Prion strain adaptation: breaking and building species barriers

2014 Spring.Prions have been an enigma to researchers and agricultural producers alike since their inception. The timing and order of prion disease discovery can be attributed to the scrutiny of the prion protein-only hypothesis. The characterization of bacteria, viruses, and the infectious qualities encoded by their genomes only confounded the hypothetical notion of protein as an infectious agent. Perhaps viral etiology theories could have been disregarded earlier if genetic prion diseases were not quickly overshadowed by experimental transmissibility of the putative infectious protein. Despite the discordant journey, mounting evidence suggests that prion pathogenesis is caused by the conversion of the normal cellular host protein, (PrPC) into a protease-resistant, abnormal disease-causing isoform devoid of nucleic acid (PrPRES). Importantly, no differences are observed in the primary sequence of PrPC as compared to PrPRES indicating that observable differences between the normal and disease-causing proteins must be conformational. Additionally, even in the absence of nucleic acid, prions are able to infect various hosts differently, suggesting the phenomenon of prion strains. Characteristically long incubation periods and incomplete attack rates, as consequence of primary passage of prion infected material between differing species, but often even within the same species, have been defined as the species and transmission barrier respectively. Conversion efficiency of infectious prions is most efficient when host and donor PrPC are identical leading some researchers to believe that heterologous PrP blocks conversion, extending the days to onset of clinical disease. Evidence also suggests that prion protein primary sequence predisposes PrPC to fold in an un-infectious normal conformation but interaction with a PrPRES conformer, enciphering biological strain characteristics, provides a template for misfolding PrPC into an infectious conformation. Protein misfolding cyclic amplification (PMCA) has provided additional evidence that PrPRES acts as a template that can convert normal prion protein (PrPC) into the infectious misfolded PrPRES isoform. PMCA utilizes sonication to break up PK resistant aggregates into smaller prion seeds that may interact and template PrPC substrate present in the uninfected brain homogenate. Uniquely, prion disease can be inherited, transmitted, or occur spontaneously. Recently, several investigators have reported spontaneous generation of infectious prions using in vitro methods such as PMCA. Additional investigations into host factors needed for efficient conversion and replication has led to the discovery of differences in the propensity of PrPC misfolding among different species. Several groups have recently suggested that cervid prion protein has a higher propensity for misfolding in vitro and in vivo as a result of a unique rigid loop identifiable in cervid PrPC secondary structure. It has been proposed that increased transmission efficiency of cervid prions can be attributed to the presence of this rigid loop. The principle interest in the current research of this dissertation is to gain deeper knowledge about what fundamental factors play a role in prion strain adaptation, to challenge current theories about prion strain fidelity and to assess species barriers and prion strain dynamics with the aid of differential mouse models of prion disease. The comprehensive hypothesis of this dissertation is that host factors, including but not solely PrPC, mediate prion strain adaptation and determine host range and strength of species barriers. We used PMCA, bioassay using transgenic mice expressing variable amounts of PrPC from mouse and cervid species, and cell culture lines expressing different host PrPC to address these questions. We challenged the efficiency and congruency of PMCA by characterizing strain properties of amplified material in parallel with mouse bioassay by: incubation period, PK resistance, glycoform ratios, lesion profiles, and conformational stability. We further wanted to test if PMCA de novo generated prions were infectious and what strain properties they would emulate. We hypothesized that the PK resistant material generated with PMCA was infectious and transmissible and possess strain properties reminiscent of other cervid prion strains. Finally, our lab hypothesized that PrPRES conformation enciphers prion strain properties by acting as a template for nascent PrPRES but that host factors also play a role in adapting prion strains derived from a different host and that species barriers can be overcome through this adaptation

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200xg and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed \u3e=95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols

Prion Amplification and Hierarchical Bayesian Modeling Refine Detection of Prion Infection

Prions are unique infectious agents that replicate without a genome and cause neurodegenerative diseases that include chronic wasting disease (CWD) of cervids. Immunohistochemistry (IHC) is currently considered the gold standard for diagnosis of a prion infection but may be insensitive to early or sub-clinical CWD that are important to understanding CWD transmission and ecology. We assessed the potential of serial protein misfolding cyclic amplification (sPMCA) to improve detection of CWD prior to the onset of clinical signs. We analyzed tissue samples from free-ranging Rocky Mountain elk (Cervus elaphus nelsoni) and used hierarchical Bayesian analysis to estimate the specificity and sensitivity of IHC and sPMCA conditional on simultaneously estimated disease states. Sensitivity estimates were higher for sPMCA (99.51%, credible interval (CI) 97.15–100%) than IHC of obex (brain stem, 76.56%, CI 57.00–91.46%) or retropharyngeal lymph node (90.06%, CI 74.13–98.70%) tissues, or both (98.99%, CI 90.01–100%). Our hierarchical Bayesian model predicts the prevalence of prion infection in this elk population to be 18.90% (CI 15.50–32.72%), compared to previous estimates of 12.90%. Our data reveal a previously unidentified sub-clinical prion-positive portion of the elk population that could represent silent carriers capable of significantly impacting CWD ecology

Intranasal Inoculation of White-Tailed Deer (\u3ci\u3eOdocoileus virginianus\u3c/i\u3e) with Lyophilized Chronic Wasting Disease Prion Particulate Complexed to Montmorillonite Clay

Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure

Incidence and infectivity titers of prion inocula before and after adsorption.

a<p>number of terminally ill mice/number infected.</p>b<p>DPI, days post infection.</p>c<p>×10⁴ mean LD₅₀ after 24 h @ 23°C. All SDs ≤0.001×10^4.</p>d<p>Initial titer of inocula prior to adsorption.</p>e<p>NA, not applicable.</p>f<p>below linear range of bioassay.</p

Soil Component Analysis.

a<p>% weight of whole SLS.</p>b<p>clay classification.</p>c<p>% of total clay weight.</p>d<p>clay weight % of total.</p>e<p>electrical conductivity (EC), measurement of salinity.</p>f<p>NA, not applicable.</p

Disease status and detection of prions in non-clinical mice inoculated with Mte-treated-inocula.

<p>Disease status and detection of prions in non-clinical mice inoculated with Mte-treated-inocula.</p

Representative Histology of TgA20 indicator mice.

<p>Selected examples of histological analysis using immunohistochemistry with PrP specific BAR224 Ab (reddish-brown staining, panels A–F) and anti-GFAP antibody staining activated astrocytes (bright red, panels G–L) in hippocampal sections. (<b>A and G</b>) Negative control sections from mice inoculated with SLS-treated NBH exhibited no PrP^Sc staining or spongiosis and limited astrocyte activation. (<b>B and H</b>) Positive control sections from mice inoculated with RML5 revealed diffuse PrP^Sc staining and significant spongiosis and astrogliosis. (<b>C and I</b>) Sections from mice inoculated with SLS-treated TgA20RML resulted in limited PrP^Sc deposits, spongiosis and astrogliosis, while (<b>D and J</b>) sections from mice inoculated with Mte-treated TgA20RML revealed little or no scrapie neuropathology. (<b>E and K</b>) sections from mice inoculated with SLS-treated RML5 resulted in neuropathology similar to sections from TgA20RML treated mice (Band H). (<b>F and L</b>) Hippocampal sections from the only mouse to become ill with Mte treated RML5 showed limited PrP^Sc spongiosis and astrogliosis.</p

Genetic Depletion of Complement Receptors CD21/35 Prevents Terminal Prion Disease in a Mouse Model of Chronic Wasting Disease

The complement system has been shown to facilitate peripheral prion pathogenesis. Mice lacking complement receptors CD21/35 partially resist terminal prion disease when infected i.p. with mouse-adapted scrapie prions. Chronic wasting disease (CWD) is an emerging prion disease of captive and free-ranging cervid populations that, similar to scrapie, has been shown to involve the immune system, which probably contributes to their relatively facile horizontal and environmental transmission. In this study, we show that mice overexpressing the cervid prion protein and susceptible to CWD (Tg(cerPrP)5037 mice) but lack CD21/35 expression completely resist clinical CWD upon peripheral infection. CD21/35-deficient Tg5037 mice exhibit greatly impaired splenic prion accumulation and replication throughout disease, similar to CD21/35-deficient murine prion protein mice infected with mouse scrapie. TgA5037;CD21/35(-/-) mice exhibited little or no neuropathology and deposition of misfolded, protease-resistant prion protein associated with CWD. CD21/35 translocate to lipid rafts and mediates a strong germinal center response to prion infection that we propose provides the optimal environment for prion accumulation and replication. We further propose a potential role for CD21/35 in selecting prion quasi-species present in prion strains that may exhibit differential zoonotic potential compared with the parental strains