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From: TSS ()
Subject: CONFERENCE ON PRION DISEASES OF NEUROPRION, NETWORK OF EXCELLENCE, TURIN, ITALY, 3-6 OCTOBER 2006
Date: February 15, 2007 at 11:37 am PST

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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