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JoVE Journal Medicine

Medicine

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

Published: March 10, 2015 doi: 10.3791/52522
Automatic Translation
1, 2, 3, 4, 5
1USGS National Wildlife Health Center, 2Department of Soil Science, University of Wisconsin–Madison, 3Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin–Madison, 5Department of Neurology, University of British Columbia

Summary

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.

Abstract

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’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’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.

Introduction

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases with extended incubation periods that affect a variety of animals and humans. The putative etiological agent of TSEs is comprised of a misfolded isomer of the host prion protein (PrP) that is capable of self-propagation by template-driven conversion of the normal cellular form of PrP (PrPC) into an infectious, disease-associated form (PrPTSE) that accumulates in central nervous system tissues of the infected host1. Infectious mammalian prions generally transmit from host-to-host in a species-specific manner, which has given rise to the concept of the “TSE species barrier” limiting interspecies transmission events2. The biological determinants of the prion species barrier are not well understood. Amino acid sequence similarity between the infectious PrPTSE and the host PrPC can strongly influence whether conversion takes place3-5, but remains insufficient to explain all prion transmission events observed in vivo6,7.

Thus, characterization of TSE species barriers has largely relied upon animal challenge studies: exposing naïve animals from a given species to prions from another and measuring the resulting incubation time to disease onset and attack rate as indicators of transmission efficiency. Mice expressing PrPC from heterologous species are also used for this type of study8. The costs associated with transgenic mouse production and protracted prion bioassays, as well as ethical considerations of animal use, are obstacles to experimental investigation of TSE species barriers. Assessment of the human species barrier to TSEs relies on mice engineered to express human PrPC. These mice require long incubation periods to succumb to human TSEs or modifications to the human PrPC molecule for rapid disease onset9. Incubation periods for non-human TSEs in these mice may extend beyond the normal mouse lifespan making interpretation of negative results challenging. Non-human primates have also been used as proxies for studying human species barriers, but these studies are fraught with the same challenges as other types of animal experimentation and non-human primates may not precisely recapitulate disease as it proceeds in the human host.

Animal bioassays remain the “gold standard” method for measuring the susceptibility of a species to a TSE, but the obstacles, costs and ethics of these live animal studies have compelled investigation into alternatives. A number of in vitro assays, based on assessing the conversion of host PrPC to a proteinase K (PK)-resistant state (PrPres) when seeded by PrPTSE, have been developed and used to investigate TSE species barriers10-12. Examples of in vitro assays include cell-free conversion assays, protein misfolding cyclic amplification (PMCA), and the conversion efficiency ratio (CER) assay10-14. While none of these assays take into account peripheral factors involved in species barriers after natural infection, all can be useful to identify potentially susceptible hosts for TSEs.

Here we present the protocol for the CER assay, in which two denatured PrPC substrates derived from normal brain homogenates are used in bench top prion conversion reactions (Figure 1)13. PrPC in the substrate denatured at pH 7.4 can only be converted to PrPres by PrPTSE seeded into the reaction in the absence of a species barrier14. In contrast, denaturation of PrPC in the other substrate at pH 3.5 allows it to be converted to PrPres following incubation with PrPTSE from any species and serves as a control for conversion. The ratio of conversion of PrPC to PrPres in pH 7.4 substrate relative to that of the pH 3.5 substrate provides a measure of the species barrier. We have found that the CER assay predicts known species barriers of laboratory mice to various TSEs and have used the assay in efforts to predict the species barrier of numerous mammalian species, including bighorn sheep, to chronic wasting disease (CWD) and other TSEs14,15. Investigators interested in a tool allowing rapid screening of TSE species barriers or assessment of PrPC-to-PrPres conversion will find this methodology useful.

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Protocol

Animal work conducted at the USGS National Wildlife Health Center was performed in accordance with the NIH Office of Laboratory Animal Welfare guidelines and under institutional animal care and use committee protocol #EP080716. Tissues from hunter-harvested animals were gifts from the Wisconsin or Michigan Departments of Natural Resources.

1. Solution Preparation

NOTE: The solutions listed below are required for preparation of the CER assay substrate in Section 2 below. Prepare each of these solutions from higher concentration stock solutions; recommended concentrations for stock solutions will be given in parentheses after each respective chemical name listed. Prepare all solutions fresh upon substrate preparation and store at 4 °C until used.

  1. For lysis buffer, prepare a solution of the following in 10 mM Tris, pH 7.5 (1 M Tris, pH 7.5 stock solution recommended): 100 mM sodium chloride (NaCl, 1 M stock solution), 10 mM ethylenediaminetetraacetic acid (EDTA, 500 mM EDTA, pH 8.0 stock solution), 0.5% NP-40 (10% stock solution), 0.5% deoxycholate (DOC, 10% stock solution).
  2. For conversion buffer, prepare a solution of 0.05% sodium dodecyl sulfate (SDS, 10% stock solution) and 0.5% Triton X-100 (10% stock solution) in 1 phosphate buffered saline (PBS, 10, pH 7.4 stock solution).
  3. For chaotropic solutions, prepare two 3 M solutions of guanidine hydrochloride (GdnHCl, 8 M stock) in 1x PBS (10x stock solution). Adjust the pH of one of the solutions to 3.5 ± 0.05 using concentrated hydrochloric acid (HCl). Check that the pH of the second solution remains at pH 7.4 ± 0.05 and adjust using concentrated HCl or sodium hydroxide (NaOH) if necessary.

2. Prepare CER Assay Substrate Pairs

  1. Obtain healthy, non-TSE-infected brain tissue from species of interest and prepare a 10% weight-per-volume (w/v) homogenate in lysis buffer using any of the following methods: dounce, bead-mill, or mortar and pestle homogenization. Further refine resulting homogenates by subjecting them to syringe-and-needle homogenization, using needles of increasing gauge until substrate can be aspirated and expelled with ease through a 27 G syringe needle.
    1. For substrates prepared from rodents and other small mammals, homogenize whole brain(s); for substrates prepared from larger mammals, use obex (brainstem) tissue to prepare homogenates. When selecting the quantity of brain homogenate, prepare no more than 50% of the capacity of the centrifuge used for protein precipitation in step 2.6.
    2. If the quality of acquired brain tissue is questionable, assess PrPC levels by SDS-PAGE16 and immunoblot17 prior to substrate preparation.
  2. Divide brain homogenate into two equal volumes in separate labelled plastic conical tubes. Add GdnHCl (3 M solutions in 1 PBS) at either pH 3.5 or 7.4 to each tube in a 1:1 volume ratio to brain homogenate.
  3. Check the pH values of the two solutions. Adjust the pH of the pH 3.5 substrate to pH ± 0.05 using concentrated HCl. Ensure the pH of the other substrate remains at pH 7.4 ± 0.05 and adjust pH if necessary with HCl or NaOH as needed. Avoid overshooting pH values by judicious addition of acid or base.
  4. Rotate solutions on an end-over-end mixer at room temperature (RT) for 5 hr.
  5. Precipitate protein by adding four volumes of methanol to the substrate solutions in each conical tube, vortex to mix well, and incubate at -20 °C for 16-18 hr.
  6. Sediment samples by centrifugation at 13,000 × g for 30 min at 4 °C. Carefully decant and discard the supernatants and allow methanol to evaporate from the pellets. Use cotton-tipped applicators to assist in absorbing methanol clung to the inside of the tube. Do not allow pellets to over-dry and once methanol is no longer visible, proceed to the next step.
  7. Resuspend protein pellets in conversion buffer. Use a quantity of conversion buffer equal to the amount of brain homogenate starting material dispensed into each tube in step 2.2.
    NOTE: It is critical that the conversion buffer used to resuspend protein pellets from both pH 3.5 and 7.4-treated substrate preparations is the solution defined in step 1.2 above (which contains low levels of detergents and a physiological pH).
  8. Briefly sonicate substrate solutions in a cuphorn sonicator (10 sec at ~30% maximal power), aliquot into 0.5 - 1.0 ml volumes in labeled 1.5 ml microcentrifuge tubes. Perform a quality control immunoblot (step 2.9) on an aliquot of each substrate. Store other aliquots at -80 °C until ready to perform CER assay (Section 4).
  9. Perform quality control on CER substrate pairs prior to use (Figure 2). These steps ensure that CER substrates will provide optimal results in conversion studies.
    1. Ensure comparable amounts of PrPC in the two substrates. Compare PrP levels in 10-25 µl of each substrate by SDS-PAGE16 and immunoblotting17. If protein levels in the pH 7.4 and 3.5 substrates are within ~10% of each other, the substrate pairs are appropriate for use in CER studies.
      NOTE: The PrP immunoblots should not show evidence of extensive PrPC degradation. The PrP immunoreactivity should be mainly >20 kDa. Bands less than this molecular mass can be degradation products. Banding patterns should be roughly equivalent between substrate pairs.
    2. As the PrPC in both substrates should be PK sensitive, treat 10-25 µl of each substrate with PK at a final concentration of 100 μg/ml and digest samples at 1,000 rpm at 37 °C for 1 hr in the thermo-shaker. Assess any remaining PrP signal by SDS-PAGE16 and immunoblotting17. Substrates with PrPres are not appropriate for use in CER studies.

3. Prepare TSE Agent Seeds

  1. Obtain TSE-infected brain tissue from species of interest and prepare a 10% (w/v) homogenate in 1x PBS pH 7.4 using the methods listed in step 2.1 above.
    NOTE: Seeds could also be produced from other TSE-infected tissues outside the central nervous system, however, efficiency of conversion of brain derived substrates by seeds derived from TSE-infected peripheral tissues has not yet been experimentally assessed in the published literature.
  2. Aliquot resulting homogenate(s) into 100 - 300 µl volumes in 0.5 ml microcentrifuge tubes and store at -80 °C until ready to perform the CER assay.

4. CER Assay

NOTE: The CER assay involves addition of a relatively small amount of PrPTSE (provided in the “seed”) from one species into a relative excess of PrPC (provided in the uninfected brain “substrate”) from another species that has been partially denatured at either pH 3.5 or 7.4. Following an extended shaking incubation period, template-driven conversion of PrPC by PrPTSE in each reaction is assessed by the densitometric signal of PrPres remaining after PK digestion and immunoblot detection. Comparing PrPres levels in the substrates previously denatured at pH 3.5 vs. 7.4 provides a measure of the species barrier. An experimental overview of this assay procedure is provided in Figure 1.

  1. Slowly thaw uninfected CER substrate pairs (assay requires equal volumes of substrates previously denatured at both pH 3.5 and 7.4) and TSE-infected seed solutions by placing them on top of a bed of ice (approximately 1 hr thaw time).
  2. Once fully thawed, aspirate and then expel each substrate solution through a 27 G syringe needle several times to re-homogenize it. Briefly sonicate each TSE-infected seed solution for 10 sec at ~30% maximal power. Keep all solutions on ice while preparing conversion reactions.
  3. Label 5 individual low binding, thin-walled PCR tubes per TSE agent being tested.
  4. Prepare conversion reactions in these tubes by adding 5 μl of 10% TSE-infected seed solution to 95 μl of (a) CER substrate prepared at pH 7.4 and (b) CER substrate prepared at pH 3.5.
    1. For each experimental sample, prepare parallel reactions in CER substrate pairs previously denatured at both pH 3.5 and 7.4.
    2. Optimize ratios of TSE agent seed to uninfected brain substrate through empirical determination. Common seed:substrate ratios employed maintain a total reaction volume of 100 μl and include 1:99 μl, 2:98 μl and 5:95 μl. For most applications, the 5:95 μl seed:substrate volume ratio is appropriate and is what is presented in this protocol.
  5. Prepare three control samples per TSE agent being tested as follows: (a) add 5 µl of 10% TSE-infected seed solution to 95 µl conversion buffer as a control for input PrPres; add 5 µl conversion buffer to 95 µl substrate prepared at (b) pH 3.5 and (c) pH 7.4 as controls for non-specific, non-templated conversion.
  6. Briefly vortex each sample in its closed PCR tube to mix seed and substrate well. Follow with a momentary pulse in a low-speed bench top mini-centrifuge to remove any volume of the reaction mixture trapped in the PCR tube lid.
  7. Load samples in individually-labeled PCR tubes into a bench top thermo-shaker equipped to accept PCR tubes. Shake samples at 1,000 rpm at 37 °C for 24 hr.
  8. Following shaking, add sarkosyl to a final concentration of 2% (w/v) and PK to a final concentration of 100 μg/ml. Digest samples at 1,000 rpm at 37 °C for 1 hr in the thermo-shaker.
    NOTE: Addition of sarkosyl to samples post-conversion aids in PK digestion of PrPC. False positive signals in unseeded samples (Step 4.5) are virtually eliminated by addition of sarkosyl.
  9. Add SDS-PAGE sample buffer, heat samples to 95°C for 5 minutes and resolve remaining PrPres in each sample by SDS-PAGE16 followed by immunoblotting17.
    NOTE: After addition of sample buffer and heating, samples may be immediately processed or samples can be stored at -20 °C or -80 °C prior to immunoblotting. All data presented here were generated using the Novex NuPAGE gel and transfer systems. Individual samples from the CER assay were treated with sample buffer and reducing agent according to manufacturer’s instructions. Samples were subsequently heated at 95 °C for 5 min and 30 µl of each sample was loaded into a 12% bis-tris precast gels.
  10. Calculate the conversion efficiency ratio (CER) as a measure of species barrier. Measure levels of PrPres by densitometry17 and divide the total density of the pH 7.4 sample by that of the pH 3.5 sample. Multiply by 100 to produce a percentage.
  11. Compile data from independent experimental replicates to generate a mean CER ± standard deviation for a species with a given TSE agent.

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Representative Results

Successful use of the CER assay is largely dependent upon the quality of the substrate pairs used in conversion reactions. For this reason, following procedures to prepare CER substrate pairs, a quality control immunoblot should be performed (Figure 2). For both substrates, assay 10-25 μl by immunoblotting. PrPC should be easily detectable in each substrate and PrPC levels must be approximately the same between the two. Immunoreactivity will be mainly visible above 20 kDa, but smaller bands may also be evident. In our experience, the presence of smaller bands is species dependent. If lower molecular weight products are observed, their presence should be confirmed in both substrate pairs.

It is important to ensure that CER substrates are free from endogenous PrPres by treating substrates with PK (Figure 2). If PrPres is present, the animal used for substrate preparation may have been TSE-infected or subclinically-infected. Alternatively, the PrPC in the substrate may have misfolded to a PK-resistant state during denaturation or precipitation procedures. Independent from the reason, CER substrate containing PrPres is not acceptable for use in the conversion assay.

Laboratory mice are one of the most frequently used experimental animals in TSE research; as such, their susceptibility to most TSEs is well-characterized8, providing an in vivo benchmark against which to test the fidelity of the CER assay. Figure 3A presents results of CER assay conversion of PrPC from mouse (CD1 strain) substrate to PrPres by 1) the RML strain of mouse-passaged scrapie, an agent adapted to the mouse host18, 2) domestic sheep classical scrapie, an agent that can transmit to mice following a lengthy incubation period18, and 3) white-tailed deer (Odocoileus virginianus) CWD and 263K strain of hamster-passaged scrapie, agents to which mice are minimally or not susceptible19,20. Resulting PrPres levels were variable across mouse substrates denatured at pH 7.4 and seeded with the various TSE agents while PrPres was found in all substrates denatured at pH 3.5 and seeded with a TSE agent. Conversion ratios comparing the PrPres levels in the pH 7.4 and 3.5 substrates were independently calculated at least three times and means ± SD are shown in Figure 3B. For reactions seeded by RML, conversion ratios between pH 7.4 and 3.5 substrates were approximately 100%. For those seeded by scrapie, conversion in the pH 7.4 substrate was approximately 75% of that in the pH 3.5 substrate. CWD or 263K-induced conversion of the pH 7.4 substrate was minimal. A one-way analysis of variance could not distinguish a difference in the conversion ratios of RML and scrapie or CWD and 263K, however conversion ratios of RML and scrapie were significantly different from those of CWD and 263K.

The CER assay can be adapted for use with human tissues or with animal species where bioassays are too challenging or not ethical and transgenic mouse production is not desired. We have been using this assay to characterize the susceptibility of various wildlife species to CWD. As an example of the use of this assay, we present some preliminary results in Figure 4. Brains from hunter-trapped bobcats (Lynx rufus) were found to still contain detectable levels of PrPC and PrP breakdown products were not extensive, suggesting that these tissues could be an acceptable source for CER substrate (Figure 4A). Substrate pairs generated from bobcat brain showed PrP immunoreactivity of an appropriate molecular weight and did not contain endogenous PrPres (Figure 4B). When bobcat CER substrate pairs generated from two separate animals were incubated with the same white-tailed deer CWD agent used in the mouse CER assay described in Figure 3, we found substrates prepared at pH 7.4 had levels of PrPres compared to substrates prepared at pH 3.5 (Figure 4C), indicative of a minimal species barrier of bobcat PrPC to conversion by CWD. The CER for conversion of white-tailed deer PrPC by the same CWD isolate used to convert bobcat PrPC here, as a positive control, was previously reported as 95.2 ± 18.4% in Morawski et al. (2013)15.

Figure 1
Figure 1: Experimental overview of the CER assay. Two assay substrates are prepared by partially denaturing PrPC in non-prion infected brain tissue with chaotropic solutions at either pH 3.5 or pH 7.4. Substrates are seeded with PrPTSE from prion agent of interest and experimental samples are subjected to 24 hr incubation with shaking to allow PrPC to PrPTSE conversion in both pH 3.5 and 7.4 substrates. Following incubation, conversion is assessed by SDS-PAGE and immunoblotting. Signal densities are read by densitometry and the CER is calculated by the ratio of pH 7.4 to pH 3.5 signal densities for each experimental sample.

Figure 2
Figure 2: Quality control on CER substrates. (A) Prior to their use in the CER assay, mouse substrates prepared at either pH 7.4 or 3.5 are assayed by immunoblot in the 1) absence of PK treatment to assess PrPC content of each substrate pair (PrPC levels should be equivalent between pH 7.4 and 3.5 substrates for use in CER assay) and 2) presence of PK (100 µg/ml) treatment to test for pre-existing PrPres in tissues used to prepare substrates (any substrates that display pre-existing PrPres are not suitable for use in the CER assay). (B) To test for non-specific PrPres formation during the conversion assay, mouse PrPC substrates prepared at either pH 7.4 or 3.5 are seeded with conversion buffer, shaken for 24 hr at 1,000 rpm, 37 °C, and subsequently PK-treated (100 µg/ml) and assayed for PrPres by immunoblotting (right two lanes). Non-shaken, non-PK treated mouse substrates represent total PrPC content in assay reactions (left two lanes). For (B), irrelevant lanes have been cropped for clarity, but each immunoblot panel derives from the same gel, membrane and exposure. Immunoblots in all panels used monoclonal antibody SAF 83.

Figure 3
Figure 3: CER assay using laboratory mouse substrate. (A) Mouse CER assay substrate prepared at either pH 7.4 or 3.5 was incubated with RML mouse-adapted scrapie, domestic sheep classical scrapie, white-tailed deer chronic wasting disease (CWD) or 263K strain of hamster-adapted scrapie in the CER assay. Control samples (labeled “none”) contained only an equal amount of infectious agent and no mouse substrate. Samples were analyzed for the presence of PK-resistant prion protein (PrPres) by immunoblot with monoclonal antibody SAF 83. Raw densitometric values for each sample are displayed below each lane. (B) Bar graph indicating the average ratios (± standard deviation) between pH 7.4 and 3.5 mouse substrates for each infectious agent based on at least 3 independent assay runs. Lower-case letters refer to statistically homogenous subsets (analysis of variance with Tukey-Kramer minimum significance differences method; p <0.05). This figure has been modified from Morawski et al. 201315.

Figure 4
Figure 4: Sample use of CER assay to investigate bobcat susceptibility to CWD. Bobcat brain homogenate (A) and CER assay substrate (B) was tested by immunoblotting in the absence and presence of PK (100 µg/ml) to assess existing PrPC levels and the presence of pre-existing PrPres, respectively. (C) Bobcat substrates were seeded with conversion buffer and assayed in the CER assay to assess non-specific PrPres formation (left two lanes). Non-shaken, non-PK treated bobcat substrates represent total PrPC content in assay reactions (right two lanes). (D) Bobcat substrate prepared at either pH 7.4 or 3.5 was incubated with white-tailed deer CWD using the CER assay. The control sample (labeled “none”) contained an equal amount of CWD agent but no bobcat substrate. Raw densitometric values for each sample are displayed below each lane. Immunoblots in all panels used monoclonal antibody 3F4, which reacts with the feline prion protein21.

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Discussion

For successful completion of this protocol, attention should be paid to PrPC levels in uninfected brain tissue used for substrate preparation (step 2.1.2) and the seed to substrate ratio for conversion reactions (step 4.4.2). In our experience, brains can be extracted for use as CER substrate after a substantial period post-mortem, as long as PrPC is present by immunoblotting (Figure 4). In fact, some autolysis was observed in the brains of the bobcats used for CER studies. Nevertheless, incubation of substrates derived from these brains still resulted in the formation of PrPres. We speculate that, in part, the robustness of the CER assay is derived from the denaturation of CER substrates in 1.5 M GdnHCl, which will inactivate many proteases.

Seed to substrate ratios may need to be empirically determined to obtain acceptable PrPres signal by immunoblotting. Too much or too little PrPres to perform densitometry, high background PrPres levels in control samples lacking substrate and inconsistent conversion ratios are all reasons to optimize seed:substrate ratios. Variations maintain a total reaction volume of 100 μl and include 1:99 μl, 2:98 μl and 5:95 μl. On almost all occasions, we have found that 5:95 μl seed:substrate volume ratio is optimal. No matter what ratio is employed, equal amounts of TSE agent are used and the assay is internally consistent. However, seed concentration of the prion-infected brain homogenate used to template reactions likely affects the outcome of the CER assay in that varying seed titers or dilutions could result in varying levels of PrPC-to-PrPres conversion. This effect has been demonstrated in other in vitro prion conversion assays22 and may complicate the comparison of different prion isolates. To address this in the CER assay, seeds could be titrated in each PrPC substrate prepared at pH 3.5 and 7.4 to identify a range of dilutions that template PrPC conversion. Ideal seed dilutions avoid saturating the solution with excess seed prions and yet provide insight into the sensitivity of the conversion reaction. Alternatively, PrPC-to-PrPres conversion in CER assay reactions could be measured at multiple, regular timepoints throughout the duration of the assay and plotted as rate-of-conversion curves, akin to the readouts for real-time polymerase chain reaction (qPCR)23 and real-time quaking induced conversion (RT-QuIC)24 assays. Such readouts could allow more comprehensive interpretations of species barriers and are an interesting focus of future development of this methodology.

In the protocol presented here, we use low binding, thin-walled PCR tubes, but we have also used standard 1.5 ml tubes in a thermo-shaker equipped for this type of tube. No other variations to the protocol are needed to use the larger sized tubes. In our experience, however, we have had more PrPres production and more reproducible results in low-binding PCR tubes compared to 1.5 ml tubes. While explanations for the improved performance of the assay in PCR tubes are only speculative at this time, based on results indicating that the air-water interface is critical for PrP fibrillization25, we hypothesize that the smaller tubes provide a more optimal surface tension for PrP conversion.

Perhaps the chief limitation to use of the CER assay is in understanding what conversion ratios represent in terms of in vivo species barriers determinants, such as disease penetrance or length of incubation period. While currently unknown, translation between in vitro and in vivo species barrier factors should become clearer with continued use of the CER assay in testing additional TSE species barriers that have already been established in vivo and will help to qualify results in species where bioassay data are not available. An advantage of the CER assay compared to PMCA is the presence of an internal control for PrP conversion, namely the pH 3.5 substrate, which can be converted by any TSE agent. This control is of value when it is unclear which, if any, TSE agents should cause misfolding of an exotic host’s PrPC. The inability of a given host substrate to amplify a specific TSE agent in PMCA could be interpreted as evidence of a species barrier or could be due to technical failings. Disadvantages of the CER assay compared to PMCA include limited amplification of PrPres, additional steps in CER substrate preparation and no known infectivity in the PrPres produced by CER reactions. It is to be noted that in a previous study, however, we found the CER assay resulted in a similar pattern of PrPres formation as that of PMCA15. The results presented here also indicate that the CER assay correctly predicts the species barriers for transmission of various TSEs to laboratory mice (Figure 3) and suggest that bobcats may have susceptibility to CWD (Figure 4), in agreement with reports that domestic cats (Felis catus) can acquire prion disease after experimental CWD challenge26,27. Studies using the CER assay could compliment PrP sequencing efforts to understand wildlife susceptibility to CWD28.

The CER assay enables rapid, low-cost, reliable assessment of TSE species barriers in vitro. While not a complete replacement for the “gold standard” of animal bioassay, the CER assay can be used as an initial assessment of the existence of a TSE species barrier that may justify or obviate further studies in living animals. For this reason, and because the assay relies only on brain tissue which can be acquired from non-experimental animals, the CER is in keeping with efforts to replace, reduce and refine animal experimental procedures29. In addition to assessing species barriers, the CER assay can also provide a simplified platform with which to investigate the mechanisms of the fundamental event defining prion disease biology: the conversion of normal, functional PrPC to a misfolded form.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

We thank John Olsen (Wisconsin Department of Natural Resources), Tom Cooley, Daniel O'Brien and Steve Schmitt (Michigan Department of Natural Resources) and Dr. Daniel Walsh (USGS National Wildlife Health Center) for assistance with tissue acquisition. We also thank the Wisconsin State Laboratory of Hygiene for diagnostic testing services, and Dr. Tonie Rocke and her staff (USGS National Wildlife Health Center) for use of equipment. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the U.S. Government.

Materials

Name Company Catalog Number Comments
12% Bis-Tris SDS-PAGE gels Life Technologies NP0342
0.5 mm Zirconium oxide beads Next Advance ZROB05 Other varieties of beads are also effective
Antibodies various suppliers Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest
Bead homogenizer Next Advance BBY24M
Centrifuge Beckman Coulter 369434 High speed with temperature control
Conical tubes any brand
Cotton-tipped applicator Uline S-18991
Cuphorn sonicator Heat Systems-Ultrasonics W-380 Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC
Densitometry software program UVP Vision Works LS Image Acquisition and Analysis software Other programs, such as NIH ImageJ, will also work
Dounce homogenizer Kimble Chase 885300
End-over-end mixer Labnet International H5600
Ethylenediaminetetra acetic acid Boston Bioproducts P-770 Hazardous chemical: eye irritation
Guanidine hydrochloride, 8 M Thermo Fisher Scientific 24115 Hazardous chemical: acute toxicity, skin irritation, eye irritation
Heating block Fisher Scientific 11-718
Hydrochloric acid Sigma-Aldrich 435570 Hazardous chemical: strong acid
Lithium dodecyl sulfate sample buffer, 4x Life Technologies NP0008 Hazardous chemical: skin & respiratory irritation, serious eye damage, flammable solid
Methanol Fisher Scientific A454-4 Hazardous chemical: acute toxicity, flammable liquid
Microcentrifuge tubes any brand
Mini-centrifuge Labnet International C1301
N-lauroyl-sarcosine (sarkosyl) Sigma-Aldrich  L-5125 Hazardous chemical: acute toxicity, skin irritation, eye damage
Nonidet P-40 Amresco M158 Hazardous chemical: skin irritation, eye damage
SDS-PAGE gel system Life Technologies NuPAGE electrophoresis system Other SDS-PAGE systems will also work
PCR tubes (low-binding) Axygen PCR-02-L-C
Pestle homogenizer Fisher Scientific 03-392-106
pH meter Sentron SI600
Polyvinyldifluoride membrane Millipore IPVH00010
Proteinase K Promega V3021 Hazardous chemical: skin & eye irritation, respiratory sensitisation, organ toxicity
Reducing agent for SDS-PAGE samples, 10x Life Technologies NP0009
Sodium chloride Fisher Scientific 7647-14-5
Sodium deoxycholate Sigma-Aldrich  D6750 Hazardous chemical: acute toxicity
Sodium dodecyl sulfate Thermo Fisher Scientific 28364 Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid
Sodium hydroxide Sigma-Aldrich S5881 Hazardous chemical; strong base
Syringe BD Biosciences various Use syringe size appropriate to volumes of substrate to be homogenized
Syringe needles BD Biosciences various
Thermoshaker (PCR tube shaker) Hangzhou All Sheng Instruments MS-100
Tris base Bio Basic 77-86-1 Hazardous chemical: skin, eye, respiratory irritation
Triton X-100 Integra Chemical Company T756.30.30 Hazardous chemical: acute toxicity, eye irritation
Vortexer Fisher Scientific 12-812

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References

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Transmissible Spongiform Encephalopathy TSE Prion Diseases In Vitro Assay Prion Protein Conversion Conversion Efficiency Ratio CER Assay PH 7.4 Substrate PH 3.5 Substrate Proteinase K (PK)-resistant State Species Barrier Laboratory Mice Bobcats White-tailed Deer Chronic Wasting Disease Agent
Assessing Transmissible Spongiform Encephalopathy Species Barriers with an <em>In Vitro</em> Prion Protein Conversion Assay
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Johnson, C. J., Carlson, C. M.,… More

Johnson, C. J., Carlson, C. M., Morawski, A. R., Manthei, A., Cashman, N. R. Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay. J. Vis. Exp. (97), e52522, doi:10.3791/52522 (2015).

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