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

Miyazawa, Kohtaro; Emmerling, Kaitlin; Manuelidis, Laura

doi:10.4161/viru.2.3.15880

Cited by 19 publications

(39 citation statements)

References 46 publications

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“…To rule out preparative artifacts we tested PK digestion on total brain and cell homogenates from high titer FU-CJD mouse brain and FU-CJD infected GT1 cells by quantitative Western blotting. Infectivity was titrated by a rapid and verified incubation end-point cell culture assay [Liu et al, 2008; Miyazawa et al, 2011]. This study of total PrP has become most pertinent because the newly modified prion proposal now posits that only a possibly minor and still uncharacterized PK-sensitive form of PrP is the real infectious entity [Colby and Prusiner, 2011; Cronier et al, 2008].…”

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High CJD infectivity remains after prion protein is destroyed

2011

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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, PrPsc) has been assumed to be the infectious form, with speculative types of misfolding encoding the many unique 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 2hr 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 2hr, yet decreased titer by >2.5logs; few residual protein bands remained. FU-CJD infected cells with 10x the infectivity of brain by both animal and cell culture assays were also evaluated. NAP again significantly reduced cell infectivity (>3.5 logs). Extreme PK digestions were needed to reduce cell PrP to <0.2%, yet a very high titer of ≥7.8 logs remained. Our FU-CJD brain results are in good accord with the only other report on maximal PrP digestion and titer. It is likely that one or more residual non-PrP proteins may protect agent nucleic acids in infectious particles.

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High CJD infectivity remains after prion protein is destroyed

2011

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“…In this TCID assay, a 4 log reduction of the FU-CJD agent shows a positive signal at culture passage 13. 19,50 The insoluble PrP and its clearly present PrP-res (>1%) also did not predict the FU-CJD titer losses which were >10,000 fold, or <0.01% of the control infectivity. In sum, neither the PrP-res amount or solubility can be used to assess titer.…”

Section: Urea and Thiourea Reduce Fu-cjd Infectivitymentioning

confidence: 99%

“…7,44,50 Briefly, control and treated cell equivalent (CE) subcellular fractions were serially diluted, then applied to target GT1 cells, and passaged. Replicate wells of at least 3 different passages in the linear range (at least 6 samples for Western blot) were used to determine TCID, and replicate wells all showed comparable results (<10% TCID differences).…”

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“…The TCID assay has been verified in parallel animal inoculations, and indicator cells infected by different agents also recapitulate agent-specific titer differences by animal assay. 3,7,12,19,20,[42][43][44]50 Figure 6 shows 2 representative assays at early passages after infecting indicator cells. Figure 6A shows the urea and Figure 6B the GdnHCl effects on titer.…”

Section: Representative Tcid Determinationsmentioning

confidence: 99%

“…As in animal incubation time assays, and in others' scrapie cell assays, 8,54 a low titer requires more time to induce PrP-res pathology. The TCID is calculated from published standard dilution curves that give a progressively increasing linear PrP-res response in indicator cells at sequential passages, as determined for the FU-CJD and 22L agents, e.g.. 7,50 Even though an infectivity assay is less precise than a direct analysis of cellular molecules, note the comparable %PrP-res elicited in duplicate wells exposed to the same sample. In Figure 6A, the non-infectious mock control sample induces no PrP-res (C PK lane 2, 10x load) in indicator cells.…”

Section: Representative Tcid Determinationsmentioning

confidence: 99%

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

Botsios

Tittman²,

Manuelidis

2015

Virulence

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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 lateonset 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.

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

et al. 2014

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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.

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

Cited by 19 publications

References 46 publications

High CJD infectivity remains after prion protein is destroyed

High CJD infectivity remains after prion protein is destroyed

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

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

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