Evidence for preexisting prion substrain diversity in a biologically cloned prion strain

Prion diseases are a group of inevitably fatal neurodegenerative disorders affecting numerous mammalian species, including Sapiens. Prions are composed of PrPSc, the disease specific conformation of the host encoded prion protein. Prion strains are operationally defined as a heritable phenotype of disease under controlled transmission conditions. Treatment of rodents with anti-prion drugs results in the emergence of drug-resistant prion strains and suggest that prion strains are comprised of a dominant strain and substrains. While much experimental evidence is consistent with this hypothesis, direct observation of substrains has not been observed. Here we show that replication of the dominant strain is required for suppression of a substrain. Based on this observation we reasoned that selective reduction of the dominant strain may allow for emergence of substrains. Using a combination of biochemical methods to selectively reduce drowsy (DY) PrPSc from biologically-cloned DY transmissible mink encephalopathy (TME)-infected brain resulted in the emergence of strains with different properties than DY TME. The selection methods did not occur during prion formation, suggesting the substrains identified preexisted in the DY TME-infected brain. We show that DY TME is biologically stable, even under conditions of serial passage at high titer that can lead to strain breakdown. Substrains therefore can exist under conditions where the dominant strain does not allow for substrain emergence suggesting that substrains are a common feature of prions. This observation has mechanistic implications for prion strain evolution, drug resistance and interspecies transmission.


Introduction
Prion diseases are transmissible neurodegenerative disorders that affect mammals and are inevitably fatal.In humans, prion diseases include Creutzfeldt-Jakob disease (CJD), Gerstmann-Stra ¨ussler-Scheinker syndrome, fatal familial insomnia, and Kuru.Prion diseases in other animals are comprised of scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, transmissible mink encephalopathy (TME) in ranch-raised mink, chronic wasting disease (CWD) in cervids, and camel prion disease.Prions can be zoonotic as evidenced by the interspecies transmission of BSE to humans resulting in the emergence of variant CJD [1,2,3].CWD is an emerging prion disease that was first identified in Colorado in the 1960's and is currently found in 30 US states, 4 Canadian provinces, South Korea and has recently been identified in Norway, Sweden, and Finland [4,5,6,7].
Prions are comprised of PrP Sc , the self-templating, disease-specific conformation of the host-encoded prion protein, PrP C [8,9,10,11].PrP C is a glycosylphosphatidylinositol anchored cell surface protein with two N-linked glycosylation sites that is required for prion conversion and neurotoxicity [12,13,14,15,16,17].Prion conversion occurs at the cell surface and/or in the endosomal lysosomal system resulting in a complete restructuring of PrP C from a monomeric alpha helical structure to that of fibrillar parallel in-register intermolecular β-sheet (PIRIBS) structure [18,19,20,21].Recent near-atomic resolution cryo-electron microscopy (EM) studies have also provided important structural evidence for the interaction of PrP Sc with polyanionic cellular cofactors that facilitate prion conversion [20,21].
Prion strains are operationally defined by heritable differences in the phenotype of disease upon defined transmission conditions [22].The prion strain-specific phenotype of disease can include incubation period, clinical signs of infection, strain mutation rate, tropism of prion conversion within and between tissues and zoonotic potential [23,24,25,26,27,28,29,30,31,32,33].Strain-specific differences in the biochemical features of PrP Sc include migration on SDS-PAGE following proteinase K (PK) digestion, conformational stability in chaotropic agents and in vitro conversion efficiency [34,35,36,37,38].These biochemical features of PrP Sc are consistent with the hypothesis that strain-specific conformations of PrP Sc encode prion strain diversity [34,39].Cryo-EM analysis of PrP Sc of the murineadapted scrapie strains RML and ME7 indicate that while they both share PIRIBS architecture, there are strain-specific differences in the subfolding of PrP rungs providing the most direct evidence to date in support of this hypothesis [21,40].
Prions exist as mixtures of strains.Scrapie-infected sheep and patients with sporadic CJD can contain mixtures of prion strains as determined by strain-specific Western blot migration profiles of PrP Sc [41,42,43,44,45].Passage of these field isolates to transgenic mice expressing either ovine or human PrP C can result in the isolation of distinct prion strains consistent with the hypothesis that an individual can be simultaneously infected with more than one prion strain [42,46].Experimental inoculation of rodents with more than one prion strain indicates that prion strains can compete for PrP C and that the dominant strain can suppress, but not eliminate, the minor strain [47,48,49,50].Interestingly, treatment of rodents with anti-prion therapies can result in the emergence of drug-resistant prion strains that revert to a drug sensitive state following removal of the anti-prion drug [51,52,53,54,55,56,57].These observations

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Preexisting prion substrain diversity are consistent with the hypothesis that prion strains are comprised of a dominant strain and substrains [58,59,60].While a wealth of experimental evidence supports this hypothesis, direct observation of substrains has not been documented.Here we investigated if the well-characterized biologically cloned drowsy (DY) strain of hamster-adapted TME contained substrains.Input samples were ten-fold serially diluted, subjected to one round of PMCA followed by PK digestion, and probed by immunoblotting using antibodies 3F4 (detects all strains) and 12B2 (specific for an epitope present on HY PrP Sc but not DY PrP Sc ).Both strains amplify independently, with HY PrP Sc having higher replication efficiency compared to DY PrP Sc .When mixed at a constant HY to DY ratio of 1:1000, the 500:0.5 μg eq mixture shows complete suppression of HY PrP Sc amplification, while the 50:0.05μg eq.mixture shows incomplete suppression, with some HY PrP Sc being detectable in the sample using the 12B2 antibody.When DY PrP Sc is below 50 μg eq, HY PrP Sc amplifies without interference.

DY TME is not a class III prion strain
Biological stability of prion strains ranges from class I strains being highly stable to class III strains breaking down to a shorter incubation period strain [61].Breakdown of class III strains occurs more frequently when passaged at high prion titer compared to low titer [27,28].We routinely passage DY TME inoculum at low titer (>10 −4 dilution of brain homogenate) and have not observed changes in the strain properties of DY TME [32,37,62,63,64,65,66].To more rigorously investigate if DY TME is a class III strain, we serially passaged DY TME at high titer (10 −1 dilution of brain homogenate) by the intracerebral (i.c.) route of infection for five serial passages.Each serial passage of DY TME was accompanied with an uninfected negative control group.In all (n = 5) of the animals for each serial passage, the DY TME-infected animals maintained clinical signs, incubation period, PrP Sc migration and guanidine hydrochloride (Gdn-HCl) conformational stability properties of DY TME (Table 1).None (n = 5) of the negative control group animals included for each serial passage developed clinical signs of prion infection by 250 days post infection (dpi).DY TME is not lymphotropic and does not cause infection by extraneural routes of infection [32,63].To investigate if lymphotropic strains are present in the 4 th i.c.serial high titer hamster passage of DY TME-infected brain, groups (n = 5) of hamsters were inoculated by either the intraperitoneal (i.p.) or extranasal (e. n.) routes of infection.None (n = 5) of the DY TME or uninfected negative control group animals i.p. or e.n.inoculated developed clinical signs of prion infection by 650 dpi (Table 1 and S2 Fig) .Overall, these data indicate that DY TME is a stable prion strain.

Detection of PrP Sc substrains in DY TME-infected brain
DY can suppress replication of short incubation period, highly pathogenic strains (Fig 1).DY PrP Sc is more susceptible to digestion with proteinase K (PK) compared to other known hamster prion strains (S3 Fig) [34,37,67].We reasoned that extended PK digestion of DY TMEinfected brain homogenate would reduce the suppressive pressure of the dominant strain that may allow for detection of PrP Sc from substrains with relatively higher PK resistance.

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Preexisting prion substrain diversity The conformational stability of DY PrP Sc is lower compared to other hamster-adapted prion strains [37,38].Based on this observation, we reasoned that denaturation and degradation of the relatively low conformational stability DY PrP Sc could reduce the suppressive pressure of the dominant strain and allow for interrogation of the sample for substrains with higher PrP Sc conformational stabilities that are below the limit of Western blot detection by using PMCA.Uninfected brain homogenate that was subjected to the conformational strain selection assay (CSSA) at either 2M or 4M Gdn-HCl did not result in PMCA detection of PrP

Hamsters infected with CSSA products have a bona fide prion infection
Hamsters were inoculated with the products of the CSSA to determine if they were infectious.All (n = 5) hamsters i.c.inoculated with either 2 or 4M Gdn-HCl uninfected CSSA reactions failed to cause disease by 280 dpi (Table 2) and did not contain detectable PrP Sc in PK-digested brain homogenates (      ).The [Gdn-HCl] 1/2 value of first and second hamster passage significantly differed compared to HY TME (p<0.05), while the third passage value did not significantly (p>0.05)differ compared to HY TME.Overall, the [Gdn-HCl] 1/2 value of hamsters infected with 2M DY TME CSSA reaction products was consistent with infection with DY TME, while hamsters infected with 4M DY TME CSSA reaction products had [Gdn-HCl] 1/2 values that were higher than HY TME on first and second hamster passage that, by third hamster passage, were similar to HY TME-infected animals.

Discussion
Replication of the dominant prion strain can suppress replication of prion substrains.It is known that when two prion strains infect the same host one strain can interfere with or completely block another strain from causing disease [32,47,68,69].The relative onset of prion replication between the two strains, in a common population of cells, dictates which strain will emerge [49,50,70].Altering either the relative ratios of the two strains that are infected at the same time (co-infection), or the time interval between inoculation of the first and second prion strain (superinfection) will determine which strain emerges [48,66].Mechanistically, strains compete for PrP C , however, it is unclear if the blocking strain PrP Sc simply binds to PrP C rendering it inaccessible for the other strain (site blocking) or if prion replication is required for strain interference to occur [25,49,50,70].To discriminate between these two possibilities, we used a ratio of DY and HY where DY can block HY from emerging in PMCA and 10-fold serial dilutions of the DY and HY mixture were subjected to PMCA.This experimental approach keeps the ratio of DY and HY PrP Sc the same in all dilutions tested but since DY has a lower PMCA conversion activity per unit PrP Sc compared to HY, as the strain mixture is diluted, DY conversion is reduced at a proportionally faster rate [37].We found that the ability of DY to interfere with HY was strong when DY conversion is robust, but, as DY conversion decreased, HY was able to emerge despite having the same ratio of DY to HY PrP Sc (Fig 1).
Based on this observation, we hypothesize that DY conversion may contribute to the strain interference effect.Prions exhibit properties of quasispecies.Treatment of rodents with anti-prion therapies can result in the emergence of drug-resistant prion strains and subsequent removal of the antiprion drug results in reversion to a drug-sensitive state [51,52,53,54,55,56,57].Serial repeated passage of prions at low titer (i.e., bottlenecking) results in a decrease in prion fitness [71].This observation is consistent with Muller's ratchet, where populations with a high mutation rate (i.e., quasispecies) undergo a reduction in fitness during bottlenecking events [72,73,74].These observations led to the hypothesis that prions are quasispecies; a population of similar, but not identical conformations of PrP Sc [58,59,74].The emergence of drug-resistant prions is hypothesized to be the result of the suppression of the dominant strain by the anti-prion therapy allowing for the emergence of a preexisting drug resistant substrain, analogous to what occurs in conventional microorganisms [75].It is unclear, however, if the treatments select for a preexisting substrain or, alternatively, change the conformation of PrP Sc during prion formation comparable to what has been observed with prion conversion cofactors [76].While the existence of prion substrains is supported by much evidence, direct observation of substrains has not been documented.
Prions are comprised of a dominant strain and substrains.Building upon our observation that PK digestion of a mixture of DY and HY allows for a more rapid emergence of HY PrP Sc [77], we found that extended PK digestion of DY TME resulted in the amplification of PrP Sc with different biochemical properties compared to the parental strain, DY TME (Fig 2).Since PK digestion does not change strain properties and is independent of prion conversion, we interpret this finding as evidence of a preexisting substrain [78].The conformational stability of PrP Sc is strain specific [36,37] and we reasoned that denaturation and PK digestion of relatively low conformational stability PrP Sc would reduce the suppressive pressure of the dominant strain, allowing for the emergence of substrains with relatively higher PrP Sc conformational stabilities.Uninfected brain homogenate that was subjected to the conformational strain selection assay (CSSA) at either 2M or 4M Gdn-HCl did not result in detection of PrP Sc or prion infectivity (Fig 3 , panels A, B, E, F, I, J, M, N; Table 1), indicating that PrP Sc was not introduced into the CSSA reaction either via exogenous sources (e.g., contamination) or by de novo prion formation by the process itself.DY TME seeded 2M CSSA reactions resulted in detection of DY PrP Sc that, upon passage into hamsters, had an incubation period, clinical signs, PrP Sc migration and conformational stability properties of DY TME (Tables 2 and S1 and Figs 4 and 5).Taken together, these data suggest that in the 2M DY TME Gdn-HCl CSSA reactions, DY PrP Sc abundance is reduced, but not to a sufficient level to allow for the emergence of substrains.As transmission of this material to hamsters results in the maintenance of DY TME strain characteristics, this indicates that the CSSA assay and subsequent PMCA is not modifying DY TME strain properties.This is consistent with previous studies where treatment of prion strains with Gdn-HCl altered infectivity, but not the prion strain [79,80,81] and PMCA generated prions maintain the properties of the strain they are seeded with [49,70].CSSA reactions seeded with DY TME-infected brain homogenate treated at 4M Gdn-HCl resulted in detection of PrP Sc only after the second round of PMCA in subset of replicates that was immunoreactive with both 3F4 and 12B2 (Fig 3 , panels L, P, replicates a and d).These observations suggest the treatment conditions in the 4M DY TME CSSA reactions reduced the suppressive effect of DY PrP Sc sufficiently to allow for detection of substrains present in the DY TME-infected brain.The selection methodology occurred in the absence of prion formation; therefore, we hypothesize that the substrains are preexisting.Transmission of this material to hamsters resulted in the development of clinical signs of hyperexcitability, PrP Sc that was immunoreactive with both of the anti-PrP antibodies 3F4 (  2).These observations suggest that

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this is a preexisting substrain present in the DY TME-infected brain homogenate with properties consistent with the selection criteria (i.e., relatively higher PrP Sc conformational stability) and not contamination (Table 2).Second and third serial hamster passage of the 4M DY TME CSSA material resulted in a shortening of the incubation period, maintenance of the clinical signs and PrP Sc electrophoretic mobility and 12B2 immunoreactivity (Table 2 and Fig 4).Interestingly, by third hamster passage the PrP Sc conformational stability was comparable to that of short incubation period strains in hamsters [37,38] (Table 2).
The overrepresentation of prion strains with similar properties from diverse transmission histories has long been observed [35,82,83].In hamsters, short incubation period, high PrP Sc conformational stability strains with clinical signs of hyperexcitability and ataxia have been isolated following the interspecies transmission of TME, scrapie and CWD [35,66,82,83].In mice, the ME7 strain was isolated in approximately over half of the mice inoculated with various sources of sheep scrapie [84,85,86].It is hypothesized that a given primary amino acid sequence of PrP will have a thermodynamically favored conformation (e.g.strain) of PrP Sc [58].The transmission history of the 4M DY TME CSSA product suggests that this material contained a mixture of strains that, upon serial passage in hamsters, evolved to a strain with PrP Sc properties resembling other overrepresented short-incubation period hamster strains consistent with this hypothesis.
We hypothesize that substrains are a common feature of prion strains.DY TME is biologically stable and not prone to strain breakdown.The identification of substrains in DY TMEinfected brain suggests that substrains can exist under conditions where the dominant strain does not allow for substrain emergence.The two complementary methodologies for substrain identification allowed for exploration of only a portion of the possible substrain repertoire and restricted the properties of the substrains that could be identified.Additionally, PMCA may only identify a subpopulations of existing strains whereas a newly described method of PMCA utilizing shaking in place of sonication can identify metastable PrP Sc conformations [87].Despite the bias in strain selection and PMCA, substrains were identified and we hypothesize that the diversity of substrains is much greater than what is reported here.Overall, these findings provide important mechanistic insight into prion strain biology, the selection of drug resistant prion strains, and interspecies transmission.

Ethics statement
All procedures involving animals were approved and in compliance with the Guide for the Care and Use of Laboratory Animals (protocol numbers 880 and 1030) by the Creighton University Institutional Animal Care and Use Committee.

Animal bioassay
Male Syrian hamsters (Harlan-Sprague-Dawley, Indianapolis, IN) were i.c.inoculated with 25 μl of either a 1% w/v brain homogenate or a 1:10 dilution of PMCA generated material in

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Preexisting prion substrain diversity DPBS.Hamsters were observed three times per week for the onset of clinical signs of prion disease and the incubation period was calculated as the number of days between inoculation and onset of clinical signs.Two tail Student's T test (Prism Version 8.4.3, for Mac; GraphPad Software Inc., La Jolla, CA) with a p value of 0.01 was used to compare incubation periods.All tissues were collected with strain dedicated tools that are decontaminated between animals by immersion in bleach (neat) for 15 minutes at room temperature.

Conformational stability assay
The PrP Sc conformational stability assay was performed as described previously [89].Briefly, brain homogenate (1% w/v) was incubated in Gdn-HCl (Sigma-Aldrich, St. Louis, MO) ranging from 0 M to 3.5 M while shaking for one hour at room temperature.The concentration of Gdn-HCl was adjusted to 0.5 M prior to transferring to a 96-well filter plate with a PVDF membrane bottom (Merck Millipore, Co. Cork, Ireland).Samples were digested with PK (5 μg/mL; 1:100 PK:BH) for one hour at 37˚C (5 μg/ml; Roche Diagnostics, Mannheim, Germany) followed by incubation with phenylmethane sulfonyl fluoride (PMSF; MP Biomedicals, LLC, Salon, OH) for 20 minutes at room temperature.Endogenous peroxidases were inhibited with 0.3% H 2 O 2 in methanol and the PVDF membrane blocked using 5% w/v nonfat dry milk in TTBS (BioRad Laboratories, Hercules, CA).The hamster prion protein was immunodetected using the mouse monoclonal anti-PrP antibody 3F4 (final concentration of 0.1 μg/mL; EMD Millipore, Billerica, MA).The membrane was developed with the Pierce SuperSignal West Femto system (Pierce, Rockford, IL) and imaged on a Li-Cor Odyssey Fc Imager (Li-Cor, Lincoln, NE).PrP Sc signal intensity was determined using Li-cor Image Studio Software v.5.2.5 (Lincoln, NE).The point where half of PrP Sc is in a PK resistant state and half is in a PK sensitive state (i.e.[Gdn-HCl] 1/2 ) was determined by calculating the log IC 50 of the non-linear curve fitted to the normalized data (GraphPad Software, San Diego, CA).PrP Sc denaturation curves were generated using GraphPad Prism (GraphPad Software, San Diego, CA).Statistical comparison of the [GdnHCl] 1/2 values were performed using Student's t-test (GraphPad Software, San Diego, CA).

Proteinase strain selection assay
250 μl of 10% brain homogenate is digested at 37˚C for 24 hours with 250 μl of 200 μg/ml proteinase K solution.(Roche Diagnostics, Mannheim, Germany).To remove PK from the sample prior to PMCA, the PK digested brain homogenate is incubated at 37˚C for 1 hour with 1 μl benzonase (MilliporeSigma, Burlington, MA).The sample is then incubated at room temperature for one hour with 250 μl of sarkosyl solution (20% N-lauroylsarcosine in 10 mM Tris Buffer pH 7.5), 1 μl DL-dithiothreitol 250 mM (Sigma-Aldrich, Burlington, MA), and 1 μl of Antifoam (Sigma-Aldrich, Burlington, MA).After incubation, the sample is centrifuged at 10,000 x g for 30 minutes, the pellet is discarded, and the supernatant centrifuged at 100,000 x g for 1 hour.The supernatant is discarded, the pellet resuspended in DPBS (Corning, Corning, NY) and centrifuged at 100,000 x g for 1 hour.The supernatant is discarded, the pellet resuspended in 0.1% sarkosyl solution (0.1% N-lauroylsarcosine in DPBS) and stored at -80˚C.

Conformational strain selection assay
10% w/v brain homogenate is diluted in detergent buffer (5% sodium deoxycholate and 5% Igepal in Dulbecco's Phosphate Buffered Saline [(DPBS), Corning, Corning, NY] and centrifuged at 15,000 x g for 5 minutes.Supernatant is collected and the pellet discarded.20 μl of the supernatant is treated with increasing concentrations of Gdn-HCl (Millipore Sigma, Burlington, MA) (0M, 2M, or 4M) at room temperature for 2 hours.Each sample tube is normalized

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Preexisting prion substrain diversity to 0.5M Gdn-HCl prior to digestion with 20 μg/ml of PK (Roche Diagnostics, Mannheim, Germany) for 1 hour at 37˚C.The PK digestion is stopped with 2 mM phenylmethane sulfonyl fluoride (PMSF; Millipore Sigma, Burlington, MA) for 10 minutes and adjusted to 2% w/v Nlauroylsarcosine and incubate for 10 minutes on ice.Samples are then centrifuged at 100,000 x g for 1 hour at 4˚C and the supernatant discarded.The pellet is resuspended in 0.1% w/v sarkosyl solution in DPBS and stored at -80˚C.
Protein misfolding cyclic amplification (PMCA) reactions seeded with 10-fold serial dilutions of either DY or HY PrP Sc separately reveals differential amplification efficiency between the two strains while maintaining their strain-specific migration pattern of 19-and 21-kDa, respectively (Fig 1, panels A and B).To investigate prion strain interference during prion coinfection, serial 10-fold dilutions of a 1000:1 ratio DY to HY TME were seeded into PMCA reactions (Fig 1, panel C).Following one round of PMCA, DY TME suppressed the replication of HY TME as evidenced by migration of PrP Sc and immunoreactivity to the anti-PrP antibody 12B2 that recognizes HY but not DY PrP Sc (S1 Fig).Strain interference was observed under concentrations where robust DY PrP Sc amplification was detected (500:0.5 μg eq and 50:0.05μg eq DY to HY; Fig 1, panel C, lanes 3 and 4).As replication of DY PrP Sc diminishes following serial dilution a corresponding increase in HY PrP Sc was detected as evidenced by the migration of PrP Sc and the emergence of 12B2 immunoreactive PrP Sc (Fig 1, panel C, lanes 4-6).Overall, these data suggest that DY PrP Sc replication suppresses HY PrP Sc formation.

Fig 1 .
Fig 1. Suppression of HY PrP Sc formation by DY TME.(A-C) Western blot analysis of representative PMCA serial dilution samples (n = 3) of DY PrP Sc alone (A), HY PrP Sc alone (B) and mixtures of HY and DY PrP Sc(C).Input samples were ten-fold serially diluted, subjected to one round of PMCA followed by PK digestion, and probed by immunoblotting using antibodies 3F4 (detects all strains) and 12B2 (specific for an epitope present on HY PrP Sc but not DY PrP Sc ).Both strains amplify independently, with HY PrP Sc having higher replication efficiency compared to DY PrP Sc .When mixed at a constant HY to DY ratio of 1:1000, the 500:0.5 μg eq mixture shows complete suppression of HY PrP Sc amplification, while the 50:0.05μg eq.mixture shows incomplete suppression, with some HY PrP Sc being detectable in the sample using the 12B2 antibody.When DY PrP Sc is below 50 μg eq, HY PrP Sc amplifies without interference.
Uninfected brain homogenate was subjected to the proteinase strain selection assay (PSSA) did not result in PMCA detection of PrP Sc (Fig 2, panels A, C, E, G).PMCA reactions seeded with PSSA of biologically-cloned DY-infected brain homogenate resulted in detection of PrP Sc that was immunoreactive with 3F4 in both first and second serial round of PMCA (Fig 2, panel B and F, respectively) and was immunoreactive to 12B2 only in a subset of reactions following the second serial round of PMCA (Fig 2, panel H, lanes e).Non-PK digested uninfected or DY TME brain homogenate seeded PMCA reactions either failed to amplify PrP Sc or maintained DY PrP Sc properties, respectively (S4 Fig).This pattern of 3F4 and 12B2 immunoreactivity is inconsistent with the DY PrP Sc that was added to the PSSA reaction suggesting it is a non-DY conformation of PrP Sc .[37] (S1 Fig).
Sc (Fig 3, panels A, B, E, F, I, J, M, N).CSSA reactions seeded with DY-infected brain homogenate treated at 2M Gdn-HCl resulted in PrP Sc that was immunoreactive with 3F4 in both PMCA round 1 (Fig 3, panel C) and round 2 (Fig 3, panel K) in all (n = 6) of the replicates but was not immunoreactive with 12B2 (Fig 3, panels G, O) consistent with DY PrP Sc (S1 Fig).

Fig 2 .
Fig 2. Extended PK digestion of DY TME-infected brain homogenate reveals the presence of non-DY PrP Sc species.Western blot analysis of proteinase K strain selection assay products seeded with uninfected (UN; panel A,C,E,G) or drowsy (DY) brain homogenate (panels B, D, F, H) after one (panels A-D) or two (panels E-H) rounds of PMCA probed with either the anti-PrP antibody 3F4 (panels A-B, E-F) or 12B2 (panels C-D, G-H).https://doi.org/10.1371/journal.ppat.1011632.g002 Fig 4, lanes 4 and 5).All (n = 4, one intercurrent death at 191 dpi) hamsters inoculated with second round PMCA reaction from 2M Gdn-HCl DY TME seeded CSSA reactions (Fig 3, panel H, K, replicate d) developed clinical signs of progressive lethargy at 214±5 dpi and contained PrP Sc that was immunoreactive with the anti-PrP antibody 3F4 (Fig 4, lane 6, top panel) with a 19 kDa migration of the unglycosylated PrP Sc polypeptide.The anti-PrP antibody 12B2 failed to detect PrP Sc from this sample (Fig 4, lane 6, bottom panel).A second serial hamster passage of this brain homogenate resulted in all (n = 5) of the hamsters developing clinical signs of progressive lethargy at 174±3 dpi (Table2) with these animals maintaining the PrP Sc immunoreactivity and migration properties from first hamster passage (Fig 4, lane 8).Hamsters inoculated with second round PMCA reaction from a 4M Gdn-HCl

Fig 4 .
Fig 4. Hamsters infected with CSSA products have a bona fide prion infection.Western blot analysis of proteinase K digested brain homogenate from mock infected hamster (UN; lane 1), DY TME infected hamster (DY; lane 2), HY TME infected hamster (HY; lane 3) or CSSA products from mock-infected reactions (lanes 4 and 5) or DY CSSA reactions with either 2M (lane 6) or 4M (lane 7) Gdn-HCl.Second (lanes 8 and 9) and third (lane 10) serial hamster passage of brain material from hamsters infected with CSSA products from lanes 6 and 7. Western blots were probed with either the anti-PrP antibody 3F4 (top panel) that recognizes both the 19 (lane 2) and 21 kDa (lane 3) unglycosylated PrP Sc polypeptide or the anti-PrP antibody 12B2 which recognizes the 21 kDa (lane 3) but not the 19 kDa (lane 2) unglycosylated PrP Sc polypeptide.The migration of the 19 and 21 kDa unglycosylated PrP Sc polypeptide are indicated at the left of the panel.https://doi.org/10.1371/journal.ppat.1011632.g004

Fig 5 .
Fig 5. Conformational stability of PrP Sc from hamsters infected with brain-derived prion strains and the CSSA isolated substrain differ.Representative PrP Sc conformational stability curves from hamsters infected with either HY TME, DY TME, 2M DY CSSA reaction products (panel A), or 4M DY CSSA reaction products (panel B).The conformational stability curves were repeated a minimum of 8 times with similar results.https://doi.org/10.1371/journal.ppat.1011632.g005 Fig 4, lane 7, top panel) and 12B2 (Fig 4, lane 7, bottom panel) and PrP Sc with conformational stability higher relative to other known hamster prion strains [37,38] (Fig 5 and Table

Table 2 )
and contained PrP Sc that was immunoreactive with both the anti-PrP antibodies 3F4 (Fig 4, lane 7, top panel) and 12B2 (Fig 4, lane 7, bottom panel) with a 21 kDa migration of the unglycosylated PrP Sc polypeptide.Second and third serial hamster passage of this brain homogenate resulted in all (n = 5) hamsters developing clinical signs of hyperexcitability at 65±3 and 59±3 dpi, respectively, and retained the PrP Sc immunoreactivity and migration patterns from first hamster passage (Fig 4, lanes 9 and 10).All (n = 5) groups of mock-infected controls included for second and third hamster passage remained clinically normal by 250 dpi (S5 Fig and S1 Table).Overall, the CSSA products are infectious, and the properties of the hamsters infected with the 2M DY TME CSSA products are consistent with infection with DY TME.In contrast, hamsters infected with the 4M DY TME CSSA products have clinical signs, incubation periods and PrP Sc Western blot migration properties that differ from the parental strain, DY TME.