP 129 polymorphism, is a crucial determinant of cervid prion transmission and has a profound controlling effect on PrPSc-related prion strain properties. Our transgenic approaches modeling trans-species prion susceptibility in cervids also speak to the possible origins of CWD since cervid transgenic mice are also vulnerable, to varying degrees, to sheep scrapie prions, the degree of susceptibility being strain related. One particularly well-characterized sheep scrapie isolate, SSBP/1, caused disease as efficiently as CWD prions from diseased deer or elk. Finally, while transmissions in transgenic mice based on the protein X model of prion propagation produced chimeric prions, passage of which resulted in novel cervid prions with an extended host range compared to CWD-cervid prions, the unexpected susceptibilities of such mice to CWD and mouse prions are inconsistent with the previously hypothesized role of protein X in prion propagation. snip... ORAL-21 ROLE OF PrP MEMBRANE ANCHORING IN BRAIN AND EXTRANEURAL PATHOGENESIS OF PRION DISEASES. B.Chesebro1, M.Trifilo2, K. Meade-White1, R.Race1, E.Masliah3 K.Knowlton3, M.Oldstone2 1Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, Hamilton, Montana, USA; 2The Scripps Research Institute, LaJolla, California, USA; 3University of California at San Diego, LaJolla, California USA. Prion protein (PrP) conversion from the normal protease-sensitive form (PrPsen) to the diseaseassociated protease-resistant form (PrPres) is an important event in the pathogenesis of prion diseases. Normal PrP exists primarily bound to the cellular plasma membrane by a glycosylphosphatidylinositol (GPI) linkage, and conversion to the disease-associated form is believed to occur mainly as a membrane-associated event. Our previous experiments demonstrated that PrP lacking the GPI anchor could be converted to protease-resistant PrP in tissue culture cells and in cellfree in vitro conditions. To test the in vivo influence of GPI anchoring on prion disease infection and disease, we generated and studied transgenic (tg) mice which express only the anchorless GPInegative PrP. Following scrapie infection of such tg mice we observed replication of scrapie infectivity, accumulation of PrPres in brain mainly as an amyloid form, and vacuolation primarily in white matter areas. Surprisingly there was no typical fatal clinical disease over an observation period of 600-700 days; however, in the later phases minor clinical defects were detectable by neurobiological testing. The lack of fatal prion disease in these tg mice suggests that either the amyloid form of PrPres has a reduced level of neurotoxicity and/or that membrane anchored PrP is required for the usual neurotoxic effects. Further analysis indicated that PrPres could be detected in the heart and blood of infected tg mice, and cardiac studies showed deficits consistent with restrictive cardiomyopathy typical of the early stages of amyloid heart disease in humans. snip... ORAL-31 PRION-INDUCED AMYLOID HEART DISEASE WITH HIGH BLOOD INFECTIVITY M.J. Trifilo1, T. Yajima2, M. Sanchez1, K.L. Peterson2, R.E. Race3, K. White3, K. Knowlton2, B. Chesebro3, and M.B.A. Oldstone1 1Viral-Immunobiology Laboratory, Departments of Molecular and Integrative Neurosciences and Infectology, Scripps Research Institute, La Jolla, CA. 2Department of Medicine, University of California, San Diego, La Jolla, CA. 3Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT. 4Departments of Neurosciences and Pathology, University of California, San Diego, CA. mtrifilo@scripps.edu Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of infectious diseases that cause neurodegeneration and death. The majority (>95%) of normal host derived prion protein (PrPsen) exists as a membrane bound, glycophosphatydilinositol (GPI) anchored protein. We recently (Science 2005, vol.308) constructed transgenic (tg) mice where the C-terminal 21 amino acids were not transcribed, resulting in a secreted form of PrPsen in which >98% of that molecule existed in a soluble non-membrane bound form. Intracerebral inoculation of these tg mice with numerous murine scrapie strains (RML, ME7, 22L) resulted in amyloid deposition in the brain and excessive buildup of abnormal folded prion protein (PrPres) in the absence of overt disease manifestations normally associated with scrapie over 700 days post infection (dpi). However, aberrant physiologic events in terms of delayed neural transmission and loss of long term potentiation could be found within 150 days post infection. Novel distribution patterns of PrPres within and around endothelial cells lining blood vessels occurred within the brain. Examination of blood demonstrated that both infectivity and PrPres could be readily detected by 280 dpi in both serum and cellular fractions of RML scrapie infected tg mice. Moreover, multiple extraneural tissues, such as spleen heart lunge, pancreas, and kidney, also showed PrPres deposition. Focus on the heart indicated that similar to brain, deposits of both PrPres and amyloid that was infectious occurred. The deposition of amyloidogenic PrPres within the hearts of scrapie-infected tg mice resulted in disordered cardiac function including dramatic alterations in both systolic (reduced compliance) and diastolic (stiffening of the heart) function. ORAL-32 INFECTIVITY IN URINE OF HAMSTERS INFECTED WITH SCRAPIE AND IMPLICATIONS ON MECHANISMS OF HORIZONTAL TRANSMISSION Gregori and RG Rohwer; Veterans Affairs Medical Center, Baltimore and University of Maryland at Baltimore. USA lgreg002@umaryland.edu Horizontal transmission from animal to animal of transmissible spongiform encephalopathies (TSE) has been documented in the wild and in laboratory animals. The mechanism underlining these transmissions is not fully understood although several studies implicated environmental contamination. Urine and feces could be responsible for environmental contamination since they are excreted and due to the resistant nature of the TSE agent their infectivity could persist indefinitely. Previous studies have indicated that urine from TSE affected animals and humans is not infectious. However, those studies were conducted with a small number of animals and in some cases transmissions were attempted across the specie barrier. We investigated urinary excretion as a potential source of secondary exposure in the environmental. Urine from hamsters infected with the 263K strain of scrapie was collected using metabolic cages and titered by intracerebral inoculation in the same animal specie with the limiting dilution method. Five-ml equivalents of urine (diluted 1:3) were inoculated. After 267 days post inoculation, 4 animals developed scrapie. Although the study is still on-going, the current data indicate that urine from infected hamsters contains low but measurable levels of infectivity (at least 0.8 + 0.4 infectious doses per ml). These results are in contrast with previously reported studies. We argue that similarly to blood infectivity, quantitation of infectivity in urine requires inoculation of a few milliliters of urine into the same animal specie. Ten percent bladder and kidney tissue homogenates from infected hamsters were also titered using the end point titration method. At 253 days, the titers are 104.5 and 103.6 ID50/ml for bladder and kidney, respectively. Furthermore, in a look-back investigation of previous titrations conducted in our laboratory, we observed a pattern of clustering of positive cases consistent with infectivity been transmitted by secondary exposure in the cage environment. Our results highlight a new pathway of TSE infection in animals and may be in humans. This pathway involves the organs of the urinary system and directly affects the environment via urine infectivity excretion. Exposure to chronic low doses of infectivity in the environment may be responsible for TSE horizontal transmission. snip... ORAL 46B WHO TSE REFERENCE MATERIALS (Importance of standardized reference materials) At several WHO TSE Consultations (1), issues related to the development of TSE Reference Materials in general and TSE Blood Reference Materials in particular have been discussed. WHO CJD brain-derived Reference Materials have been prepared (2). The preparations are maintained and distributed by the National Institute of Biological Standards (NIBSC), a WHO Collaborating Centre for Biological Standards. Several groups recently claimed to have developed promising and even partially validated bloodbased methods to identify animals infected with TSE agents during incubation period and humans with CJD. Some of those tests may be presented for regulatory review, requesting marketing authorization from national regulatory authorities. WHO has been asked to take a leading role in developing TSE Blood Reference Materials suitable to evaluate blood-based TSE diagnostic tests and screening tests for donors of blood and tissues. Materials to evaluate blood-based tests of animal TSEs important for human health would also be useful. As for other reference materials, a TSE Blood Reference Material need not be offered as a working reagent for test developers but as a calibrant to prepare in-house reference materials. Reference materials can also be suitable to assemble panels of replicate TSE-derived and control materials (in dilutions) coded and randomized. Both the original TSE Blood Reference Materials and the panels would be intended for preliminary characterization in international collaborative studies, just as the TSE Brain Reference Materials were studied previously. Any suitable biological reference material should be available in sufficient quantity to allow comparison of performance characteristics of candidate tests at a global level. If this effort proceeds, WHO would consider reconvening the WHO Working Group on TSE Reference Materials to assist in coordinating the selection of TSE materials and organizing international collaborative studies. References: (1) WHO Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies: www.who.int/bloodproducts/tse/en (2) Proposal to establish WHO Reference Reagents for in vitro assays of CJD specimens. WHO/BS/03.1965 Rev.1: www.who.int/bloodproducts/tse/en 45 snip... ORAL-53 VARIANT CREUTZFELDT-JAKOB DISEASE AND BLOOD INFECTIVITY IN ANIMAL MODELS Cervenakova Transmissible Diseases Department, American Red Cross Holland Laboratory, Rockville, Maryland, USA cervenakl@usa.redcross.org Patients with variant Creutzfeldt-Jakob disease (vCJD) accumulate infectivity and disease-associated prion protein (PrPTSE) in lymphoreticular tissues more extensively than patients with classical CJD. These findings suggest that the blood of vCJD patients may also carry higher levels of infectivity. Three instances of secondary transmission of infection associated with blood transfusion have recently been reported in the UK, increasing the concern about blood safety. We have shown that during the clinical phase of the disease, buffy coat from mice inoculated with mouse-adapted human strains of Gerstmann-Sträussler-Scheinker disease (GSS) had a higher level of infectivity than buffy coat from mice inoculated with vCJD. In contrast, during the preclinical stage of disease, both buffy coat and plasma from vCJD-inoculated mice had higher levels of infectivity than mice inoculated with GSS. We also showed that infection is efficiently transmitted by i.c. and i.v. routes for buffy coat and platelet poor plasma. Here, we present data on rates of transmission by i.v. inoculation of whole blood from animals infected with vCJD, and discuss the results in connection with other investigations of TSE transmission through blood or blood components using a variety of animal models. snip... ORAL-55 SOIL CLAY CONCENTRATES PRION PROTEIN AND PRION INFECTIVITY P.Rigou1, S.Noinville1, Y.Quenet1, H.Rezaei1, S.Staunton2, H.Quiquampoix2, P.Sarradin3, C. Barc3, F.Lantier3, P.Berthon3, S.Bernard3, J.Grosclaude1. 1 INRA, Virologie et Immunologie Moléculaires, Jouy-en-Josas, France. jeanne.grosclaude@jouy.inra.fr; 2 INRA, Rhizosphère et Symbiose, Montpellier, France. 3 INRA, Infectiologie Animale et Santé Publique, Nouzilly, France. Better control of domestic animal prion diseases is currently achieved through sanitary provisions and genetic selection for resistant sheep.The persistence of an environmental reservoir for infectivity would counteract this progress, particularly in the wild. Abiotic mechanisms of prion retention and dissemination in soil were first investigated by studying interaction between a pure clay (Montmorillonite or Mt) and a model oligomeric prion protein mimicking the pathological isoform; these physico-chemical approaches (mainly FTIR-spectrometry, RMN studies, biochemical and immunochemical depletion studies) allowed to reason infectivity studies with infected brain homogenates adsorbed on Mt. Clays are known for displaying a high adsorption capacity for proteins, due to their negative permanent charge and their high specific area (800 square metres for one gramme of sodic montmorillonite). Adsorption of PrPrec to Mt ranged from 1g protein / g Mt at pH 3-5 to more than 2 g protein / g Mt at neutral/alcaline pH. The mechanism relied mainly on the two-domain structure of the prion protein, allowing a strong interaction of the positively charged N-terminal domain and a hydrophobic-like interaction of the full-length protein with the Mt surface (Revault et al., 2005, BBA, 1724,367). Protein desorption from Mt could not be achieved by saline, detergent or chaotropic reagents supporting irreversibility of adsorption in environmental conditions. Moreover when competing with other biological material such as serum proteins, PrPrec was selectively adsorbed by Mt. So Mt behaves as a strong concentrating agent for prion protein (Rigou et al., 2006, Environ.Sci.Technol, 40,1479). In natural conditions Mt might constitute "infectivity hot points" from trace amounts of infected biological material recurrently deposited on soil like in whelping areas. Mt efficiency in capturing infectivity from infected organs was checked in bioassays on transgenic mice. Infectivity experiments emphasized the infectious capacity of contaminated Mt devoid of any biological debris after one night contact with infected brain homogenates. Fate of Mt-adsorbed labelled PrP in brain after inoculation or in gut after ingestion was followed by dynamic imaging techniques. Implications of these results for environmental control of prion diseases will be discussed taking into account recent publications from other research groups. snip...end...full text 234 pages, lot more...TSS http://www.neuroprion.com/pdf_docs/conferences/prion2006/abstract_book.pdf TSS
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