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High CJD infectivity remains after the prion protein is destroyed

High CJD infectivity remains after the prion protein is destroyed. By Sylvester Gates. High CJD infectivity remains after prion protein is destroyed (2011) - Kohtaro Miyazawa, Kaitlin Emmerling and Laura Manuelidis. Goals:

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High CJD infectivity remains after the prion protein is destroyed

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  1. High CJD infectivity remains after the prion protein is destroyed By Sylvester Gates

  2. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin Emmerlingand Laura Manuelidis • Goals: • Determine if infectivity is linked to PrP load. Infectivity decreases as direct proportion to decrease of PrP with proteinase K. • Experiments: • Mouse passaged FU-CJD • PrP Protease digestion (Keratinase = NAP and proteinase K = PK) at different concentrations of Tx100 (detergent) http://onlinelibrary.wiley.com/doi/10.1002/jcb.23286/full

  3. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis • Tested NAP effectiveness under specific conditions • Digestion of FU-CJD brain with NAP • Ln5&6 NAP digestion • Ln7 under PK digestion – reveals max PrP-res

  4. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis 99.5% digested 99.8% digested 95.5% digested • PrP takes longer to digest in cells

  5. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis Tissue Culture Infectious Dose

  6. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis Tissue Culture Infectious Dose 2hrs 2hrs • PK at 6 and 8 hrs only yield <2 log reduction • NAP reduced infectious dosage by >3.5 logs

  7. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis 99.8% digested 97.9% digested 99.5% digested in 10x • Showed no loss of infectivity after virtually complete PK digestion of PrP • Suggests PrP-res regenerates to normal levels after p13

  8. High CJD infectivity remains after prion protein is destroyed (2011) - KohtaroMiyazawa, Kaitlin EmmerlingandLauraManuelidis • “Virtually complete digestion of all PrP with preservation of infectivity lead… to the conclusion that no form of prion protein is infectious” • PrP-res levels climb back up to normal even after PrP digestion by • PK digestion left ≤0.3% PrP after 2hr, yet there was no reduction in titer. • NAP digestion left 0.8% residual PrP after 2hr, yet decreased titer by >2.5logs • “GdnSCN… shown to reduce infectivity by >4logs in brain [Manuelidis, 1997], practical and complete sterilization of precious instruments should be further effected by a subsequent digestion with NAP, and probably other keratinases.”

  9. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity(2012) - Jodi D Smith, Eric M Nicholson, Gregory H Foster and Justin J Greenlee • Goals: • Evaluate effectiveness of a commercial product containing sodium percarbonate to inactivate prions. • Experiments: • Mouse brain with mouse-adapted scrapie agent (RML) • Exposed to sodium percarbonate-based product (SPC-P). • Western blots to test immunoreactivity for abnormal prion protein • Residual infectivity tested by mouse bioassay

  10. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity(2012) - Jodi D Smith, Eric M Nicholson, Gregory H Foster and Justin J Greenlee 2.5% SDS Ladder RML +ctrl 30 min 90 min 180 min http://www.biomedcentral.com/1746-6148/9/8

  11. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity(2012) - Jodi D Smith, Eric M Nicholson, Gregory H Foster and Justin J Greenlee Brain treatment with 0.35 M sodium hydrogen phosphate buffered solution • PrPSc undetectable after PK digestion ladder ctrl 30m 90m 180m ladder ctrl 30m 90m 180m

  12. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity(2012) - Jodi D Smith, Eric M Nicholson, Gregory H Foster and Justin J Greenlee Avg. survival time RML ctrl

  13. Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity(2012) - Jodi D Smith, Eric M Nicholson, Gregory H Foster and Justin J Greenlee • Exposure of RML (scrapie agent) to an SPC-containing product alone or in combination with SDS does not eliminate prion infectivity • Small effect of SPC-P alone, but an 2–3 log10 reduction observed with the addition of SDS • exposure to SDS alone resulted in an approximate 2 log10 reduction.

  14. BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle(2010) - Anne Balkema-Buschmann, Martin Eiden, Christine Hoffmann, Martin Kaatz, Ute Ziegler, Markus Keller and Martin H. Groschup • “Challenged transgenic mice overexpressing the bovine prion protein with homogenates prepared from a wide variety of tissue samples collected from BSE-infected cattle” • Various detection methods: purification, immunohistochemistry, and the protein misfolding cyclic amplification technique http://vir.sgmjournals.org/content/92/2/467.full

  15. BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle(2010) - Anne Balkema-Buschmann, Martin Eiden, Christine Hoffmann, Martin Kaatz, Ute Ziegler, Markus Keller and Martin H. Groschup http://vir.sgmjournals.org/content/92/2/467.full

  16. BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle(2010) - Anne Balkema-Buschmann, Martin Eiden, Christine Hoffmann, Martin Kaatz, Ute Ziegler, Markus Keller and Martin H. Groschup • protein misfolding cyclic amplification (PMCA) http://vir.sgmjournals.org/content/92/2/467.full

  17. BSE infectivity in the absence of detectable PrPSc accumulation in the tongue and nasal mucosa of terminally diseased cattle(2010) - Anne Balkema-Buschmann, Martin Eiden, Christine Hoffmann, Martin Kaatz, Ute Ziegler, Markus Keller and Martin H. Groschup • Detect BSE infectivity in tongue and nasal mucosa of terminally diseased BSE cases as well as experimentally challenged cattle by transgenic-mouse bioassay. • This shows that BSE infectivity can be present in the peripheral tissues. http://vir.sgmjournals.org/content/92/2/467.full

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