The work was supported by a grant from the Creutzfeldt Jacob Disease Foundation. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

All procedures involving animals were approved and in compliance with the Guide for the Care and Use of Laboratory Animals by the Creighton University Institutional Animal Care and Use Committee.
NOTE: A schematic overview of the prion safety laboratory swipe test is shown in Figure 1.
1. Selection of swabbing sites and preparation for surface swabbing
<ol>
	<li>Identify and label appropriate surveillance areas for swabbing. Refer to the template provided for documentation and reference (Figure 2 and Supplementary Figure 1).</li>
	<li>Prepare two 1.5 mL microcentrifuge tubes for each of the identified areas. Add 250 &#181;L of Dulbecco&#39;s phosphate-buffered saline to the first set of microcentrifuge tubes for use in step 3.2. Let the second set of microcentrifuge tubes remain empty for use in step 4.3.</li>
	<li>Retrieve foam-tipped swabs from storage in an area free of prion contamination.</li>
	<li>Prepare a squeeze bottle with Milli-Q water (MQ H2O) for use in step 2.1.</li>
</ol>
2. Positive and negative control swab preparation
<ol>
	<li>Prepare dilutions for positive and negative swab controls using relevant control types. Example: For positive control, prepare a 1% dilution of prion-infected brain homogenate in Dulbecco&#39;s phosphate-buffered saline (DPBS). For negative control, prepare a 0.1% dilution of uninfected (UN) brain homogenate (BH) from the same species as the positive control in DPBS.</li>
	<li>Using clean gloves, retrieve the appropriate number of swabs from the clean packaging and place the handle side down into a tube rack, taking care to space out foam swabs so that the tips do not contact other swabs or surfaces. For each control sample, prepare three swabs. Change gloves between each sample.</li>
	<li>Holding a clean foam swab by the handle, apply 50 &#181;L of respective positive and negative control samples to foam swab tips. Make sure to apply to both sides of the swab tip to ensure complete absorption. 
	NOTE: Always change gloves prior to removing a clean swab from the tube rack.</li>
	<li>Using scissors, cut off the excess handle of the swab (approximately &#189; of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.</li>
	<li>Continue applying control samples after changing gloves until all samples have been applied.</li>
</ol>
3. Surface swabbing
<ol>
	<li>Holding a clean foam tip swab by the handle, prewet the foam tip with MQ H2O and shake off the excess. Place the moistened foam tip of the swab onto the area chosen for surveillance and swab the area back and forth approximately ten times while simultaneously rotating the tip of the swab on the surface.</li>
	<li>Using scissors, cut off the excess handle of the swab (approximately &#189; of the length) and place the swab into the preloaded microcentrifuge tube with the foam tip portion pointed down. Ensure that the foam tip is submerged in the DPBS and that the handle is cut adequately to close the lid of the microcentrifuge tube completely.</li>
	<li>Discard gloves and place new gloves on before each respective swabbing site to minimize the probability of cross contamination.</li>
	<li>Repeat the procedure until all areas chosen for surveillance have been swabbed.</li>
</ol>
4. Swab extraction and vacuum concentration
NOTE: Turn on the vacuum concentrator 30 min prior to use to allow the instrument to warm up.
<ol>
	<li>Place microcentrifuge tubes into a circular tube rack. Place the microfuge tube rack into the cup horn sonicator water bath. Ensure the foam swabs in DPBS within the microcentrifuge tubes are below the surface of the water in the cup horn (the handle portion does not have to be submerged). Apply the following settings: 15 s total run time (5 s on, 5 s off) at ~75-85 watts.</li>
	<li>Following sonication, centrifuge the tubes for approximately 15 s to collect DPBS in the bottom of the tube prior to transfer.</li>
	<li>Using a P1000 pipette set to 250 &#181;L, carefully collect all liquid (swab extract) from the bottom of the microcentrifuge tube and transfer into the corresponding microcentrifuge tube from the second, empty prelabeled set. Use the pipette tip to squeeze any excess liquid from the foam tip. Discard empty tubes containing swabs.</li>
	<li>Set the following settings on the vacuum concentrator: Temperature: 45 &#176;C, Heat time: 15 min, Run time: 2 h, Vacuum: 5.1.</li>
	<li>Place the microcentrifuge tubes containing swab extracts into the vacuum concentrator, ensuring the tubes are balanced and all tube caps are open.</li>
	<li>Upon cycle completion, ensure that samples are completely concentrated (only the pellet remains). Store the pellets at -80 &#176;C until utilized for RT-QuIC. 
	NOTE: In some instances, additional vacuum concentration time may be required to ensure complete concentration. Remove all concentrated samples, leaving only the tubes that still contain liquid. Rebalance the remaining tubes within the concentrator and run at additional 1-h increments until samples are completely concentrated.</li>
</ol>
5. Preparation of swabbing controls for use in RT-QuIC
NOTE: The RT-QuIC controls should be performed prior to the assay of environmental swab extracts to ensure that contamination has not been introduced during the swabbing, extraction, or concentration procedures. For example layouts, see Figure 3 and Figure 4.
<ol>
	<li>Prepare an appropriate negative RT-QuIC plate control (e.g., uninfected brain homogenate [UN BH]) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare a positive plate control dilution by diluting prion-infected brain homogenate to a dilution of 10-3 in tissue dilution solution.</li>
	<li>Remove previously stored control swab extract pellets from -80 &#176;C and resuspend each with 50 &#181;L of MQ H2O by pipetting up and down approximately 10 times, followed by vortexing briefly. Allow samples to sit at room temperature (RT) while performing the remaining steps.</li>
	<li>Load 2 &#181;L of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate).</li>
	<li>Load 2 &#181;L of each positive and negative swab extract technical replicate into a minimum of four replicate RT-QuIC plate wells. 
	NOTE: If negative swabbing controls exhibit RT-QuIC seeding above a laboratory&#39;s standard for a positive determination, this would indicate that contamination had been introduced during the swabbing, extraction, and concentration process and that the swabbing experiment should be performed again</li>
</ol>
6. Preparation of samples for use in RT-QuIC
<ol>
	<li>Prepare an appropriate negative plate control (e.g., uninfected brain homogenate, uninfected lymph node, etc.) by diluting to the appropriate concentration in tissue dilution solution (N2-0.1%SDS/PBS). Prepare positive plate controls by diluting infected brain homogenate to a dilution of 10-3 in tissue dilution solution.</li>
	<li>Remove previously stored swab extract pellets from -80 &#176;C and resuspend with 20-50 &#181;L of tissue dilution solution (depending on the suspected amount of contamination) by pipetting up and down approximately ten times, followed by vortexing briefly. Allow samples to sit at RT while performing the remaining steps.</li>
	<li>Load 2 &#181;L of specified plate controls, including negative plate controls (tissue dilution solution alone and uninfected brain homogenate) and a positive plate control (infected brain homogenate) into the RT-QuIC plate wells.</li>
	<li>Load 2 &#181;L of each swab extract into four replicate wells.</li>
</ol>
7. RT-QuIC analysis and results
<ol>
	<li>Perform RT-QuIC according to individual laboratory protocols (optional protocols38,40).</li>
	<li>Define the parameters required for determining the positivity of a sample (see representative results, Figure 5). 
	NOTE: These parameters are defined by each lab.</li>
	<li>Record results in the provided table and take appropriate action based on laboratory best practices (Table 1 and Supplementary Figure 2).</li>
</ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+determination+of+residual+prion+infectivity+from+prion-decontaminated+surfaces.">Rapid and sensitive determination of residual prion infectivity from prion-decontaminated surfaces.</a> mSphere. 9, e00504-e00524 (2024).</li><li>Orru, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+pathological+seeds+of+alpha-synuclein,+tau+and+prion+protein+on+solid+surfaces.">Sensitive detection of pathological seeds of alpha-synuclein, tau and prion protein on solid surfaces.</a> PLoS Pathog. 20 (4), e1012175 (2024).</li><li>Wilham, J. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+prions+and+amyloid+seeds+with+hypochlorous+acid.">Inactivation of prions and amyloid seeds with hypochlorous acid.</a> PLoS Pathog. 12 (9), e1005914 (2016).</li><li>Williams, K., Hughson, A. G., Chesebro, B., Race, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+chronic+wasting+disease+prions+using+sodium+hypochlorite.">Inactivation of chronic wasting disease prions using sodium hypochlorite.</a> PLoS One. 14 (10), e0223659 (2019).</li><li>Taylor, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoclaving+standards+for+Creutzfeldt-Jakob+disease+agent+[letter].">Autoclaving standards for Creutzfeldt-Jakob disease agent [letter].</a> Ann Neurol. 22 (4), 557-558 (1987).</li><li>Brown, P., Rohwer, R. G., Gajdusek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newer+data+on+the+inactivation+of+scrapie+virus+or+Creutzfeldt-Jakob+disease+virus+in+brain+tissue.">Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob disease virus in brain tissue.</a> J Infect Dis. 153 (6), 1145-1148 (1986).</li></ol>

J.C.B. and Q.Y. are inventors on a patent application pertaining to prion surface swabbing technology.

The described prion safety swabbing method can be used to enhance existing prion safety measures. This method can monitor prion laboratory spaces and equipment, as well as shared laboratory spaces, for potential prion contamination. Importantly, this method can be adapted to test laboratory-specific disinfection techniques to verify decontamination of prion-contaminated surfaces. As various prion strains display different sensitivities to disinfection techniques, this method can confirm that these techniques are effective for current laboratory experiments, such as treatment with sodium hydroxide or sodium hypochlorite (bleach)43,44.
Key steps for this methodology include the identification of appropriate swabbing sites that will provide a sample of both high-traffic areas as well as areas to monitor that may be involved with downstream cross-contamination of experiments. Additionally, laboratories should utilize positive and negative controls that closely mirror those commonly used in a given lab or clinic. For example, if working with rodent prions, the positive and negative controls should match that species. Finally, surveillance data should be kept up to date and organized so that trends in positivity can easily be identified, thus allowing for mitigation and retraining of laboratory staff in the event of consistent positive results for a given area.
One limitation of this protocol is the potential generation of erroneous RT-QuIC results. Given the sensitive nature of RT-QuIC, the presence of residual detergents, salt, and other substances on laboratory surfaces can impact the outcome of the reaction39. Overall, given this observation, it is advantageous that surfaces remain free of substances that can interfere with RT-QuIC, such as residual salt, detergents, and dust. As improvements to the RT-QuIC methodology become available, we anticipate that many of the current limitations of RT-QuIC will be resolved. We, therefore, encourage the end user to keep abreast of the literature to take advantage of the latest improvements in RT-QuIC.
A key benefit of this method for laboratories researching prions is the ability to minimize contamination that may impact the results of sensitive amplification assays. Necropsy tools are commonly disinfected prior to being reused for future necropsies. The described swabbing method provides a method to assess necropsy tools for residual prion infectivity that may affect downstream results. This can provide an additional level of rigor to experiments to exclude the possibility that prion detection in tissues was not due to contamination from the necropsy tools.

Prion diseases are invariably fatal neurodegenerative diseases with no known treatment or cure. Prion diseases are caused by PrPSc, the misfolded and pathogenic form of the normal cellular form of the prion protein1,2,3,4,5, PrPC. Prion diseases are known to affect humans and several other animal species. One human prion disease, Creutzfeldt-Jakob Disease, CJD, has three known etiologies: sporadic, inherited, and acquired. Acquired CJD can occur as a result of accidental transmission (iatrogenic and occupational) and is thought to be the cause of Kuru in the Fore people of Papua New Guinea6.
Prion transmission has been associated with prion-contaminated medical devices and transplant materials7,8,9,10,11,12,13,14,15,16,17. Iatrogenic transmission of CJD can occur via blood, tissue, or from prion-contaminated surfaces18,19,20. For example, iatrogenic CJD can develop in patients following an electroencephalogram with electrodes previously used on an individual in the preclinical stage of CJD who then later succumbed to CJD21. More recent laboratory-based occupational transmission has also occurred where a laboratory worker contracted prion disease via a skin puncture with forceps used to handle brain slices from an animal infected with sheep-adapted BSE22,23. Such transmission scenarios could occur within clinical, laboratory, and diagnostic laboratory settings where prion samples are handled.
Prions resist common disinfection techniques and can persist and remain infectious on surfaces for extended periods of time24,25,26,27,28,29. Common disinfection techniques such as the use of ethanol, phenolic cleaners, hydrogen peroxide, various forms of radiation, and formaldehyde are inadequate for the inactivation of prions, allowing surfaces to retain infectivity30,31,32,33,34,35,36,37. These characteristics contribute to the transmission of prions during iatrogenic and occupational exposure.
Methods for the detection of environmental prions have only recently been developed. An environmental swabbing method coupled with real-time quaking-induced conversion (RT-QuIC) can assess residual prion infectivity from environmental surfaces as well as common laboratory surfaces following ineffective disinfection38,39,40,41,42. Here, we describe how this technique can be incorporated into a broader prion safety program. Overall, this method can allow for the monitoring of laboratory-dependent disinfection protocols, the investigation and proper documentation of contamination status, which can help ensure the validity of experiments by minimizing cross-contamination, the assessment of shared use spaces for prion contamination and allows for directional retraining of personnel based on commonly contaminated areas.

<table><tbody><tr><td>Fisherbrand&amp;nbsp;PurSwab Foam Swabs</td><td>Fisher brand</td><td>Catalog #14-960-3E</td><td></td></tr><tr><td>Milli-Q IQ 7005 Ultrapure Water System</td><td>MilliporeSigma</td><td>Q7005T0C</td><td></td></tr><tr><td>Mini-centrifuge, 6000 rpm</td><td>Southern Labware</td><td>MLX-306</td><td></td></tr><tr><td>Omega plate reader</td><td>BMG Labtech</td><td>FLUOstar Omega plate reader</td><td></td></tr><tr><td>Q700 sonicator</td><td>QSonica</td><td>Q700-110</td><td></td></tr><tr><td>Recombinant hamster prion protein</td><td>MNPRO</td><td>MNPROtein-Hamster</td><td>syrian hamster, amino acids 90-231</td></tr><tr><td>Savant speedvac&amp;nbsp;</td><td>Thermo Scientific</td><td>SPD1030-230</td><td></td></tr><tr><td>Silica nanospheres (50 nm)</td><td>nano Composix&amp;nbsp;</td><td>SISN50-25M</td><td></td></tr></tbody></table>

prion safety laboratory swipe test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrPC, into the misfolded and pathogenic form PrPSc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.

Written description of positive and negative results (including positive/negative plate and swab controls)
Negative control swabs are included in the surveillance swabbing to monitor for potential prion contamination that could be introduced during the swabbing, extraction, and concentration process. The first RT-QuIC plate performed for a given monthly surveillance should include the positive and negative swab controls. Successful negative controls fail to cross the positive fluorescence threshold (Figure 6A). This result would indicate that contamination had not been introduced during the experimental procedures. Successful positive control swab extracts would exhibit positive seeding in all replicate wells for a given sample (positive control swabs were contaminated with the hamster-adapted transmissible mink encephalopathy strain Hyper (HY TME) brain homogenate. The inclusion of a positive control dilution series allows for the determination of the sensitivity of prion detection for a given experiment (Figure 6A).
When examining surface swab extract samples, surfaces that fail to show seeding above the predetermined positive fluorescence threshold can be considered to be prion negative (Figure 6B). Conversely, prion-contaminated surface swab extracts will show seeding capabilities above the positive fluorescence threshold, although the maxpoint ratio (MPR) and time to fluorescence can vary compared to the included positive plate control (Figure 6B). The ability of the method to assess adequate disinfection is highlighted by the bleach-treated prion contaminated surfaces, which now fail to seed RT-QuIC (Figure 6B).
Importantly, while our laboratory defines a positive sample as a sample that passes the set positive fluorescence threshold in at least half of the replicate wells, it is necessary for each laboratory to set its own standards. The rate of amyloid formation (RAF) and time to fluorescence can also be used to help establish laboratory-specific thresholds for positivity.
We have observed surface artifact results that pass the positive fluorescence threshold but with altered kinetic curves and after an abnormally long time to fluorescence compared to positive control samples (Figure 6C). These results should be cautiously interpreted as they may be caused by the presence of dust or residual chemicals present on a surface. These findings highlight the necessity of general laboratory cleanliness, as well as criteria for differentiating between true positive and false positive seeding.
Disposal of solid and liquid biohazard waste should be done following a given institution&#39;s current biohazard disposal guidelines. Common prion disinfection techniques include treatment with sodium hydroxide, sodium hypochlorite (bleach), or autoclaving at 134 &#176;C for 18 min43,44,45,46.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67889/67889fig01.jpg" /> 
Figure 1: Schematic overview of prion safety laboratory swipe test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67889/67889fig02.jpg" /> 
Figure 2: Sample swabbing site layout. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67889/67889fig03.jpg" /> 
Figure 3:&#160;Sample experiment design for swab controls included prion safety laboratory swipe test. (A) Sample layout for negative and positive swab extract controls. (B) Negative and positive swab extract controls should be resuspended with 50 &#181;L of H2O. A 10-fold dilution of the resuspended swab extract should be generated by diluting it into a tissue dilution solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67889/67889fig04.jpg" /> 
Figure 4:&#160;Sample experiment design for surface swab extracts from prion safety laboratory swipe test. (A) Sample layout for surface swab extracts. (B) Surface swab extract samples should be resuspended with 20 &#181;L of tissue dilution solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67889/67889fig05.jpg" /> 
Figure 5: Monthly prion safety laboratory swipe test. Flowchart used for interpreting results and determining appropriate response. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67889/67889fig06.jpg" /> 
Figure 6: Representative surface swabbing experiment. (A) Surface swab control plate containing negative swab extract controls in triplicate (DPBS, uninfected hamster brain homogenate 10-4 and 10-10) and positive swab extract controls in triplicate (HY brain homogenate 10-3). (B) Representative swab extracts for benchtop, glass, and stainless steel (S.S.) surfaces that were swabbed prior to contamination, following contamination with HY 10-3, and following bleach treatment of contaminated surfaces. (C) Fluorescence tracing comparison of uninfected brain homogenate 10-4, HY brain homogenate 10-3, and the swab extract from the benchtop coated with a fine film of dust. Negative plate controls for panels A and B include a blank (tissue dilution solution) and uninfected hamster brain homogenate 10-4. Panel B includes the addition of uninfected brain homogenate 10-4 and the positive plate control of HY brain homogenate 10-3. A positive fluorescence threshold (illustrated by a red dashed line) was determined to be at 2. The maxpoint ratio (MPR) reported is the maximum fluorescence divided by the initial fluorescence reading obtained by the plate reader. Each point represents one technical well replicate for a given sample type. The mean and standard deviation are presented. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/67889fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sample swabbing site monthly documentation form. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: Swabbing site layout template. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/Supplementary Figure 1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Swabbing site monthly documentation form. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67889/Supplementary Figure 2.pdf" target="_blank">Please click here to download this File.</a>

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Prion Safety Laboratory Swipe Test

Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to ...

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

449 Views.  Creighton University. Onze multidisciplinaire groep probeert te begrijpen hoe een infectieus eiwit, een prion, complexe biologische taken kan uitvoeren. We zijn ook geïnteresseerd in hoe prionen jaren tot decennia in het milieu kunnen blijven bestaan. Na prionontsmettingsprocedures was er geen robuuste methode beschikbaar om de werkzaamheid van prioninactivatie te bepalen.Met ons onderzoek proberen we dit probleem aan te pakken. De zwabbermethode, in combinatie met RT-QuIC...

Video: Prion Veiligheid Laboratorium Swipe-test

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Medicine

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Detection of Abnormal Prion Protein by Immunohistochemistry

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform encephalopathies (TSEs). These neurodegenerative diseases affect humans and several animal species and include scrapie, zoonotic bovine spongiform encephalopathy (BSE), chronic wasting disease of cervids (CWD), and the newly identified camel prion disease (CPD). Diagnosis of TSEs relies on immunodetection of PrPSc by application of both immunohistochemistry (IHC) and western immunoblot methods (WB) on encephalon tissues, namely, the brainstem (obex level). IHC is a widely used method that uses&#160;primary antibodies (monoclonal or polyclonal) against antigens of interest in cells of a tissue section. The antibody-antigen binding can be visualized by a color reaction that remains localized in the area of the tissue or cell where the antibody was targeted. As such, in prion diseases, as in other fields of research, the immunohistochemistry techniques are not solely used for diagnostic purposes but also in pathogenesis studies. Such studies involve detecting the PrPSc patterns and types from those previously described to identify the new prion strains. As BSE can infect humans, it is recommended that biosafety laboratory level-3 (BSL-3) facilities and/or practices are used to handle cattle, small ruminants, and cervid samples included in the TSE surveillance. Additionally, containment and prion-dedicated equipment are recommended, whenever possible, to limit contamination. The PrPSc IHC procedure consists of a formic acid epitope-demasking step also acting as a prion inactivation measure, as formalin-fixed and paraffin-embedded tissues used in this technique remain infectious. When interpreting the results, care must be taken to distinguish non-specific immunolabeling from target labeling. For this purpose, it is important to recognize artifacts of immunolabeling obtained in known TSE-negative control animals to differentiate those from specific PrPSc immunolabeling types, which can vary between TSE strains, host species, and prnp genotype, further described herein.

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Abnormal prion proteins (PrPSc) are the disease-associated isoform of cellular prion protein and diagnostic markers of transmissible spongiform ...

Neuroscience

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Biology

Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

Source: Orge, L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64560">Detection of Abnormal Prion Protein by Immunohistochemistry.</a> J. Vis. Exp. (2023).
This video demonstrates a technique to detect misfolded prion protein in brain sections using immunohistochemistry. Upon treating the brain section with formic acid to denature prions and minimize infection risk, as well as unmasking the prion aggregates using heat-induced epitope retrieval, the sections are immunolabeled for the misfolded prion protein aggregates and observed under a microscope.

1. Tissue sectioning and slide preparation

	Cut sections of formalin-fixed, paraffin-embedded (FFPE) tissues at 3-5 &#181;m thickness using a microtome.
	...

Prion Safety Laboratory Swipe Test

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