Saturday, November 2, 2013

Recommendation of the Swiss Expert Committee for Biosafety on the classification of activities using prion genes and prion protein January 2013

Recommendation of the Swiss Expert Committee for Biosafety on the classification of activities using prion genes and prion protein


January 2013




Page 2






Introduction ................................................................................................................................ 3


Legal bases................................................................................................................................ 3


Safety measures for work using TSE agents.............................................................................. 3


Risk assessment for work with prion proteins and prion genes................................................... 4


Classification of work with TSE agents....................................................................................... 4


References................................................................................................................................. 7




Page 3






Prions (proteinaceous infectious particles) cause neurodegenerative diseases such as Creutzfeldt-Jakob Disease (CJD) and Kuru in humans, and Bovine spongiform encephalopathy(BSE) and scrapie in animals. The umbrella term for these diseases is transmissible spongiform encephalopathy (TSE), which is used in this Recommendation. A prion originates in a PrP gene.The transcription and translation of this gene generates the physiologically apathogenic PrPprotein (PrPc; c = cellular). The essential, or possibly the only, component of a prion is apathologically misfolded form of the PrP protein, PrPsc (sc = scrapie-associated). PrPsc in turn leads to a further refolding of PrPc to PrPsc, producing an autocatalytic chain reaction. Thus PrPsc collects in the affected tissues, primarily in the brain, sometimes leading to irreversible lethal pathological changes. Prions can be transmitted orally, iatrogenically or via wounds. Thus persons who handle prions are at risk of becoming infected, with a TSE as a consequence.


Legal bases


• Agents causing TSE are classified under group 3** (FOEN Classification of Organisms. Module 2: Viruses; December 2011).


• The term group 3** is used for organisms that possess a limited risk of infection to workers because air-borne transmission, according to current scientific knowledge, cannot normally occur. Transmission can only take place through contact with infected tissue, body fluids or contaminated medical instruments.


• It is now certain that humans can become infected through the oral uptake of a BSE pathogen. Thus, work must be carried out in laboratories of level 3**, although the omission of particular safety measures may be requested (see below)


• No link to human TSEs has yet been shown for other animal TSEs (scrapie, chronic wasting disease (CWD)). The safety measures of a level 2 laboratory are therefore sufficient.


Safety measures for work using TSE agents


• The laboratory must satisfy the safety measurements of level 3; the omission of particular safety measures may however be requested (see below).


• An airlock with washing and disinfection facilities must be available, but a shower is not necessary.


• All studies in which aerosols may be produced must be performed in a class II microbiological safety cabinet.


• An autoclave must be able to reach temperatures of 134°C (validated). It must also be available in the same laboratory if possible, or at least in the same building.


• Wastes and waste water must be decontaminated.


• A list of the laboratory personnel who have been in contact with prions must be kept for at least the period prescribed in the Contain O and the PEMO.




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Measures that could be omitted when working with prions, since the danger to the environment is relatively small:


• Filtration of air exiting the laboratory with a HEPA filter


• Negative air pressure in the laboratory


• Ability to seal the area for gassing, since prions cannot be destroyed by gas (e.g.formaldehyde). Risk assessment for work with prion proteins and prion genes The following findings from scientific research must be borne in mind when the risks associated with handling prions are being assessed. Studies with native and with mutant recombinant prion proteins are also included in the considerations.


• Experimental studies on scrapie in mouse and in hamster have been carried out for several decades. There are no indications that the scrapie agent represents an increased risk either for occupationally exposed persons or for the public and the environment. To date, it has not been demonstrated that scrapie can be transmitted to humans. However, rodents infected with scrapie agents should be handled using the same safety precautions as rodents infected with natural, mouse-specific pathogens (classified into group 2).


• It has been shown that the primary structure of the prion protein plays an important role in transmission of prions from one species to another (Prusiner et al., 1990; Colby et al.,2011). The sequence of the bovine PrP gene shows greater homology to the sequence of the human gene than the sequence of the mouse or hamster gene (Gabriel et al., 1992).Although it is possible to transmit bovine TSE agents to humans, it appears unlikely that mouse- or hamster-specific TSE agents can be transmitted to humans.


• It is now possible to transform native recombinant PrPc or PrPc from tissue extracts into infectious prions (PrPsc). Particularly when using PMCA technology (Protein Misfolding Cyclic Amplification), infectious prions reproduce efficiently in the laboratory (Castilla et al.,2005; Wang et al., 2010). It is vital that this be taken into account in the risk assessment.


• Certain activities using PrP genes should be treated with caution. Bearing in mind the current gaps in knowledge, the following activities present an increased risk to humans and the environment: (1) cloning of PrP genes in host organisms that could replicate in/on humans, and (2) the cloning and expression of modified PrP genes in prokaryotic and eukaryotic expression systems where there is a possibility of producing infectious PrPSc.


• Mutations in the DNA sequence of the PrP gene, without a host organism, poses a negligible risk according to the current state of science. This activity may thus be classified under Class 1. Classification of work with TSE agents Based on the risk assessment above, the following recommendations can be made for work with prions and prion proteins (see also Table on p. 5):




• Autopsy of sick cattle should be done in accordance with the Epizootic Diseases Ordinance and according to special clinical regulations, in principle using the safety measures of level 2(see Diagram on p. 6). Infected cattle used for research purposes can be kept in safety level 1, since transmission via aerosol can be virtually ruled out.




Page 5




• Activities with animals infected with rodent-adapted scrapie prions, i.e. animal husbandry and care, invasive procedures (e.g. inoculation with prions), and post-mortem examinations and tissue sampling from animals, are classified as class 2.


• Activities with live mice and hamsters infected with BSE prions, BSE-related prions or human TSE agents (e.g. animal husbandry and care), are classified as class 2. The risk of bite and scratch wounds is reduced by handling the animals with long, blunt forceps.Invasive procedures (e.g. inoculation with TSE agents) as well as post-mortem examinations of infected animals and tissue sampling are classified under class 3.




All experimental studies involving lymphoid and neural animal tissue that contains rodent-adapted scrapie prions are classified as class 2. Activities using tissue from humans and animals that contains human or bovine TSE agents are classified as class 3.


Classification of work using genetically modified PrP genes and proteins


Origin of the PrP genes


sheep, rodent cattle, human


Cloning of DNA in plasmid with inactive promoter 1 1


State of the protein


PrPc PrPSc PrPc PrPSc


Expression in prokaryotic and eukaryotic expression systems that replicate in/on humans 1 2 1 2


Expression in hosts that replicate in/on humans 1 2 2 3




Page 6




Overview of the classification of pathological work using prions




Page 7






• Advisory Committee on Dangerous Pathogens, Spongiform Encephalopathy Advisory Committee. Transmissible spongiform encephalopathy agents: safe working and the prevention of infection. London (1990). HSE Books, PO Box 1999, Sudbury, Suffolk CO106FS.


• Prusiner SB, Scott M, Foster D, Pan KM, Groth D, Mirenda C, Torchia M, Yang SL, SerbanD, Carlson GA, et al. Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63, 673-86 (1990).


• Colby DW and Prusiner B. De novo generation of prions. Nature 9, 771-777 (2011).


• Gabriel JM, Oesch B, Kretzschmar H, Scott M, Prusiner SB. Molecular cloning of a candidate chicken prion protein. Proc Natl Acad Sci U S A 89, 9097-101 (1992).


• Council Directive of 26 November 1990 on the protection of workers from risks related to exposure to biological agents at work of 26 November 1990 (90/679/EEC). Official Journal of the European Communities No. L 374/1 of 31.12.1990, and modifications up to and including 6.12.1997 (97/65/EC).


• Risk assessment of laboratories involving the manipulation of unconventional agents causing TSE. (2009) isp/wiv.


• Guidance from the ACDP TSE Risk Management Subgroup


• Castilla et al. In vitro generation of infectious scrapie prions (2005) Cell 121, 195-206


• Deleault et al. Formation of native prions from minimal components in vitro (2007) PNAS104, 9741-9746


• Wang et al. Generating a prion with bacterially expressed recombinant prion protein (2010)Science, 327, 1132-1135



Classification of activities using prion genes and ... - EFBS -






TSE Transmissible spongiforme encephalopathic agents


Biological agents associated with transferable spongiform encephalopathies are classified as 3**.


Work with these agents must be done according to safety level 3, though certain level 3 measures can be omitted (see Classification of work using prion genes and prion proteins, ).


Classification of Organisms. Part 2- Viruses. Status January 2013





Sunday, October 27, 2013


A Kiss of a Prion: New Implications for Oral Transmissibility




From: Terry S. Singeltary Sr.


Sent: Monday, October 28, 2013 11:48 AM





Re: [CJDVoice] A Kiss of a Prion: New Implications for Oral Transmissibility


hi xxxxx,


no, I can’t really put your mind to rest xxxxx. wish I could. I believe the kiss of the prion was done several years ago. the deaths of cjd to man and wife, man and cat, friend friend and friend, with what Brown et al stated with the man and his wife i.e. ‘ therefore, we are left with the unanswerable alternatives of human-to-human transmission or the chance occurrence of sporadic CJD in a husband and wife.’ it is what it is, however, I would not loose any sleep over it. we are (in my opinion), exposed to the TSE prion in so many different ways in every day life. the potential for exposure and then becoming infected via taking care of a loved one with TSE prion disease, in my opinion risk factor there from is minimal, if proper precautions are taken, and even if they were not, the chance of becoming infected from a kiss, or casual contact is low, but I do not think it is zero, actually, far from it. I put this all together for a documentation of the known facts to date, of the potential casual human to human transmission. I did not put it together to scare anyone. with aerosol transmission of the TSE prion a reality now, infectivity in urine and feces and transmission there from being reality now with the TSE prion disease, I don’t see how anyone can rule _out_ the potential for transmission of the TSE prion via a kiss (a vehicle for transmission of the TSE prion via saliva), or even for a cut or open wound (all a cut is and transmission there from, is an crude inoculation of sorts, and inoculation has been proven to be an efficient mode of transmission for the TSE prion disease), even the eye, from either one of the body fluids now that how proven to be infections. I can’t see why we have such safety protocols for laboratory workers working with the TSE prion disease, but yet the same officials will say it’s o.k. for the public, friends, and or family members to do just the opposite with their loved ones when succumbing to the CJD TSE prion disease. don’t get me wrong, I did it too, and would probably do it again as far as kissing my mom. but science is science, and the transmission studies speak for themselves with the bodily fluids. simply put, which is all I was saying, we can’t say never, and or that none of these cases to date, have not been, and or will not be, a potential vehicle for transmission. I believe, and this is my opinion, that more concern for casual transmission with body fluids and materials there from, should be put forth to families with their loved ones, and I think that the safety protocols there from should be revised, to match that of the laboratory settings. again, this is my opinion. your opinion, and or others here, may read the same science and feel different out the findings. ...take care, kind regards, terry










Name: Creutzfeldt-Jakob agent, Kuru agent


Synonym or Cross Reference:: Subacute spongiform encephalopathy, Creutzfeldt-Jakob disease (CJD), Kuru, Transmissible Spongiform Encephalopathy (TSE).


Characteristics: Filterable, self-replicating agent, slow infectious pathogen, prion protein (PrP)




Containment Requirements: Biosafety level 3 facilities, practices and containment equipment for activities involving these agents; also listed under biosafety level 2 with special precautions; level of containment will depend on the nature of the manipulations and the amount of sera, bio/necropsy materials handled


Protective Clothing: Gown and gloves when handling potentially infectious materials; eye protection may also be indicated


Other Precautions: Extreme care must be taken to avoid accidental autoinoculation or other parenteral inoculations of infectious tissues and fluids




Spills: Allow any potential aerosols to settle; wearing protective clothing, gently cover spill with paper towel and apply 1N sodium hydroxide, starting at perimeter and working towards the center; allow sufficient contact time (1 hour) before clean up


Disposal: Decontaminate before disposal; steam sterilization (132·C for 1 hour), disinfection with 1N sodium hydroxide for 1 hour, incineration


Storage: In sealed containers that are appropriately labeled




The main precaution to be taken by laboratorians working with prion-infected or contaminated material is to avoid accidental puncture of the skin.3 Persons handling contaminated specimens should wear cut-resistant gloves if possible. If accidental contamination of unbroken skin occurs, the area should be washed with detergent and abundant quantities of warm water (avoid scrubbing); brief exposure (1 minute to 1N NaOH or a 1:10 dilution of bleach) can be considered for maximum safety.6 Additional guidance related to occupational injury are provided in the WHO infection control guidelines.6 Unfixed samples of brain, spinal cord, and other tissues containing human prions should be processed with extreme care in a BSL-2 facility utilizing BSL-3 practices.


Bovine Spongiform Encephalopathy Although the eventual total number of variant CJD cases resulting from BSE transmission to humans is unknown, a review of the epidemiological data from the United Kingdom indicates that BSE transmission to humans is not efficient.9 The most prudent approach is to study BSE prions at a minimum in a BSL-2 facility utilizing BSL-3 practices. When performing necropsies on large animals where there is an opportunity that the worker may be accidentally splashed or have contact with high-risk materials (e.g., spinal column, brain) personnel should wear full body coverage personal protective equipment (e.g., gloves, rear closing gown and face shield). Disposable plasticware, which can be discarded as a dry regulated medical waste, is highly recommended. Because the paraformaldehyde vaporization procedure does not diminish prion titers, BSCs must be decontaminated with 1N NaOH and rinsed with water. HEPA filters should be bagged out and incinerated. Although there is no evidence to suggest that aerosol transmission occurs in the natural disease, it is prudent to avoid the generation of aerosols or droplets during the manipulation of tissues or fluids and during the necropsy of experimental animals. It is further strongly recommended that impervious gloves be worn for activities that provide the opportunity for skin contact with infectious tissues and fluids.




The main precaution to be taken when working with prion-infected or contaminated material is to avoid puncture of the skin. If accidental contamination of skin occurs, the area is swabbed with In sodium hydroxide (NaOH) for 5 minutes and then washed with copious amounts of water. Unfixed samples of brain, spinal cord, and other tissues containing human prions should be processed with extreme care at BSL 3.


Prions are characterized by extreme resistance to conventional inactivation procedures including irradiation, boiling, dry heat, and chemicals (formalin, betapropiolactone, alcohols). Sterilization of rodent brain extracts with high titers of prions requires autoclaving at 132C for 4.5 hours. Denaturing organic solvents such as phenol or chaotropic reagents such as guanidine isothiocyanate or alkali such as NaOH can also be used for sterilization. Disposable plasticware, which can be discarded as a dry waste, is highly recommended.


Although there is no evidence to suggest that aerosol transmission occurs in the natural disease, it is prudent to avoid the generation of aerosols or droplets during the manipulation of tissues or fluids and during the necropsy of experimental animals. Formaldehyde-fixed and paraffin-embedded tissues, especially of the brain, remain infectious. Some investigators recommend that formalin-fixed tissues from suspected cases of prion disease be immersed for 30 min in 96% formic acid or phenol before histopathologic processing, but such treatment may severely distort the microscopic neuropathology.




another interesting aspect of the TSE prion disease is KURU ;


Figure 25. All cooking. including that of human flesh from diseased kinsmen. was done in pits with steam made by pouring water over the hot stones, or cooked in bamboo cylinders in the hot ashes. Children participated in both the butchery and the handling of cooked meat, rubbing their soiled hands in their armpits or hair, and elsewhere on their bodies. They rarely or never washed. Infection with the kuru virus was most probably through the cuts and abrasions of the skin. or from nose-picking, rye (eye...tss) rubbing, or mucosal injury.




These detailed descriptions will be published elsewhere but have reaffirmed the oral histories of endocannibalism in the Fore recorded previously12,22–24 and that this practice ceased abruptly at the time of Australian administrative control over the kuru areas. Although isolated events might have occurred for a few years after this prohibition, we are confident that new exposures of individuals to kuru at mortuary feasts would not have occurred after 1960. Not only have no cases of kuru been recorded in people born after 1959 (and only nine were recorded in those born after 1956); but also all the 11 last recorded cases of kuru that we report here were born before 1950. If any source of infection remained, whether from surreptitious cannibalism, possible ground contam-ination with human prions at sites where food was prepared, or other lateral routes, we would expect individuals born after this period to have kuru—especially since children are thought to have had shorter incubation periods than adults. However, no such cases have been observed. Additionally, although a fraction of hamster-adapted scrapie prions have been shown to survive in soil for at least 3 years,25 the mortuary feast practices (during which the entire body would be consumed) were undertaken so that any substantial contamination of soil would not have occurred, and traditional bamboo knives and leaf plates were burned after the feast. Furthermore, no clusters of kuru cases, as seen earlier in the epidemic,26 have been recorded for many years....




Kuru: The Science and the Sorcery


Special Jury Prize Winner, Pacific International Documentary Film Festival 2011.


This is the true story of one of the most incredible and challenging medical detective stories of the 20th Century; a history of human tragedy, adventure and discovery. It is the story of the Fore, a Papuan community immersed in cannibalistic mortuary practices and sorcery in one of the most remote regions on the planet, and the tragic disease that threatened to wipe out their entire population.


In 1961, a young Australian medical researcher, Michael Alpers, puts up his hand to work on a new and strange disease in the Eastern Highlands of Papua New Guinea. There, he teams up with an American outer, Dr Carleton Gajdusek, who has been in the local Fore region since 1957. For Michael it is the beginning of a lifelong obsession.


Together, they are amidst a major epidemic. It is killing over 200 people a year with devastating effects. It mainly targets women and children. The local people, the Fore, call the disease kuru, their word for shivering. They believe it is caused by sorcery.


Michael and Carleton are baffled by the disease. There are no scientific disciplines to guide them as they attempt to unravel its mysteries. By pure chance, a link is made to a strange transmissible animal disease in sheep, Scrapie. The two kuru researchers embark on a 10-year experiment to see if the fatal degenerative brain disease in humans could be transmissible like Scrapie.


The decision is made to perform an autopsy on a kuru victim and inoculate the kuru material into a chimpanzee. Kigea, ayoung girl in the village is identified as being in the early stages of kuru. Kigea’s family, gives Michael permission to perform an autopsy upon her death.


A brain sample taken from Kigea after her death is flown to the USA and injected into a chimpanzee called Daisy. While Michael follows the progress of the transmission experiment, he starts to collate all the recorded data on kuru and begins to suspect cannibalism as the cause of the spreadof the disease.


Within two years, he diagnoses Daisy with kuru. This is a defining moment. It confirms kuru is transmissible and can cross the species barrier. The revelation, together with epidemiological data collated with anthropologist Shirley Lindenbaum, links the Fore’s mortuary feasts (consumption of dead relatives) to the transmission of kuru. Cannibalism is the cause, and its origin is linked to a rare disease called Creutzfeldt Jakob Disease(CJD), but the story of kuru is far from over.


The infecting agent is the first new pathogen – prions – to be discovered in over 100 years. Research results in two Nobel prizes: it’s discoveries turning scientific understanding upside down, causing rifts in the beliefs ofthe science community.


Then Mad Cow Disease (Bovine Spongiform Encephalopathy or BSE) reared its head in the mid 1980s, and 10 years later the human variant CJD. All eyes turned to kuru, the only model of a prion epidemic in human populations. Many unknowns still surround prion diseases: there is no cure for kuru, or any of the prion diseases. The effects are devastating and unprecedented incubation periods can extend beyond 50 years.


Michael is the key and heart to this story, providing unique access to the Fore people, and the world’s other leading authorities on the matter; including Americans Prof. DC Gajdusek (Nobel Prize 1976), Prof. Stan Prusiner (Nobel Prize 1997), Prof Shirley Lindenbaum (Anthropologist) and British Prof. John Collinge (Director, MRC Prion Unit, UK).


Kuru: The Science and the Sorcery combines history, science and anthropology to tell a unique and ongoing ‘history of science’ documentary spanning five decades. It intertwines the thinking of great minds, locally and internationally, to reveal how this rare disease in the remote highlands of PNG exploded to international attention and how Prion research has now revealed we are all descendants of a remote past of cannibal practices.




Kuru: The Science and the Sorcery Australian scientist Michael Alpers dedicated over 50 years to researching Kuru, an obscure and incurable brain disease unique to the Fore people of New Guinea. Kuru was once thought to be a psychosomatic illness, an infection, a genetic disorder, even a sorcerer's curse, but Alpers' findings pointed to cannibalism as the culprit. Yet a recent discovery has proven to be even more disturbing: the malady is linked to mad cow disease and its human equivalent, variant CJD. With a decades-long incubation period, could a larger outbreak be on its way?





human flesh taste very sweet








Michael Alpers




First Reports




People of the Kuru region part 1


boy playing with animal bladder, blowing it up like a balloon. ...




People of the Kuru region part 2




Monday, November 19, 2012


Prion in Saliva of Bovine Spongiform Encephalopathy–Infected Cattle





kind regards, terry






*** Saturday, November 2, 2013 ***


Exploring the risks of a putative transmission of BSE to new species




Wednesday, September 25, 2013


Presence of subclinical infection in gene-targeted human prion protein transgenic mice exposed to atypical BSE




Wednesday, October 30, 2013






Monday, August 26, 2013


The Presence of Disease-Associated Prion Protein in Skeletal Muscle of Cattle Infected with Classical Bovine Spongiform Encephalopathy




Tuesday, September 24, 2013


NORDION (US), INC., AND BIOAXONE BIOSCIENCES, INC., Settles $90M Mad Cow TSE prion Contamination Suit Cethrin(R)


*** Case 0:12-cv-60739-RNS Document 1 Entered on FLSD Docket 04/26/2012 Page 1 of 15 ***




Tuesday, July 2, 2013


APHIS USDA Administrator Message to Stakeholders: Agency Vision and Goals Eliminating ALL remaining BSE barriers to export market




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





*** The discovery of previously unrecognized prion diseases in both humans and animals (i.e., Nor98 in small ruminants) demonstrates that the range of prion diseases might be wider than expected and raises crucial questions about the epidemiology and strain properties of these new forms. We are investigating this latter issue by molecular and biological comparison of VPSPr, GSS and Nor98.



Saturday, July 6, 2013


*** Small Ruminant Nor98 Prions Share Biochemical Features with Human Gerstmann-Sträussler-Scheinker Disease and Variably Protease-Sensitive Prionopathy


Research Article




Wednesday, October 09, 2013






Thursday, October 10, 2013


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




Monday, October 14, 2013


Researchers estimate one in 2,000 people in the UK carry variant CJD proteins




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 $$$



Sunday, August 11, 2013


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




Sunday, September 08, 2013


Iatrogenic Creutzfeldt-Jakob disease via surgical instruments and decontamination possibilities for the TSE prion




Thursday, September 26, 2013


Minimise transmission risk of CJD and vCJD in healthcare settings Guidance




Sunday, June 9, 2013


TSEAC March 14, 2013: Transmissible Spongiform Encephalopathies Advisory Committee Meeting Webcast




Tuesday, May 28, 2013


Late-in-life surgery associated with Creutzfeldt-Jakob disease: a methodological outline for evidence-based guidance




*** U.S.A. 50 STATE BSE MAD COW CONFERENCE CALL Jan. 9, 2001 ***





Tuesday, March 05, 2013


A closer look at prion strains Characterization and important implications Prion


7:2, 99–108; March/April 2013; © 2013 Landes Bioscience






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