Kristen A. Davenport

Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion

Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrP(C)) to a pathogenic misfolded conformation. Templated conversion has been modelled in several in vitro assays, including serial protein misfolding amplification, amyloid seeding and real-time quaking-induced conversion (RT-QuIC). As RT-QuIC measures formation of amyloid fibrils in real-time, it can be used to estimate the rate of seeded conversion. Here, we used samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed was linearly related to the rate of amyloid formation over a range of 10(-3) to 10(-8) µg. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods, we estimated that urine and saliva from CWD-infected deer both contained 1-5 LD50 per 10 ml. Thus, over the 1-2 year course of an infection, a substantial environmental reservoir of CWD prion contamination accumulates.

Prion-seeding activity in cerebrospinal fluid of deer with chronic wasting disease.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrPd) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrPd detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrPd identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.

Detection of Chronic Wasting Disease in the Lymph Nodes of Free-Ranging Cervids by Real-Time Quaking-Induced Conversion

ABSTRACT Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of deer, elk, and moose, is the only prion disease affecting free-ranging animals. Since the disease was first identified in northern Colorado and southern Wyoming in 1967, new epidemic foci of the disease have been identified in 20 additional states, as well as two Canadian provinces and the Republic of South Korea. Identification of CWD-affected animals currently requires postmortem analysis of brain or lymphoid tissues using immunohistochemistry (IHC) or an enzyme-linked immunosorbent assay (ELISA), with no practical way to evaluate potential strain types or to investigate the epidemiology of existing or novel foci of disease. Using a standardized real-time (RT)-quaking-induced conversion (QuIC) assay, a seeded amplification assay employing recombinant prion protein as a conversion substrate and thioflavin T (ThT) as an amyloid-binding fluorophore, we analyzed, in a blinded manner, 1,243 retropharyngeal lymph node samples from white-tailed deer, mule deer, and moose, collected in the field from areas with current or historic CWD endemicity. RT-QuIC results were then compared with those obtained by conventional IHC and ELISA, and amplification metrics using ThT and thioflavin S were examined in relation to the clinical history of the sampled deer. The results indicate that RT-QuIC is useful for both identifying CWD-infected animals and facilitating epidemiological studies in areas in which CWD is endemic or not endemic.

Pathways of Prion Spread during Early Chronic Wasting Disease in Deer

ABSTRACT Among prion infections, two scenarios of prion spread are generally observed: (i) early lymphoid tissue replication or (ii) direct neuroinvasion without substantial antecedent lymphoid amplification. In nature, cervids are infected with chronic wasting disease (CWD) prions by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events in prion spread during early infection remains incomplete. To investigate this knowledge gap in early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies to assess PrPCWD tissue distribution by real-time quaking-induced conversion (RT-QuIC) and tyramide signal amplification immunohistochemistry (TSA-IHC). Although PrPCWD was not detected by either method in the initial days (1 and 3) postexposure, we observed PrPCWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues at 1 and 2 months postexposure (MPE). At 3 MPE, PrPCWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrPCWD burden in all lymphoid tissues had increased and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. These results indicate the first site of CWD prion entry is in the oropharynx, and the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion. IMPORTANCE Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy affecting cervids, and natural infection occurs through oral and nasal mucosal exposure to infectious prions. Terminal disease is characterized by PrPCWD accumulation in the brain and lymphoid tissues of affected animals. However, the initial sites of prion accumulation and pathways of prion spread during early CWD infection remain unknown. To investigate the chronological events of early prion pathogenesis, we exposed deer to CWD prions and monitored the tissue distribution of PrPCWD over the first 4 months of infection. We show CWD uptake occurs in the oropharynx with initial prion replication in the draining oropharyngeal lymphoid tissues, rapidly followed by dissemination to systemic lymphoid tissues without evidence of neuroinvasion. These data highlight the two phases of CWD infection: a robust prion amplification in systemic lymphoid tissues prior to neuroinvasion and establishment of a carrier state.

Detection and Quantification of CWD Prions in Fixed Paraffin Embedded Tissues by Real-Time Quaking-Induced Conversion

Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrPCWD) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrPCWD burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrPRES in prion, and potentially other, protein misfolding disease states.

PrPC expression and prion seeding activity in the alimentary tract and lymphoid tissue of deer

The agent responsible for prion diseases is a misfolded form of a normal protein (PrPC). The prion hypothesis stipulates that PrPC must be present for the disease to manifest. Cervid populations across the world are infected with chronic wasting disease, a horizontally-transmissible prion disease that is likely spread via oral exposure to infectious prions (PrPCWD). Though PrPCWD has been identified in many tissues, there has been little effort to characterize the overall PrPC expression in cervids and its relationship to PrPCWD accumulation. We used immunohistochemistry (IHC), western blot and enzyme-linked immunosorbent assay to describe PrPC expression in naïve white-tailed deer. We used real-time, quaking-induced conversion (RT-QuIC) to detect prion seeding activity in CWD-infected deer. We assessed tissues comprising the alimentary tract, alimentary-associated lymphoid tissue and systemic lymphoid tissue from 5 naïve deer. PrPC was expressed in all tissues, though expression was often very low compared to the level in the CNS. IHC identified specific cell types wherein PrPC expression is very high. To compare the distribution of PrPC to PrPCWD, we examined 5 deer with advanced CWD infection. Using RT-QuIC, we detected prion seeding activity in all 21 tissues. In 3 subclinical deer sacrificed 4 months post-inoculation, we detected PrPCWD consistently in alimentary-associated lymphoid tissue, irregularly in alimentary tract tissues, and not at all in the brain. Contrary to our hypothesis that PrPC levels dictate prion accumulation, PrPC expression was higher in the lower gastrointestinal tissues than in the alimentary-associated lymphoid system and was higher in salivary glands than in the oropharyngeal lymphoid tissue. These data suggest that PrPC expression is not the sole driver of prion accumulation and that alimentary tract tissues accumulate prions before centrifugal spread from the brain occurs.

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

ABSTRACT The normal cellular prion protein (PrPC) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time quaking-induced conversion (RT-QuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white-tailed deer brain homogenates and found that lipid extraction enabled RT-QuIC detection of CWD prions in a 2-log10-greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated that this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process. IMPORTANCE Prion conversion is likely influenced by lipid interactions, given the location of normal prion protein (PrPC) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking-induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prion-seeded amyloid formation, suggesting that prion conversion is guided by an environment of proconversion and anticonversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

Assessment of the PrPc amino-terminal domain in prion species barriers

ABSTRACT Chronic wasting disease (CWD) in cervids and bovine spongiform encephalopathy (BSE) in cattle are prion diseases that are caused by the same protein-misfolding mechanism, but they appear to pose different risks to humans. We are interested in understanding the differences between the species barriers of CWD and BSE. We used real-time, quaking-induced conversion (RT-QuIC) to model the central molecular event in prion disease, the templated misfolding of the normal prion protein, PrPc, to a pathogenic, amyloid isoform, scrapie prion protein, PrPSc. We examined the role of the PrPc amino-terminal domain (N-terminal domain [NTD], amino acids [aa] 23 to 90) in cross-species conversion by comparing the conversion efficiency of various prion seeds in either full-length (aa 23 to 231) or truncated (aa 90 to 231) PrPc. We demonstrate that the presence of white-tailed deer and bovine NTDs hindered seeded conversion of PrPc, but human and bank vole NTDs did the opposite. Additionally, full-length human and bank vole PrPcs were more likely to be converted to amyloid by CWD prions than were their truncated forms. A chimera with replacement of the human NTD by the bovine NTD resembled human PrPc. The requirement for an NTD, but not for the specific human sequence, suggests that the NTD interacts with other regions of the human PrPc to increase promiscuity. These data contribute to the evidence that, in addition to primary sequence, prion species barriers are controlled by interactions of the substrate NTD with the rest of the substrate PrPc molecule. IMPORTANCE We demonstrate that the amino-terminal domain of the normal prion protein, PrPc, hinders seeded conversion of bovine and white-tailed deer PrPcs to the prion forms, but it facilitates conversion of the human and bank vole PrPcs to the prion forms. Additionally, we demonstrate that the amino-terminal domain of human and bank vole PrPcs requires interaction with the rest of the molecule to facilitate conversion by CWD prions. These data suggest that interactions of the amino-terminal domain with the rest of the PrPc molecule play an important role in the susceptibility of humans to CWD prions.

Assessment of CWD prion shedding in deer saliva with occupancy modeling

ABSTRACT The detection of prions is difficult due to the peculiarity of the pathogen, which is a misfolded form of a normal protein. The specificity and sensitivity of detection methods are imperfect in complex samples, including in excreta. Here, we combined optimized prion amplification procedures with a statistical method that accounts for false-positive and false-negative errors to test deer saliva for chronic wasting disease (CWD) prions. This approach enabled us to discriminate the shedding of prions in saliva and the detection of prions in saliva—a distinction crucial to understanding the role of prion shedding in disease transmission and for diagnosis. We found that assay sensitivity and specificity were indeed imperfect, and we were able to draw several conclusions pertinent to CWD biology from our analyses: (i) the shedding of prions in saliva increases with time postinoculation, but is common throughout the preclinical phase of disease; (ii) the shedding propensity is influenced neither by sex nor by prion protein genotype at codon 96; and (iii) the source of prion-containing inoculum used to infect deer affects the likelihood of prion shedding in saliva; oral inoculation of deer with CWD-positive saliva resulted in 2.77 times the likelihood of prion shedding in saliva compared to that from inoculation with CWD-positive brain. These results are pertinent to horizontal CWD transmission in wild cervids. Moreover, the approach described is applicable to other diagnostic assays with imperfect detection.

Comparative analysis of prions in nervous and lymphoid tissues of chronic wasting disease-infected cervids

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.