Evaluation of a combinatorial approach to prion inactivation using an oxidizing agent, SDS, and proteinase K.

Jodi D Smith,Justin J Greenlee,Eric M Nicholson

doi:10.1186/1746-6148-9-151

Jodi D Smith, Justin J Greenlee + Show 1 more

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https://doi.org/10.1186/1746-6148-9-151

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Abstract

BackgroundPrions demonstrate an unusual resistance to methods effective at inactivating conventional microorganisms. This has resulted in a very tangible and difficult infection control challenge to the medical and veterinary communities, as well as animal agriculture and related industries. Currently accepted practices of harsh chemical treatments such as prolonged exposure to sodium hydroxide or sodium hypochlorite, or autoclaving are not suitable in many situations. Less caustic and more readily applicable treatments to contaminated environments are therefore desirable. We recently demonstrated that exposure of the RML scrapie agent to a commercial product containing sodium percarbonate (SPC-P) with or without sodium dodecyl sulfate (SDS) rendered PrPSc sensitive to proteinase K (PK), but did not eliminate infectivity. The current study was designed to evaluate the efficacy of a combinatorial approach to inactivating prions by exposing RML-positive brain homogenate to SPC-P and SDS followed by PK. Treated samples were evaluated for PrPSc-immunoreactivity by western blot, and residual infectivity by mouse bioassay.ResultsTreatment of infected brain homogenate with SPC-P and SDS followed by PK exposure resulted in a 4–5 log10 reduction in infectivity when bioassayed in tga20 mice.ConclusionsThis study demonstrates that exposure of the RML scrapie agent to SPC-P and SDS followed by PK markedly reduces, but does not eliminate infectivity. The results of this study encourage further investigation into whether consecutive or concomitant exposure to sodium percarbonate, SDS, and a protease may serve as a viable and non-caustic option for prion inactivation.

Highlights

Prions demonstrate an unusual resistance to methods effective at inactivating conventional microorganisms
In a prior study [29], we found that exposure of RML-positive brain homogenate to a sodium percarbonate-containing product (SPC-P) with or without sodium dodecyl sulfate (SDS) was unsuccessful at eliminating infectivity, but did increase sensitivity of Diseaseassociated isoform of the prion protein (PrPSc) to proteinase K (PK) digestion
Immunoblotting Residual PrPSc in brain homogenate treated with a low or high concentration of the SPC-containing product (SPC-PL and High concentration SPC-P (SPC-PH), respectively) in combination with 2.5% w/v SDS followed by exposure to PK was assayed via western blot (WB)

Summary

Introduction

Prions demonstrate an unusual resistance to methods effective at inactivating conventional microorganisms. The exact nature of the infectious agent remains open to debate with the identification of protease sensitive forms of disease-associated PrP [2,3,4] and retention of infectivity after complete PrP digestion [5] Regardless of their specific identity, prions are notoriously difficult to inactivate, withstanding methods effective against conventional pathogens, such as moderate heating, and reasonably sensitive to ecological systems when applied to contaminated outdoor environments. A genetically engineered variant of subtilisin applied under alkaline conditions was shown to reduce infectivity of a mouse-adapted BSE strain by > 7 logs [21] While these reagents have demonstrated anti-prion activity on their own, combining proteases and SDS appears to enhance their prion inactivating abilities. More recent studies have demonstrated significant reductions in infectivity for hamster-adapted scrapie and mouse-adapted BSE strains using vaporized hydrogen peroxide [25,28]

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