Wednesday, January 01, 2014

Molecular Barriers to Zoonotic Transmission of Prions

Molecular Barriers to Zoonotic Transmission of Prions


*** chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.



Volume 20, Number 1—January 2014




Molecular Barriers to Zoonotic Transmission of Prions


Suggested Citation


Marcelo A. Barria, Aru Balachandran, Masanori Morita, Tetsuyuki Kitamoto, Rona Barron, Jean Manson, Richard Knight, James W. Ironside, and Mark W. HeadComments to Author


Author affiliations: The University of Edinburgh, Edinburgh, Scotland, UK (M.A. Barria, R. Knight, J.W. Ironside, M.W Head); Canadian Food Inspection Agency, Ottawa, Ontario, Canada (A. Balachandran); Japan Blood Products Organization, Kobe, Japan (M. Morita); Tohoku University Graduate School of Medicine, Sendai, Japan (T. Kitamoto); University of Edinburgh, Easter Bush, Scotland, UK (R. Barron, J. Manson)




The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.






Multiple factors govern the transmission of prions in experimental settings. In addition to infectious dose and route, a species or transmission barrier phenomenon is well recognized. Within the theoretical confines of the prion hypothesis, the most obvious basis of a species barrier effect would be dissimilarity in PRNP sequence between the infectious source and the exposed individual. However, PRNP sequence similarity alone does not seem to accurately predict whether prions are transmissible between species, perhaps because interactions between PrPC and PrPSc occur as native PrPC and misfolded and aggregated PrPSc conformers. The possible effects of species-specific sequence difference on PrPC folding are not well understood. Neither is the secondary and higher order structure of PrPSc, except for clear evidence that different prion strains are associated with different PrPSc conformers and glycotypes (reviewed in 4,18) and that these might exist as a quasispecies or molecular cloud (19). Under such a scenario molecular compatibility might be difficult to predict.


To isolate and study molecular effects, we have previously conducted cell-free PrP conversion experiments by PMCA using homogenates of bovine and ovine prion disease brain samples to seed brain homogenates containing human PrP, assessing the extent of conversion by detection of human PrPres. These studies showed that samples of C-type BSE (which is a known human pathogen and the cause of vCJD) efficiently converted human PrP, with a codon 129 preference similar to that of vCJD (MM>MV>VV), whereas samples of classical scrapie (which is not thought to be a human pathogen) failed to convert human PrP to a measurable extent. Equally importantly, a sheep BSE isolate resembled C-type BSE and vCJD in its ability to convert human PrP, thus underscoring influence of strain over sequence similarity in determining what might be termed a molecular transmission barrier (10).


Here we applied the same approach to a series of animal prion diseases whose risk to human health is poorly characterized. Our results show that under the PMCA conditions used, L-type BSE, H-type BSE, and atypical scrapie isolates fail to produce detectable human PrPres. The CWD isolate used converted human PrPC, albeit less efficiently than C-type BSE. This observation remained true whether the input animal prion disease brain homogenate was normalized by tissue weight or by PrPres abundance and whether the PMCA substrate was from human brain, PRNP humanized murine brain, or a human-derived and human PrPC overexpressing cell line. The conversion of human PrPC by CWD brain homogenate in PMCA reactions was less efficient when the amino acid at position 129 was valine rather than methionine. Furthermore, the form of human PrPres produced in this in vitro assay when seeded with CWD, resembles that found in the most common human prion disease, namely sCJD of the MM1 subtype.


Previous attempts to determine the transmissibility of these prion diseases to humans and thus assess their zoonotic potential have used experimental challenge of nonhuman primates, humanized PrP transgenic mice, and cell-free assays with sometimes conflicting results. Successful transmission of CWD and L-BSE to certain nonhuman primates has been reported: L-type BSE showing a different pathologic profile and a shorter incubation period than C-type BSE (20–23). However, Kong et al. (24) reported that CWD failed to transmit to humanized PrP 129M overexpressing mice inoculated with an elk brain homogenate. In contrast, Beringue et al. (25) reported that humanized PrP 129M overexpressing mice were susceptible to L-type BSE and suggested that L-type BSE was more virulent than C-type BSE and presented a zoonotic risk. H-type BSE reportedly failed to transmit to these same mice. Sandberg et al. (26) and Tamgüney et al. (27) confirmed the previous report of Kong et al. that CWD fails to transmit to transgenic mice, irrespective of whether 1) the mice expressed bovine, ovine, or human PrP; 2) the mice expressed the human 129M or 129V PrP allelic variants; or 3) the CWD isolates were from mule deer, elk, or white-tailed deer.


Cell-free approaches to modeling human susceptibility to animal prion prion diseases also have been published (8,10,28–31). Raymond et al. (28) compared the ability of CWD, C-type BSE, sheep scrapie, and CJD brain homogenates to convert human PrPC metabolically labeled and purified from transfected cells. These experiments obtained limited conversion of human PrPC by CWD, C-type BSE, and scrapie. In contrast to our study, this early cell-free system failed to distinguish between scrapie and C-type BSE in their ability to convert human PrPC; however, it indicated a substantial molecular barrier to conversion of human PrPC by CWD PrPSc (28,29), which agrees with this report. Kurt et al. (31) reported that PMCA using human PrPC overexpressing transgenic mice brain (both 129M and 129V lines) as substrate failed to support amplification when seeded with CWD cervine brain homogenate. Cervidized Prnp transgenic mouse brain homogenate can support CWD prion replication (32), and extensive in vitro conditioning of a CWD isolate by PMCA in a cervidized substrate (or passage in cervidized mice) was sufficient to overcome the barrier and enable efficient in vitro amplification in a humanized transgenic mouse substrate (33). Direct comparison of these studies is made difficult by the differences in approach (in vivo vs. in vitro), the different transgenic constructs used, and the technical details of the cell-free conversion assays undertaken (Table). An additional possibly significant difference between these studies is the nature of the CWD isolate used. CWD affects different deer species (some of which show allelic variation in their Prnp sequence), but CWD also occurs as different biologic strains of agent (34–36). Different strains of CWD may have a role in determining transmissibility and conversion efficiency. Recently, Meyerett et al. (37) reported the in vitro strain adaptation of a CWD isolate by serial PMCA, similar to that produced by in vivo subpassage.


The most directly comparable in vivo study to that reported here is Wilson et al. (11), in which a similar series of atypical animal prion diseases were used to challenge transgenic mice expressing physiologic levels of human PrPC. Atypical scrapie; C-, H-, and L-type BSE; and CWD all failed to produce disease (or signs of infection) on first passage in these mice (11). The use of different animal prion disease isolates (and possibly differing species and strains of CWD) might explain this discrepancy; however, a more fundamental difference might be that the in vivo and in vitro model systems assess different aspects of the agent and its replication. The in vivo model is undoubtedly more complex and arguably more physiologically relevant, and the readout is disease; however, it remains disease in a mouse, in which the PRNP sequence alone is human. The in vitro cell-free model does not assess disease as such, only the compatibility of particular combinations of seed and substrate homogenates (some of which, in these examples, were entirely of human origin) to produce PrPres. Differences between the in vivo and in vitro models are exemplified by the comparison of C-type BSE, and vCJD. Both amplify well in PMCA using humanized (129MM) brain homogenate as a substrate (10), whereas intracranial inoculation of C-type BSE into humanized (129MM) mice fails to produce disease (12), unless first experimentally transmitted to sheep or goats (13,38,39).


The interpretation of different amplification efficiencies as a semiquantitative measure of relative risk is tempting but is probably premature and almost certainly an oversimplification. The testing of more isolates, especially of CWD in deer and elk, is advisable before any firm conclusions can be drawn. Additionally, possible strain-specific effects on amplification efficiency by the precise PMCA experimental conditions are difficult to discount and might complicate interpretation. The relative amplification efficiencies of C-, H-, and L-type BSE might differ intrinsically because certain strains of sheep scrapie appear to, even when amplified in homologous sheep substrates (40). However, we can say with confidence that under the conditions used here, none of the animal isolates tested were as efficient as C-type BSE in converting human PrPC, which is reassuring. Less reassuring is the finding that there is no absolute barrier to the conversion of human PrPC by CWD prions in a protocol using a single round of PMCA and an entirely human substrate prepared from the target organ of prion diseases, the brain.


Mr Barria is a PhD student at the National CJD Research and Surveillance Unit, University of Edinburgh (UK). His main research interest is the molecular basis of human prion diseases.







*** chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.

 *** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, and there may be asymptomatic individuals infected with the CWD equivalent. These circumstances represent a potential threat to blood, blood products, and plasma supplies.






Sunday, August 25, 2013


***Chronic Wasting Disease CWD risk factors, *humans*, domestic cats, blood, and mother to offspring transmission




Sunday, July 21, 2013


*** As Chronic Wasting Disease CWD rises in deer herd, what about risk for humans?



Thursday, October 10, 2013

*** CJD REPORT 1994 increased risk for consumption of veal and venison and lamb

Thursday, January 2, 2014

*** CWD TSE Prion in cervids to hTGmice, Heidenhain Variant Creutzfeldt-Jacob Disease MM1 genotype, and iatrogenic CJD ???


Wednesday, January 01, 2014


APHIS-2006-0118-0100 Chronic Wasting Disease Herd Certification Program and Interstate Movement of Farmed or Captive Deer, Elk, and Moose




Friday, November 22, 2013


*** Wasting disease is threat to the entire UK deer population CWD TSE PRION DISEASE Singeltary submission to Scottish Parliament




Sunday, December 29, 2013


Impacts of wildlife baiting and supplemental feeding on infectious disease transmission risk: A synthesis of knowledge




Sunday, November 3, 2013


*** Environmental Impact Statements; Availability, etc.: Animal Carcass Management [Docket No. APHIS-2013-0044]




Wednesday, September 04, 2013


***cwd - cervid captive livestock escapes, loose and on the run in the wild...




Saturday, February 04, 2012


Wisconsin 16 MONTH age limit on testing dead deer Game Farm CWD Testing Protocol Needs To Be Revised






Thursday, August 08, 2013


Characterization of the first case of naturally occurring chronic wasting disease in a captive red deer (Cervus elaphus) in North America




Friday, August 09, 2013


***CWD TSE prion, plants, vegetables, and the potential for environmental contamination




Sunday, September 01, 2013


hunting over gut piles and CWD TSE prion disease




Monday, October 07, 2013


The importance of localized culling in stabilizing chronic wasting disease prevalence in white-tailed deer populations




Friday, December 14, 2012


DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced into Great Britain? A Qualitative Risk Assessment October 2012




Saturday, March 10, 2012


CWD, GAME FARMS, urine, feces, soil, lichens, and banned mad cow protein feed CUSTOM MADE for deer and elk




----- Original Message -----


From: "Terry S. Singeltary Sr."




Sent: Friday, July 12, 2002 8:43 PM


Subject: Re: CWD AMERICA ???


hello Dr. Jebara,


many thanks for your swift and kind reply.


if i am not mistaken, it was the same email address. it was 3 or 4 weeks ago i wrote, as it is, i don't save 'sent' emails anymore, unless very important.


my main concern (besides the fact that a potential TSE has been in the USA cattle for some time, but the APHIS do not test to find), is that the CWD could very well be transmitting to humans, and i just did not see to much posted about it on OIE site.


Coming back to your question, Chronic Wasting Disease is not an OIE


listed disease. Please see OIE disease lists at




why is this TSE (CWD) not listed and followed as with BSE ?'



Article 1. Countries shall make available to other countries, through the OIE, whatever information is necessary to minimise the spread of important animal diseases and to assist in achieving better worldwide control of these diseases.




The USA CWD is an important animal disease.


why is it not followed?


The decision to add or delete a disease from the OIE lists, come through proposals made by Member Countries and it has to be adopted by the International Committee.


i _urgently_ suggest a proposal to the OIE to follow this disease very closely, and to propose _more_ testing in the USA for TSEs in the USA cattle...


kindest regards, terry


 Tuesday, July 17, 2012


O.I.E. BSE, CWD, SCRAPIE, TSE PRION DISEASE Final Report of the 80th General Session, 20 - 25 May 2012






(Adopted by the International Committee of the OIE on 23 May 2006)


11. Information published by the OIE is derived from appropriate declarations made by the official Veterinary Services of Member Countries. The OIE is not responsible for inaccurate publication of country disease status based on inaccurate information or changes in epidemiological status or other significant events that were not promptly reported to the Central Bureau,




Sunday, December 15, 2013






Saturday, December 21, 2013


**** Complementary studies detecting classical bovine spongiform encephalopathy infectivity in jejunum, ileum and ileocaecal junction in incubating cattle ****




Wednesday, December 4, 2013


*** Bovine Spongiform Encephalopathy; Importation of Bovines and Bovine Products; Final Rule Federal Register / Vol. 78 , No. 233 / Wednesday, December 4, 2013




Saturday, November 2, 2013


*** APHIS Finalizes Bovine Import Regulations in Line with International Animal Health Standards while enhancing the spread of BSE TSE prion mad cow type disease around the Globe





Thursday, December 05, 2013


National Scrapie Eradication Program October 2013 Monthly Report Fiscal Year 2014 TSE PRION REPORT




Tuesday, October 29, 2013






Wednesday, October 09, 2013






Thursday, October 10, 2013


CJD REPORT 1994 increased risk for consumption of veal and venison and lamb




Friday, August 16, 2013


*** Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K. and Contaminated blood products induce a highly atypical prion disease devoid of PrPres in primates




WHAT about the sporadic CJD TSE proteins ?



WE now know that some cases of sporadic CJD are linked to atypical BSE and atypical Scrapie, so why are not MORE concerned about the sporadic CJD, and all it’s sub-types $$$


Creutzfeldt-Jakob Disease CJD cases rising North America updated report August 2013


*** Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing an extreme increase of 48% between 2008 and 2010 ***




Sunday, October 13, 2013


*** CJD TSE Prion Disease Cases in Texas by Year, 2003-2012








Tuesday, December 03, 2013 4:49 AM


To: Terry Singeltary Sr


Subject: Written submission to House of Commons Science and Technology Committee inquiry


Parliament UK


Thank you for your written submission to the House of Commons Science and Technology Committee inquiry on Blood, tissue and organ screening.


We will be in touch if we have any further questions.


From: Terry S. Singeltary Sr.


Sent: Monday, December 02, 2013 9:18 PM


To: CJDVOICE CJDVOICE Cc: bloodcjd bloodcjd


Subject: [BLOODCJD] A parliamentary inquiry has been launched today into the safety of blood, tissue and organ screening following fears that vCJD – the human form of ‘mad cow’ disease – may be being spread by medical procedures



Monday, December 02, 2013


*** A parliamentary inquiry has been launched today into the safety of blood, tissue and organ screening following fears that vCJD – the human form of ‘mad cow’ disease – may be being spread by medical procedures




Wednesday, December 11, 2013


Detection of Infectivity in Blood of Persons with Variant and Sporadic Creutzfeldt-Jakob Disease







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