Reduced Expression of Prion Protein With Increased Interferon-ß Fail to Limit Creutzfeldt-Jakob Disease Agent Replication in Differentiating Neuronal Cells.

Immortalized uninfected septal (SEP) neurons proliferate but after physiological mitotic arrest they express differentiated neuronal characteristics including enhanced cell-to-cell membrane contacts and ≥ 8 fold increases in host prion protein (PrP). We compared proliferating uninfected and Creutzfeldt-Jakob Disease (CJD) agent infected cells with their arrested counterparts over 33 days by quantitative mRNA and protein blot analyses. Surprisingly, uninfected arrested cells increased interferon-ß (IFN-ß) mRNA by 2.5-8 fold; IFN-ß mRNA elevations were not previously associated with neuronal differentiation. SEP cells with high CJD infectivity titers produced a much larger 40-68-fold increase in IFN-ß mRNA, a classic host anti-viral response that is virucidal for RNA but not DNA viruses. High titers of CJD agent also induced dramatic decreases in host PrP, a protein needed for productive agent replication. Uninfected arrested cells produced large sustained 20-30-fold increases in PrP mRNA and protein, whereas CJD arrested cells showed only transient small 5-fold increases in PrP. A > 10-fold increase in infectivity, but not PrP misfolding, induced host PrP reductions that can limit CJD agent replication. In contrast to neuronal lineage cells, functionally distinct migratory microglia with high titers of CJD agent do not induce an IFN-ß mRNA response. Because they have 1/50th of PrP of an average brain cell, microglia would be unable to produce the many new infectious particles needed to induce a large IFN-ß response by host cells. Instead, microglia and related cells can be persistent reservoirs of infection and spread. Phase separations of agent-associated molecules in neurons, microglia and other cell types can yield new insights into the molecular structure, persistent, and evasive behavior of CJD-type agents.

Prokaryotic SPHINX replication sequences are conserved in mammalian brain and participate in neurodegeneration.

A new class of viral mammalian Slow Progressive Hidden INfections of variable (X) latency ("SPHINX") DNAs, represented by the 1.8 and 2.4 kb nuclease-protected circular elements, were discovered in highly infectious cytoplasmic particles isolated from Creutzfeldt-Jakob Disease (CJD) and scrapie samples. These DNAs contained replication initiation sequences (REPs) with approximately 70% homology to those of environmental Acinetobacter phage. Antibodies against REP peptides from the 1.8 kb DNA highlighted a 41 kDa protein (spx) on Western blots, and in situ studies previously revealed its peripheral tissue expression, for example, in pancreatic islet cells, keratinocytes, kidney tubules, and oocytes but not pancreatic exocrine cells, alveoli, and striated muscle. To determine if spx concentrated in specific neurons and synapses, and also maintained a conserved pattern of architectural organization in mammalian brains, we evaluated mouse, rat, hamster, guinea pig (GP), and human samples. Most outstanding was the cross-species concentration of spx in huge excitatory synapses of mossy fibers and small internal granule neuron synapses, the only excitatory neuron within the cerebellum. Spx also localized to excitatory glutamate type synapses in the hippocampus, and both cerebellar and hippocampal synaptic spx was demonstrable ultrastructurally. Studies of two well-characterized models of sporadic CJD (sCJD) revealed novel spx pathology. Vacuolar loss of cerebellar synaptic complexes, thinning of the internal granule cell layer, and fibrillar spx accumulations within Purkinje neurons were prominent in sCJD GP brains. In rats, comparable spx fibrillar changes appeared in hippocampal pyramidal neurons, and they preceded prion protein misfolding. Hence, spx is an integral player in progressive neurodegeneration. The evolutionary origin, spread, and neuropathology of SPHINX 1.8 REP sequences opens another unanticipated chapter for mammalian symbiotic interactions with environmental microbes.

Prokaryotic SPHINX 1.8 REP protein is tissue-specific and expressed in human germline cells.

Small circular DNAs of 1.8 and 2.4 kb were initially discovered in highly infectious Creutzfeldt-Jakob Disease (CJD) and scrapie particles from mammalian brain and cultured cells. Surprisingly, these protected cytoplasmic "SPHINX" DNAs contained replication (REP) initiation sequences resembling those of Acinetobacter phage viruses. An antibody was generated against a REP peptide encoded by the SPHINX 1.8 open reading frame (ORF) that was not present in mammals. It bound to a 41kd "spx1" protein on Western blots. Cytologically, spx1 concentrated in spinal cord synapses and pancreatic islet, but not exocrine cells. We hypothesized that circular SPHINX DNAs are ancient symbiotic elements that can participate in functional differentiation and neurodegeneration. Cell and tissue-specific patterns of spx1 expression shown below implicate somatic cell-to-cell communication and differentiation functions that would favor conservation of SPHINX 1.8 in evolution. Remarkably, primary human oocytes and spermatogonia, but not mature sperm, displayed intense cytoplasmic spx1 signals that underscore the maternal inheritance of SPHINX 1.8. These findings should encourage investigations of unexplored networks of incorporated environmental infectious agents that can be key actors in progressive neurodegeneration, immunity, and cancer.

A prokaryotic viral sequence is expressed and conserved in mammalian brain.

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.

CJD and Scrapie Require Agent-Associated Nucleic Acids for Infection.

Unlike Alzheimer's and most other neurodegenerative diseases, Transmissible Spongiform Encephalopathies (TSEs) are all caused by actively replicating infectious particles of viral size and density. Different strain-specific TSE agents cause CJD, kuru, scrapie and BSE, and all behave as latent viruses that evade adaptive immune responses and can persist for years in lymphoreticular tissues. A foreign viral structure with a nucleic acid genome best explains these TSE strains and their endemic and epidemic spread in susceptible species. Nevertheless, it is widely believed that host prion protein (PrP), without any genetic material, encodes all these strains. We developed rapid infectivity assays that allowed us to reproducibly isolate infectious particles where >85% of the starting titer separated from the majority of host components, including PrP. Remarkably, digestion of all forms of PrP did not reduce brain particle titers. To ask if TSE agents, as other viruses, require nucleic acids, we exposed high titer FU-CJD and 22L scrapie particles to potent nucleases. Both agent-strains were propagated in GT1 neuronal cells to avoid interference by complex degenerative brain changes that can impede nuclease digestions. After exposure to nucleases that are active in sarkosyl, infectivity of both agents was reproducibly reduced by ≥99%. No gold-stained host proteins or any form of PrP were visibly altered by these nucleases. In contrast, co-purifying protected mitochondrial DNA and circular SPHINX DNAs were destroyed. These findings demonstrate that TSE agents require protected genetic material to infect their hosts, and should reopen investigation of essential agent nucleic acids. J. Cell. Biochem. 117: 1947-1958, 2016. © 2016 Wiley Periodicals, Inc.

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms.

Neurodegenerative human CJD and sheep scrapie are diseases caused by several different transmissible encephalopathy (TSE) agents. These infectious agents provoke innate immune responses in the brain, including late-onset abnormal prion protein (PrP-res) amyloid. Agent particles that lack detectable PrP sequences by deep proteomic analysis are highly infectious. Yet these agents, and their unusual resistance to denaturation, are often evaluated by PrP amyloid disruption. To reexamine the intrinsic resistance of TSE agents to denaturation, a paradigm for less resistant viruses and microbes, we developed a rapid and reproducible high yield agent isolation procedure from cultured cells that minimized PrP amyloid and other cellular proteins. Monotypic neuronal GT1 cells infected with the FU-CJD or 22L scrapie agents do not have complex brain changes that can camouflage infectious particles and prevent their disruption, and there are only 2 reports on infectious titers of any human CJD strain treated with chemical denaturants. Infectious titers of both CJD and scrapie were reduced by >4 logs with Thiourea-urea, a treatment not previously tested. A mere 5 min exposure to 4M GdnHCl at 22°C reduced infectivity by >5 logs. Infectious 22L particles were significantly more sensitive to denaturation than FU-CJD particles. A protocol using sonication with these chemical treatments may effectively decontaminate complicated instruments, such as duodenoscopes that harbor additional virulent microbes and biofilms associated with recent iatrogenic infections.

Proteomic analysis of host brain components that bind to infectious particles in Creutzfeldt-Jakob disease.

Transmissible encephalopathies (TSEs), such as Creutzfeldt-Jakob disease (CJD) and scrapie, are caused by infectious agents that provoke strain-specific patterns of disease. Misfolded host prion protein (PrP-res amyloid) is believed to be the causal infectious agent. However, particles that are stripped of PrP retain both high infectivity and viral proteins not detectable in uninfected mouse controls. We here detail host proteins bound with FU-CJD agent infectious brain particles by proteomic analysis. More than 98 proteins were differentially regulated, and 56 FU-CJD exclusive proteins were revealed after PrP, GFAP, C1q, ApoE, and other late pathologic response proteins were removed. Stripped FU-CJD particles revealed HSC70 (144× the uninfected control), cyclophilin B, an FU-CJD exclusive protein required by many viruses, and early endosome-membrane pathways known to facilitate viral processing, replication, and spread. Synaptosomal elements including synapsin-2 (at 33×) and AP180 (a major FU-CJD exclusive protein) paralleled the known ultrastructural location of 25 nm virus-like TSE particles and infectivity in synapses. Proteins without apparent viral or neurodegenerative links (copine-3), and others involved in viral-induced protein misfolding and aggregation, were also identified. Human sCJD brain particles contained 146 exclusive proteins, and heat shock, synaptic, and viral pathways were again prominent, in addition to Alzheimer, Parkinson, and Huntington aggregation proteins. Host proteins that bind TSE infectious particles can prevent host immune recognition and contribute to prolonged cross-species transmissions (the species barrier). Our infectious particle strategy, which reduces background sequences by >99%, emphasizes host targets for new therapeutic initiatives. Such therapies can simultaneously subvert common pathways of neurodegeneration.

Highly infectious CJD particles lack prion protein but contain many viral-linked peptides by LC-MS/MS.

It is widely believed that host prion protein (PrP), without nucleic acid, converts itself into an infectious form (PrP-res) that causes transmissible encephalopathies (TSEs), such as human sporadic CJD (sCJD), endemic sheep scrapie, and epidemic BSE. There are many detailed investigations of PrP, but proteomic studies of other proteins in verified infectious TSE particles have not been pursued, even though brain homogenates without PrP retain their complete infectious titer. To define proteins that may be integral to, process, or protect an agent genome, we developed a streamlined, high-yield purification of infectious FU-CJD mouse brain particles with minimal PrP. Proteinase K (PK) abolished all residual particle PrP, but did not reduce infectivity, and viral-size particles lacking PrP were ∼70S (vs. 90-120S without PK). Furthermore, over 1,500 non-PrP proteins were still present and positively identified in high titer FU-CJD particles without detectable PrP by mass spectrometry (LC-MS/MS); 114 of these peptides were linked to viral motifs in the environmental-viral database, and not evident in parallel uninfected controls. Host components were also identified in both PK and non-PK treated particles from FU-CJD mouse brain and human sCJD brain. This abundant cellular data had several surprises, including finding Huntingtin in the sCJD but not normal human brain samples. Similarly, the neural Wiskott-Aldrich sequence and multivesicular and endosome components associated with retromer APP (Alzheimer amyloid) processing were only in sCJD. These cellular findings suggest that new therapies directed at retromer-vesicular trafficking in other neurodegenerative diseases may also counteract late-onset sCJD PrP amyloid pathology.

Infectious particles, stress, and induced prion amyloids: a unifying perspective.

Transmissible encephalopathies (TSEs) are believed by many to arise by spontaneous conversion of host prion protein (PrP) into an infectious amyloid (PrP-res, PrP (Sc) ) without nucleic acid. Many TSE agents reside in the environment, with infection controlled by public health measures. These include the disappearance of kuru with the cessation of ritual cannibalism, the dramatic reduction of epidemic bovine encephalopathy (BSE) by removal of contaminated feed, and the lack of endemic scrapie in geographically isolated Australian sheep with susceptible PrP genotypes. While prion protein modeling has engendered an intense focus on common types of protein misfolding and amyloid formation in diverse organisms and diseases, the biological characteristics of infectious TSE agents, and their recognition by the host as foreign entities, raises several fundamental new directions for fruitful investigation such as: (1) unrecognized microbial agents in the environmental metagenome that may cause latent neurodegenerative disease, (2) the evolutionary social and protective functions of different amyloid proteins in diverse organisms from bacteria to mammals, and (3) amyloid formation as a beneficial innate immune response to stress (infectious and non-infectious). This innate process however, once initiated, can become unstoppable in accelerated neuronal aging.

Continuous production of prions after infectious particles are eliminated: implications for Alzheimer's disease.

Rat septal cells, induced to enter a terminal differentiation-like state by temperature shift, produce prion protein (PrP) levels 7x higher than their proliferative counterparts. Host PrP accumulates on the plasma membrane, newly elaborated nanotubes, and cell-to-cell junctions, important conduits for viral spread. To find if elevated PrP increased susceptibility to FU-CJD infection, we determined agent titers under both proliferating and arresting conditions. A short 5 day arrest and a prolonged 140 day arrest increased infectivity by 5x and 122x (>2 logs) respectively as compared to proliferating cells. Total PrP rapidly increased 7x and was even more elevated in proliferating cells that escaped chronic arrest conditions. Amyloid generating PrP (PrP-res), the "infectious prion" form, present at ~100,000 copies per infectious particle, also increased proportionately by 140 days. However, when these highly infectious cells were switched back to proliferative conditions for 60 days, abundant PrP-res continued to be generated even though 4 logs of titer was lost. An identical 4 log loss was found with maximal PrP and PrP-res production in parallel cells under arresting conditions. While host PrP is essential for TSE agent spread and replication, excessive production of all forms of PrP can be inappropriately perpetuated by living cells, even after the initiating infectious agent is eliminated. Host PrP changes can start as a protective innate immune response that ultimately escapes control. A subset of other neurodegenerative and amyloid diseases, including non-transmissible AD, may be initiated by environmental infectious agents that are no longer present.

High CJD infectivity remains after prion protein is destroyed.

The hypothesis that host prion protein (PrP) converts into an infectious prion form rests on the observation that infectivity progressively decreases in direct proportion to the decrease of PrP with proteinase K (PK) treatment. PrP that resists limited PK digestion (PrP-res, PrP(sc)) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique transmissible spongiform encephalopathy (TSE) agent strains. Recently, a PK sensitive form of PrP has been proposed as the prion. Thus we re-evaluated total PrP (sensitive and resistant) and used a cell-based assay for titration of infectious particles. A keratinase (NAP) known to effectively digest PrP was compared to PK. Total PrP in FU-CJD infected brain was reduced to ≤0.3% in a 2 h PK digest, yet there was no reduction in titer. Remaining non-PrP proteins were easily visualized with colloidal gold in this highly infectious homogenate. In contrast to PK, NAP digestion left 0.8% residual PrP after 2 h, yet decreased titer by >2.5 log; few residual protein bands remained. FU-CJD infected cells with 10× the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 log). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of 8 logs survived. Our FU-CJD brain results are in good accord with the only other report on maximal PrP destruction and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.

Replication and spread of CJD, kuru and scrapie agents in vivo and in cell culture.

Transmissible Spongiform Encephalopathy (TSE) agents are defined by their virulence for particular species, their spread in the population, their incubation time to cause disease, and their neuropathological sequelae. Murine adapted human agents, including sporadic CJD (sCJD), New Guinea kuru, and Japanese CJD agents, display particularly distinct incubation times and maximal infectious brain titers. They also induce agent-specific patterns of neurodegeneration. When these TSE agents are transmitted to cultured hypothalamic GT1 cells they maintain their unique identities. Nevertheless, the human kuru (kCJD) and Japanese FU-CJD agents, as well as the sheep 22L and 263K scrapie agents display doubling times that are 8x to 33x faster in cells than in brain, indicating release from complex innate immune responses. These data are most consistent with a foreign viral structure, rather than an infectious form of host prion protein (PrP-res). Profound agent-specific inhibitory effects are also apparent in GT1 cells, and maximal titer plateau in kCJD and FU-CJD differed by 1,000-fold in a cell-based assay. Remarkably, the lower titer kCJD agent rapidly induced de novo PrP-res in GT1 cells, whereas the high titer FU-CJD agent replicated silently for multiple passages. Although PrP-res is often considered to be toxic, PrP-res instead may be part of a primal defense and/or clearance mechanism against TSE environmental agents. Limited spread of particular TSE agents through nanotubes and cell-to-cell contacts probably underlies the long peripheral phase of human CJD.

Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD.

In transmissible encephalopathies (TSEs), it is commonly believed that the host prion protein transforms itself into an infectious form that encodes the many distinct TSE agent strains without any nucleic acid. Using a Ф29 polymerase and chromatography strategy, highly infectious culture and brain preparations of three different geographic TSE agents all contained novel circular DNAs. Two circular "Sphinx" sequences, of 1.8 and 2.4 kb, copurified with infectious particles in sucrose gradients and, as many protected viruses, resisted nuclease digestion. Each contained a replicase ORF related to microviridae that infect commensal Acinetobacter. Infectious gradient fractions also contained nuclease-resistant 16 kb mitochondrial DNAs and analysis of >4,000 nt demonstrated a 100% identity with their species-specific sequences. This confirmed the fidelity of the newly identified sequences detailed here. Conserved replicase regions within the two Sphinx DNAs were ultimately detected by PCR in cytoplasmic preparations from normal cells and brain but were 2,500-fold less than in parallel-infected samples. No trace of the two Sphinx replicases was found in enzymes, detergents, or other preparative materials using exhaustive PCR cycles. The Sphinx sequences uncovered here could have a role in TSE infections despite their apparently symbiotic, low-level persistence in normal cells and tissues. These, as well as other cryptic circular DNAs, may cause or contribute to neurodegeneration and infection-associated tumor transformation. The current results also raise the intriguing possibility that mammals may incorporate more of the prokaryotic world in their cytoplasm than previously recognized.

Transmissible encephalopathy agents: virulence, geography and clockwork.

Transmissible spongiform encephalopathies (TSEs) are caused by infectious agents with stable virulence characteristics that are not encoded by the host. Agent-specific features of virulence include variable disease latency and tissue pathology in a given host, as well as the ability to spread to many species. Such cross-species infections contradict predictions based on the prion hypothesis. Recent transmissions of several human agents to normal mice and to monotypic neural cells in culture, underscore the existence of unique agent clades that are prevalent in particular geographic regions. Examples include the epidemic UK bovine agent (BSE) and the New Guinea kuru agent. The virus-like biology of unique TSE agents, including epidemic spread, mutation, and superinfection, can be used to systematically define features of virulence that distinguish common endemic from newly emerging strains.

Proliferative arrest of neural cells induces prion protein synthesis, nanotube formation, and cell-to-cell contacts.

Host prion protein (PrP) is most abundant in neurons where its functions are unclear. PrP mRNA transcripts accumulate at key developmental times linked to cell division arrest and terminal differentiation. We sought to find if proliferative arrest was sufficient to cause an increase in PrP in developing neurons. Rat neuronal precursor cells transduced with the temperature sensitive SV-40 T antigen just before terminal differentiation (permissive at 33 degrees C but not at 37.5 degrees C) were analyzed. By 2 days, T antigen was decreased in all cells at 37.5 degrees C, with few DNA synthesizing (BrdU+) cells. Proliferative arrest induced by 37.5 degrees C yielded a fourfold PrP increase. When combined with reduced serum, a sevenfold increase was found. Within 2 days additional neuritic processes with abundant plasma membrane PrP connected many cells. PrP also concentrated between apposed stationary cells, and on extending growth cones and their filopodia. Stationary cells were maintained for 30 days in their original plate, and they reverted to a proliferating low PrP state at 33 degrees C. Ultrastructural studies confirmed increased nanotubes and adherent junctions between high PrP cells. Additionally, some cells shared cytoplasm and these apparently open regions are likely conduits for the exchange of organelles and viruses that have been observed in living cells. Thus PrP is associated with dynamic recognition and contact functions, and may be involved in the transient formation of neural syncytia at key times in embryogenesis. This system can be used to identify drugs that inhibit the transport and spread of infectious CJD particles through the nervous system.

Agent-specific Shadoo responses in transmissible encephalopathies.

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by an infectious agent with viral properties. Host prion protein (PrP), a marker of late stage TSE pathology, is linked to a similar protein called Shadoo (Sho). Sho is reduced in mice infected with the RML scrapie agent, but has not been investigated in other TSEs. Although PrP is required for infection by TSE agents, it is not known if Sho is similarly required. Presumably Sho protects cells from toxic effects of misfolded PrP. We compared Sho and PrP changes after infection by very distinct TSE agents including sporadic CJD, Asiatic CJD, New Guinea kuru, vCJD (the UK epidemic bovine agent) and 22L sheep scrapie, all passaged in standard mice. We found that Sho reductions were agent-specific. Variable Sho reductions in standard mice could be partly explained by agent-specific differences in regional neuropathology. However, Sho did not follow PrP misfolding in any quantitative or consistent way. Tga20 mice with high murine PrP levels revealed additional agent-specific differences. Sho was unaffected by Asiatic CJD yet was markedly reduced by the kuru agent in Tga20 mice; in standard mice both agents induced the same Sho reductions. Analyses of neural GT1 cells demonstrated that Sho was not essential for TSE infections. Furthermore, because all infected GT1 cells appeared as healthy as uninfected controls, Sho was not needed to protect infected cells from their "toxic" burden of abundant abnormal PrP and intracellular amyloid.

The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt-Jakob disease and scrapie agents.

Human sporadic Creutzfeldt-Jakob disease (sCJD), endemic sheep scrapie, and epidemic bovine spongiform encephalopathy (BSE) are caused by a related group of infectious agents. The new U.K. BSE agent spread to many species, including humans, and clarifying the origin, specificity, virulence, and diversity of these agents is critical, particularly because infected humans do not develop disease for many years. As with viruses, transmissible spongiform encephalopathy (TSE) agents can adapt to new species and become more virulent yet maintain fundamentally unique and stable identities. To make agent differences manifest, one must keep the host genotype constant. Many TSE agents have revealed their independent identities in normal mice. We transmitted primate kuru, a TSE once epidemic in New Guinea, to mice expressing normal and approximately 8-fold higher levels of murine prion protein (PrP). High levels of murine PrP did not prevent infection but instead shortened incubation time, as would be expected for a viral receptor. Sporadic CJD and BSE agents and representative scrapie agents were clearly different from kuru in incubation time, brain neuropathology, and lymphoreticular involvement. Many TSE agents can infect monotypic cultured GT1 cells, and unlike sporadic CJD isolates, kuru rapidly and stably infected these cells. The geographic independence of the kuru agent provides additional reasons to explore causal environmental pathogens in these infectious neurodegenerative diseases.

Strain-specific viral properties of variant Creutzfeldt-Jakob disease (vCJD) are encoded by the agent and not by host prion protein.

Human CJD, endemic sheep scrapie, epidemic bovine spongiform encephalopathy (BSE), and other transmissible spongiform encephalopathies (TSEs), are caused by a group of related but molecularly uncharacterized infectious agents. The UK-BSE agent infected many species, including humans where it causes variant CJD (vCJD). As in most viral infections, different TSE disease phenotypes are determined by both the agent strain and the host species. TSE strains are most reliably classified by incubation time and regional neuropathology in mice expressing wild-type (wt) prion protein (PrP). We compared vCJD to other human and animal derived TSE strains in both mice and neuronal cultures expressing wt murine PrP. Primary and serial passages of the human vCJD agent, as well as the highly selected mutant 263K sheep scrapie agent, revealed profound strain-specific characteristics were encoded by the agent, not by host PrP. Prion theory posits that PrP converts itself into the infectious agent, and thus short incubations require identical PrP sequences in the donor and recipient host. However, wt PrP mice injected with human vCJD brain homogenates showed dramatically shorter primary incubation times than mice expressing only human PrP, a finding not in accord with a PrP species barrier. All mouse passage brains showed the vCJD agent derived from a stable BSE strain. Additionally, both vCJD brain and monotypic neuronal cultures produced a diagnostic 19 kDa PrP fragment previously observed only in BSE and vCJD primate brains. Monotypic cultures can be used to identify the intrinsic, strain-determining molecules of TSE infectious particles.

A rapid accurate culture assay for infectivity in Transmissible Encephalopathies.

The molecular and structural features of infectious agents that cause CJD, scrapie and BSE remain controversial. A major impediment for agent resolution is the very long and expensive animal assays of infectivity. It is crucial to develop a rapid and broadly applicable cell culture assay to titer and compare different TSE agent strains. Because we found GT1 hypothalamic cells, unlike neuroblastoma N2a clones, were highly susceptible to a variety of TSE agents, and could stably produce high agent titers for >1 year, we studied the progressive display of abnormal prion protein (PrP-res) in GT1 cells following exposure to serially diluted 22L scrapie brain homogenates; PrP-res was used as a surrogate, but non-quantitative marker of GT1 infection. Even as early as the first cell split after 22L exposure, GT1 cells produced their own PrP-res bands that were clearly different than brain bands. Plots from passages 3-7 showed a good discrimination of 3 fold differences in titer over a range of >2 logs, with the same endpoint sensitivity (2 x 10(8) LD(50)/gm) as animal assays. Interestingly, the rapid production of de novo PrP-res suggested that GT1 PrP-res might be induced by interaction with an early-intermediate form of a particle that was not fully infectious. The GT1 assay here was also invaluable for rapidly identifying cell cultures with variant titers, even after detergent lysis. Additionally, in-situ PrP amyloid staining provided an independent measure of the minimum infectious dose per cell. Standardized GT1 assays can be used for direct comparison of different agent strains, and will facilitate the rapid isolation of essential agent components.