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

Abstract

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.

CJD and Scrapie Require Agent‐Associated Nucleic Acids for Infection

“…Table summarizes the infectious particle isolation, based on >20 independent particle preparations that included complete analysis of recovered infectivity and residual host components. As previously detailed [Botsios et al, ] fractionations were reproducible with small SEMs. Line A in Table shows that 85–110% of the whole cell infectivity was recovered in the p18 washed particles.…”

“…Summarizes the recoveries of infectivity (% starting TCID, line A) along with progressive particle purification from total host proteins (B), host prion protein (PrP) and its PrP‐res component analyzed on a separate aliquot of each sample using limited PK digestion (C), and cellular nucleic acids (D). These represent titers and analyses from >20 independent preparations [Botsios et al, ]. The ASB data is new and detailed in the results, and the infectivity of the ASB‐supe was 2.1e9/3e9 TCID.…”

“…The zwitterionic detergent ASB14 (ASB), not previously evaluated on TSE agents, was here found to be most useful for further agent purification. Minimal sarkosyl (0.2%), that does not reduce particle titers [Botsios et al, 2015], was essential for effective nuclease digestions. Table I summarizes multiple analyses of ASB14 on particle sedimentation.…”

“…We here show reproducible and significant nuclease effects on two distinct TSE agent‐strains that replicate exponentially in GT1 cells: the human FU‐CJD and the sheep 22L scrapie agents. Both have high infectious titers in GT1 cells, as also verified in wt mice [Arjona et al, ; Nishida et al, ], but 22L scrapie is more sensitive than FU‐CJD to chemicals that disrupt nucleic acid‐protein complexes [Botsios et al, ]. Both agents are “solubilized” by 0.2% sarkosyl, a detergent that does not reduce titer, but that was necessary for nuclease effects.…”

“…Several prion proponents acknowledge that a host “protein X” or other undefined “cofactor” is required for infection [Manuelidis, ], but protected nucleic acids that are present in all highly infectious preparations have been largely dismissed, as has a causal viral structure [Prusiner, ; Karpuj et al, ]. Nevertheless, chemicals that destroy conventional viral particles by releasing their nucleic acid core also release TSE particle nucleic acids and simultaneously destroy infectivity while leaving PrP/PrP‐res multimers intact [Manuelidis et al, ; Botsios et al, ]. This structural destruction of TSE infectivity implies, but does not prove, that nucleic acids are required for TSE particle infectivity.…”