Saturday, January 12, 2013

Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity

Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity


BMC Veterinary Research 2013, 9:8 doi:10.1186/1746-6148-9-8 Jodi D Smith (jodi.smith@ars.usda.gov) Eric M Nicholson (eric.nicholson@ars.usda.gov) Gregory H Foster (ghfoster@mac.com) Justin J Greenlee (justin.greenlee@ars.usda.gov) ISSN 1746-6148


Article type Research article Submission date 18 September 2012 Acceptance date 8 January 2013 Publication date 11 January 2013 Article URL http://www.biomedcentral.com/1746-6148/9/8


BMC Veterinary Research


© 2013 Smith et al.



Exposure of RML scrapie agent to a sodium percarbonate-based product and sodium dodecyl sulfate renders PrPSc protease sensitive but does not eliminate infectivity



Jodi D Smith1 Email: jodi.smith@ars.usda.gov Eric M Nicholson1 Email: eric.nicholson@ars.usda.gov Gregory H Foster1 Email: ghfoster@mac.com Justin J Greenlee1* * Corresponding author Email: justin.greenlee@ars.usda.gov


1 Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Ave, Ames, IA 50010, USA



Abstract



Background



Prions, the causative agents of the transmissible spongiform encephalopathies, are notoriously difficult to inactivate. Current decontamination recommendations by the World Health Organization include prolonged exposure to 1 N sodium hydroxide or > 20,000 ppm sodium hypochlorite, or autoclaving. For decontamination of large stainless steel surfaces and equipment as in abattoirs, for example, these methods are harsh or unsuitable. The current study was designed to evaluate the effectiveness of a commercial product containing sodium percarbonate to inactivate prions. Samples of mouse brain infected with a mouse-adapted strain of the scrapie agent (RML) were exposed to a sodium percarbonate-based product (SPC-P). Treated samples were evaluated for abnormal prion protein (PrPSc)- immunoreactivity by western blot analysis, and residual infectivity by mouse bioassay.



Results



Exposure to a 21% solution of SPC-P or a solution containing either 2.1% or 21% SPC-P in combination with sodium dodecyl sulfate (SDS) resulted in increased proteinase K sensitivity of PrPSc. Limited reductions in infectivity were observed depending on treatment condition. A marginal effect on infectivity was observed with SPC-P alone, but an approximate 2–3 log10 reduction was observed with the addition of SDS, though exposure to SDS alone resulted in an approximate 2 log10 reduction.



Conclusions



This study demonstrates that exposure of a mouse-adapted scrapie strain to SPC-P does not eliminate infectivity, but does render PrPSc protease sensitive.



Keywords Inactivation, Prion, Scrapie, Sodium dodecyl sulfate, Sodium percarbonate



SNIP...



A major finding of this study was the increased sensitivity of PrPSc to PK by the SPC-based product without (SPC-PH only) or with SDS at room temperature, as judged by immunoblotting after exposure of the samples to limited proteolysis. Based on the loss of detectable PrPSc immunoreactivity after incubation at pH 11, it appears this effect may be largely pH-dependent. It is well established that prion infectivity is reduced under extremely basic conditions, such as exposure to NaOH (pH 12–14) [19-21]. While the pH generated by SPC-P is lower at 11, it appears to be a favorable characteristic of the compound with regard to PrPSc protease sensitivity. However, a solely pH-dependent effect does not explain why SPC-PL treatment alone (pH 11) did not yield similar WB results. One possible explanation is that a lower concentration of the product may have contained diminished buffering capacity resulting in a drop in pH as treatment proceeded, but serial pH evaluation of treated brain homogenate at 30, 90, and 180 min revealed that the pH remained above 10.7. Although treatment with the SPC product did render PrPSc sensitive to digestion by proteinase K, it did not eliminate infectivity. Recent studies examining prion infectivity in infected tissue and cell cultures have also demonstrated loss of detectable PrPSc on western blot, but residual infectivity [22,23]. Our results support the inference that biochemical analysis alone is insufficient for determination of prion infectivity. The observed PrPSc/infectivity mismatch in this study and in others warrants a number of considerations including WB sensitivity, epitope disruption by inactivation treatments, and alternative infectious agents to PrPSc, such as PK-sensitive forms of PrP or viruses. It is possible the amount of residual PrPSc in our treated samples was below the detection limit of our WB (0.025 mg equivalents of brain tissue for this particular inoculum [24]), or it may be that a true dissociation of PrPSc and TSE infectivity exists supporting the actuality of alternative infectious agents to PrPSc [25]. A recent study has demonstrated poor correlation between infectivity and WB results for sheep scrapie and sheep BSE [26] in line with observations that PK-sensitive PrP particles are associated with disease [27,28].



The bioassay results we present indicate that exposure to the selected SPC-based product alone or in combination with 2.5% SDS is not a viable option for the inactivation of prions. No decrease in infectivity was observed using the SPC-PL solution alone, and a modest 1 log10 reduction was achieved with the SPC-PH solution. However, recent investigations have demonstrated differential susceptibility of distinct prion strains to the same inactivation procedure [29]; therefore, we are currently investigating the efficacy of these treatment conditions in an ovine scrapie model. It should also be acknowledged that chemical treatment of the scrapie agent has been shown to delay the dose–response relationship [30,31] resulting in prolonged incubation times without a change in calculable titer. It is possible our results could be reflecting this phenomenon, but without bioassay data from serial dilutions of treated brain homogenate this cannot be definitively determined. Some caution may therefore be warranted when interpreting these results. The addition of 2.5% SDS to the SPC-P solutions resulted in a 2–3 log10 reduction in infectivity, but exposure to SDS alone resulted in an approximate 2 log10 reduction. This suggests much of the observed combinatorial effect was due to SDS. Prior studies using SDS have demonstrated minimal effects on CJD infectivity [16], but up to a 3 log10 reduction on scrapie infectivity [17]. Exposure of hamster-adapted Sc237 scrapie to room temperature SDS at pH values of ≤4.5 or ≥10 resulted in increased PK sensitivity of PrPSc, and exposure to acidic SDS resulted in decreased infectivity [11]. Since SDS at room temperature is an effective denaturant at a pH ≥10, this could have contributed to the loss of detectable PrPScimmunoreactivity we observed after proteolysis in samples treated with SPC-P and SDS. There was also enhanced reduction in infectivity with the combination of SPC-PL and SDS. This may be indicative of an enhanced effect of SDS under basic conditions or a two-step mechanism whereby denaturation of PrPSc by the relatively high pH of the solution and/or SDS is followed by exposure of sites sensitive to oxidative damage. Alternatively, the two treatment components could be acting on different PrPSc fractions in the inoculum resulting in an additive effect since the combination of SPC-PL and SDS was roughly equivalent to slightly greater than the sum of the effects of each individual component. The combination of SPC-PH and SDS did not provide an equivalent or better increase in survival time than the combination of SPC-PL and SDS. While we are confident in this result, we cannot definitively explain this observation. Perhaps disease in this group was exacerbated by oxidative damage induced by the introduction of treated brain samples containing a greater concentration of sodium percarbonate. Oxidative stress, whether a cause or consequence of disease progression, is considered an important contributor to prion neuropathology [32-34]. It is also possible that the SPC-P solution at higher concentration may somehow be interfering with the denaturing action of SDS. SDS action may be enhanced when combined with lower concentrations of SPC-P for longer exposure times, but restricted by higher concentrations, perhaps via chemical modification of SDS binding sites on the protein.



Oxidizing agents have been used with variable success in prion inactivation studies. Exposure of prions to halogens such as sodium hypochlorite at ≥ 20,000 ppm is an accepted means of decontamination [8], but chlorine dioxide is much less effective at inactivating hamster-adapted 263 K scrapie [35]. Peroxygens such as liquid hydrogen peroxide [13,35,36] and peracetic acid [37] also promote limited inactivation. However, recent studies using vaporized hydrogen peroxide to decontaminate stainless steel surfaces have demonstrated significant reductions in infectivity for hamster-adapted 263 K scrapie and mouse-adapted BSE [13,15]. A protective effect from oxidation by peracetic acid has been demonstrated with the ME7 scrapie agent and attributed to prion aggregation [37]. Peracetic acid at 2% was effective at inactivating the ME7 scrapie agent in intact brain tissue, but not homogenized tissue. Samples in the current study were homogenized, which may have imparted a degree of protection from oxidation and contributed to the ineffectiveness of SPC-P alone at decreasing infectivity. We propose that the addition of SDS would have decreased aggregation of cell membranes to which infectivity is bound, thus enhancing the activity of SPC-P and perhaps contributing to the increased survival observed with the combination.



Conclusions



This study demonstrates that exposure of the RML scrapie agent to an SPC-containing product alone or in combination with SDS does not eliminate prion infectivity, but does render PrPSc sensitive to proteinase K. Because of this, it is interesting to consider the potential viability of a combination of SPC and SDS, even at relatively low concentrations and mild temperatures, concomitant with or followed by a protease for prion decontamination. Also, because the SPC product we used contains additional proprietary ingredients, we cannot rule-out contributions to increased PK-sensitivity or increased survival by other components of the product. Studies in our laboratory are currently underway examining exposure of prions to chemical grade SPC with or without SDS followed by exposure to a protease.










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