+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: 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

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

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

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

99.5% digested

99.8% digested

PrP takes longer to digest in cells

95.5% digested

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

Tissue Culture Infectious Dose

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

Tissue Culture Infectious Dose2hrs 2hrs

PK at 6 and 8 hrs only yield <2 log reduction

NAP reduced infectious dosage by >3.5 logs

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

Showed no loss of infectivity after virtually complete PK digestion of PrP

Suggests PrP-res regenerates to normal levels after p13

99.5% digested in 10x

99.8% digested

97.9% digested

+High CJD infectivity remains after prion protein is destroyed

(2011) - Kohtaro Miyazawa, Kaitlin Emmerling andLaura Manuelidis

“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.”

+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

+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

http://www.biomedcentral.com/1746-6148/9/8

Ladder RML +ctrl 30 min 90 min 180 min

2.5% SDS

+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

ladder ctrl 30m 90m 180m ladder ctrl 30m 90m 180m

Brain treatment with 0.35 M sodium hydrogen phosphate buffered solution

PrPSc undetectable after PK digestion

+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 GreenleeRML ctrl

Avg. survival time

+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.

+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

+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

+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

protein misfolding cyclic amplification (PMCA)

+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