Time course of prion seeding activity in cerebrospinal fluid of scrapie-infected hamsters after

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1 JCM Accepts, published online ahead of print on 11 January 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Time course of prion seeding activity in cerebrospinal fluid of scrapie-infected hamsters after intratongue and intracerebral inoculations Christina D. Orrù*, Andrew G. Hughson*, Brent Race, Gregory J. Raymond and Byron Caughey Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT * These authors contributed equally to this study. Correspondence to: Byron Caughey; bcaughey@nih.gov; Tel: (406) ; FAX (406) ABSTRACT To assess prospects for early diagnosis of prion disease based on prion seeding activity in cerebrospinal fluid (CSF), we measured the activity over time in scrapie-infected hamsters by realtime quaking-induced conversion (RT-QuIC). After intracerebral inoculation, activity appeared in CSF within 1 day and plateaued weeks before the onset of clinical signs. However, after intratongue inoculation, activity first appeared in CSF with the onset of clinical signs, well after higher-level accumulation of seeds in the brain Transmissible spongiform encephalopathies (TSE) or prion diseases are fatal transmissible neurodegenerative diseases that include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in cervids and transmissible mink 1

2 encephalopathy. TSEs involve the conversion of the host s normal prion protein (PrP C ) to abnormal pathological forms (PrP Sc ) which are associated with, and largely comprise, the infectious TSE agents or prions (6) A major problem in coping with TSEs is the difficulty in diagnosing these diseases early in their incubation period. One potentially useful diagnostic specimen is cerebrospinal fluid (CSF), which has been shown by bioassays to contain prions in clinically affected goats (11), sheep (9), and humans (5). Indeed, recent studies have shown that the diagnosis of sporadic CJD in humans in the clinical phase of disease can be markedly improved by the analysis of small (2-15 µl) aliquots of CSF using the real-time quaking-induced conversion (RT-QuIC) assay (2,12). RT-QuIC is based on the ability of TSE-associated forms of PrP to seed amyloid fibril formation of bacterially derived recombinant prion protein (rprp C ). In brief, samples containing prion seeding activity are mixed with the rprp C substrate in multi-well plates and shaken intermittently in a fluorescence plate reader in the presence of thioflavin T (ThT), a compound whose fluorescence is greatly enhanced in the presence of amyloid fibrils. RT-QuIC can be as sensitive as prion animal bioassays, and, like bioassays, can be quantitative with end-point dilutions (12). In considering the further diagnostic utility of the RT-QuIC and any other assays that might in the future be based on the analysis of CSF specimens, it is important to establish the time course and circumstances under which prion seeding activity appears in CSF. Notably, one would like to know the potential for detecting TSE infections in preclinical phases of disease. Because such temporal studies are not possible in humans, we have addressed this issue with a well characterized animal model by applying end-point dilution RT-QuIC to assay CSF samples collected from Syrian golden hamsters at various time points following scrapie inoculation by either the intracerebral or intratongue routes. The intracerebral route is known to give the shortest, most predictable incubation periods in animal models and might in some ways approximate events occurring in sporadic TSE diseases arising spontaneously in 2

3 the brain. However this route introduces the potential complication of brain tissue damage caused by the needle stick and of detecting inoculum injected directly into the CSF. Intratongue inoculations circumvent these problems and should better approximate natural peripheral routes of infection. The intratongue route is highly efficient (4), and minor lingual abrasions have been shown to substantially facilitate the transmission of chronic wasting disease (8). Intracerebral inoculations. To evaluate levels of prion seeding activity in the CSF following intracerebral inoculation of hamsters with scrapie, RT-QuIC was applied to CSF samples collected at various time points throughout the incubation period following inoculation with 50 µl of 0.5% 263K scrapie brain homogenate derived from a clinically ill hamster. Protocols for using animals in these studies were reviewed and approved by the NIAID/RML Animal Care and Use Committee and comply with relevant NIH guidelines. Animals were housed at NIAID/RML facilities accredited by AAALAC International. CSF was collected from the cisterna magna after careful dissection and retraction of skeletal muscles. A small hole was made in the dura mater using a 27 gauge beveled needle and a capillary tube was placed over the hole for CSF collection. Next, brains were removed with separate instruments and immediately frozen. Seed aliquots containing 0.4 µl of neat CSF, or serial dilutions thereof, were assayed in quadruplicate RT-QuIC reactions to determine, using Spearman-Karber analysis as described previously (12), the amount of CSF required to give positive reactions in half of the replicates, i.e. the 50% seeding dose or SD50 (Figure 1A). For the RT-QuIC substrate we used Syrian golden hamster rprp C (residues 90 to 231; accession no K02234), which was purified and characterized as previously described (1,3,12). Seeding activity was detected at /-0.4 SD50 per µl within 1 day post inoculation (dpi) (Figure 1A). The level then dropped 22-fold within 10 dpi, and rebounded 103-fold within 30 dpi to ±0.8 SD50/µl. This level of seeding activity was maintained, or slightly increased, during the remainder of the disease course. Definite clinical signs of disease (e.g. head bobbing and unkempt appearance) were initially observed at 60 days, and the hamsters were euthanized at 75 dpi. Neither seeding activity nor clinical 3

4 signs were detected in a total of 7 age-matched uninoculated control hamsters (Figure 1). Although much of the early seeding activity in the scrapie-infected hamsters may have represented the inoculum itself, the fact that the CSF SD50 concentrations dropped substantially before increasing to levels exceeding those seen shortly after inoculation suggests that most of the CSF seeding activity seen later in the incubation period was due to prions propagated in the host. For comparison, we also performed RT-QuIC analyses of brain homogenates (BH) from the intracerebrally inoculated hamsters (Figure 1B). As described previously (12), 2 µl aliquots of BH dilutions were used to seed RT-QuIC reactions. Initial SD50 values of ~10 4 per mg of brain tissue were measured at 1-6 dpi. Soon thereafter, seeding activity began a nearly log-linear increase up to a maximum of ±0 SD50/mg upon sacrifice at 75 dpi. Thus, on a per weight basis, considering that 1 µl of CSF weighs ~1 mg, the brain homogenates had much higher SD50 concentrations than CSF throughout the course of the infection. Intratongue inoculations. To avoid the potential complications of detecting inoculum in the early phases of the incubation period following intracerebral inoculations, we also performed subepithelial/intramuscular scrapie inoculations (20 µl of 1.25% BH) into the tongues of hamsters. Bartz and colleagues have shown that with hamster-adapted transmissible mink encephalopathy this peripheral route of inoculation can be highly effective without directly introducing inoculum into the brain (4). In contrast to the results from intracerebral inoculations, no seeding activity was detected in the CSF until 85 dpi, which was near the onset of clinical signs of disease at ~91 dpi. Ultimately, however, the SD50 concentrations increased to ±0.3 SD50/µl, i.e. ~4-fold lower than those observed in the clinical phase of scrapie following intracerebral inoculation. This difference of the means was statistically significant (p=0.017) according to the unpaired T-test. 4

5 We also assayed the brain tissue of the intratongue-inoculated hamsters. In contrast to the brains of intracerebrally inoculated hamsters, no seeding activity was detected at <28 dpi. However, ~10 5 SD50 per mg brain tissue were detected at 54 dpi and continued to increase to a maximum of ±0.8 SD50 per mg at the near-terminal clinical phase of disease (Figure 1B). These data show that with peripheral intratongue inoculations, seeding activity accumulated to relatively high levels in the brain tissue well before it became detectable, at much lower concentrations, in the CSF. Collectively, these data provide the first indications of the kinetics of prion accumulation in the CSF of prion-infected hosts. Clearly, the accumulation time course was highly dependent upon the route of scrapie inoculation. The data from intracerebral inoculations showed that prion seeding activity can, in principle, be detected in the CSF far in advance of the onset of clinical signs of disease. Indeed, the RT- QuIC assay was sensitive enough to pick up what appears to be largely the inoculum 1 day after injection. The subsequent reduction in seeding activity over the next several days suggests that there may be a mechanism for clearing the inoculum from the CSF, a process that is likely facilitated by the rapid turnover of CSF in the brain. The reduction in seeding activity after 1 dpi was not observed in the brain tissue homogenates. Despite the apparent early clearance of the inoculum from the CSF, the subsequent increase in SD50 values between dpi indicated that prion seeding activity was accumulating in the CSF faster than it was being cleared. Whether this increase was due to prions propagating in the CSF itself, or being released into the CSF from surrounding tissue, remains unclear. The latter scenario seems more plausible given that the SD50 per unit weight values were always much higher in the brain tissue homogenates than in the CSF regardless of the route of inoculation. Moreover, the likelihood of substantial prion propagation in the CSF seems low because this fluid is largely cell-free; however, extracellular prion propagation appears to occur in at least some models such as scrapieinfected transgenic mice expressing GPI-anchorless PrP C, in which PrP Sc accumulates almost exclusively in extracellular amyloid plaques (7). 5

6 With respect to prion disease diagnostics, a key question is the extent to which these routes of scrapie inoculation in hamsters represent more natural prion diseases. The fact that with intratongue inoculations prion seeding activity was first detected in the CSF near the onset of clinical signs of disease indicates that with some peripheral routes of infection, at least, early preclinical diagnosis based on RT- QuIC analysis of small volumes of CSF may be difficult. Because only small volumes of CSF can be obtained from hamsters, a seed capture step such as that used in the equic assay (10) was not feasible. However, equic might well improve detection sensitivity in the much larger volumes of CSF that can be obtained from larger species such as humans and livestock. Detection of prion seeding activity in the clinical phase of disease should be adequate for the CSF-based diagnosis of most sporadic CJD cases in humans (2), because it is the clinical signs that initially indicate the existence of a problem. However, there are other scenarios in which preclinical detection of seeding activity in the CSF might be valuable, for instance in people who carry familial TSE-linked PrP mutations or have been exposed to potential sources of prion infection. Our present findings emphasize that the route or source of infection will likely play a major role in determining the extent to which early detection of prions in CSF will be possible. Acknowledgements: We thank Kimberly Meade-White and Katie Phillips for assistance in collecting tissues, Jeff Severson for animal care and Drs. Suzette Priola, Andrew Timmes and Kelly Barton for critical comments on the manuscript. This work was supported by the Intramural Research Program of the NIAID, NIH

7 136 FIGURE LEGENDS Figure 1. Time course of prion seeding activity detected in CSF (A) or brain (B) of hamsters after intracerebral (IC) and intratongue (IT) inoculations with scrapie (closed symbols). Open symbols designate SD50 values from mock (buffer alone) inoculated (IT experiment; red open circles) or uninoculated (IC experiment; blue open circles) hamsters, which were all negative and therefore plotted at the detection limits. Dotted lines indicate the time of appearance of clinical signs, blue for IC and red for IT. SD50 values were determined for samples collected at the designated time points. Means +/- SD of determinations from 2-12 scrapie-inoculated hamsters, or > 2 mock-inoculated hamsters at each time point (dpi) are shown. References 1. Atarashi, R., R. A. Moore, V. L. Sim, A. G. Hughson, D. W. Dorward, H. A. Onwubiko, S. A. Priola, and B. Caughey Ultrasensitive detection of scrapie prion protein using seeded conversion of recombinant prion protein. Nat.Methods 4: Atarashi, R., K. Satoh, K. Sano, T. Fuse, N. Yamaguchi, D. Ishibashi, T. Matsubara, T. Nakagaki, H. Yamanaka, S. Shirabe, M. Yamada, H. Mizusawa, T. Kitamoto, G. Klug, A. McGlade, S. J. Collins, and N. Nishida Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat.Med. 17: Atarashi, R., J. M. Wilham, L. Christensen, A. G. Hughson, R. A. Moore, L. M. Johnson, H. A. Onwubiko, S. A. Priola, and B. Caughey Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat.Methods 5:

8 Bartz, J. C., A. E. Kincaid, and R. A. Bessen Rapid prion neuroinvasion following tongue infection. J.Virol. 77: Brown, P., C. J. Gibbs, Jr., P. Rodgers-Johnson, D. M. Asher, M. P. Sulima, A. Bacote, L. G. Goldfarb, and D. C. Gajdusek Human spongiform encephalopathy: the National Institutes of Health series of 300 cases of experimentally transmitted disease. Ann.Neurol. 35: Caughey, B., G. S. Baron, B. Chesebro, and M. Jeffrey Getting a grip on prions: oligomers, amyloids, anchors and pathological membrane interactions. Annu.Rev.Biochem. 78: Chesebro, B., M. Trifilo, R. Race, K. Meade-White, C. Teng, R. LaCasse, L. Raymond, C. Favara, G. Baron, S. Priola, B. Caughey, E. Masliah, and M. Oldstone Anchorless prion protein results in infectious amyloid disease without clinical scrapie. Science 308: Denkers, N. D., G. C. Telling, and E. A. Hoover Minor oral lesions facilitate transmission of chronic wasting disease. J.Virol. 85: Hadlow, W. J., R. C. Kennedy, and R. E. Race Natural infection of Suffolk sheep with scrapie virus. J.Infect.Dis. 146: Orru, C. D., J. M. Wilham, L. D. Raymond, F. Kuhn, B. Schroeder, A. J. Raeber, and B. Caughey Prion disease blood test using immunoprecipitation and improved quakinginduced conversion. MBio. 2:e Pattison, I. H. and G. C. Millson Distribution of the scrapie agent in the tissues of experimentally inoculated goats. J.Comp.Pathol. 72:

9 Wilham, J. M., C. D. Orrú, R. A. Bessen, R. Atarashi, K. Sano, B. Race, K. D. Meade-White, L. M. Taubner, A. Timmes, and B. Caughey Rapid End-Point Quantitation of Prion Seeding Activity with Sensitivity Comparable to Bioassays. PLoS.Pathog. 6:e Downloaded from on November 9, 2018 by guest 9

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