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Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the development of TESSA (sTainlESs steel-beadSeedAmplification assay), a prion seed amplification assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmedin vivoin a murine prion infectivity bioassay, yielding a reduction of the prion titer for the bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

Serological assays can detect anti-SARS-CoV-2 antibodies, but their sensitivity often comes at the expense of specificity. Here we developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against SARS-CoV-2. Calibration was performed with 90 prepandemic and 55 virologically and clinically confirmed COVID-19 samples. Posterior probabilities were calculated from 3×8 measurements of logarithmically diluted samples against the ectodomain and the receptor-binding domain of the spike protein and the nucleocapsid protein. We then performed 948’528 assays on 5’503 prepandemic and 34’019 copandemic samples from hospital patients and healthy blood donors. The seroprevalence increased in March 2020 (0.3%; CI95%: 0.1% - 0.5%) among hospital patients but plateaued in April at 1.1-1.3%, and dropped to 0.3-0.7% in July. A dynamic transmission model describing SARS-CoV-2 transmission and seroconversion in the general population of the Canton of Zurich yielded an infection fatality ratio of 0.6% (CI95%: 0.4%-0.8%), similarly to other European areas. While the evolution of seroprevalence points to a high effectiveness of containment measures, our data highlight that antibody waning warrants a continuous seromonitoring to reliably estimate the prevalence in a population.

Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrP$^{C}$ selectively in neurons and astrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrP$^{C}$ expression led to copious brain accumulation of PrP$^{Sc}$. As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrP$^{C}$ expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrP$^{Sc}$ is innocuous to PrP$^{C}$-deficient neurons, these results show that astrocyte-born PrP$^{Sc}$ does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.

Transmission of prion infectivity to susceptible murine cell lines has simplified prion titration assays and has greatly reduced the need for animal experimentation. However, murine cell models suffer from technical and biological constraints. Human cell lines might be more useful, but they are much more biohazardous and are often poorly infectible. Here, we describe the human clonal cell line hovS, which lacks the human PRNP gene and expresses instead the ovine PRNP VRQ allele. HovS cells were highly susceptible to the PG127 strain of sheep-derived murine prions, reaching up to 90% infected cells in any given culture and were maintained in a continuous infected state for at least 14 passages. Infected hovS cells produced proteinase K–resistant prion protein (PrPSc), pelletable PrP aggregates, and bona fide infectious prions capable of infecting further generations of naïve hovS cells and mice expressing the VRQ allelic variant of ovine PrPC. Infection in hovS led to prominent cytopathic vacuolation akin to the spongiform changes observed in individuals suffering from prion diseases. In addition to expanding the toolbox for prion research to human experimental genetics, the hovS cell line provides a human-derived system that does not require human prions. Hence, the manipulation of scrapie-infected hovS cells may present fewer biosafety hazards than that of genuine human prions.

The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endo-lysosomal organelles. Understanding the intracellular trafficking of PrPC may therefore help elucidating the conversion process. Here we describe a time-resolved fluorescence resonance energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z'-factor > 0.5). RNA interference assays showed that suppression of AP2M1, RAB5A, VPS35 and M6PR blocked PrPC internalization, whereas downregulation of GIT2 and VPS28 increased it. PrPC cell surface expression was reduced by downregulation of RAB5A, VPS28 and VPS35, and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking, and demonstrates the power of this assay for identifying modulators of PrPC trafficking.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.

Human genetic prion diseases have invariably been linked to alterations of the prion protein (PrP) gene PRNP. Two sisters died from probable Creutzfeldt-Jakob disease (CJD) in Switzerland within 14 y. At autopsy, both patients had typical spongiform change in their brains accompanied by punctuate deposits of PrP. Biochemical analyses demonstrated proteinase K-resistant PrP. Sequencing of PRNP showed 2 wild-type alleles in both siblings. Retrospectively, clinical data revealed a history of dural transplantation in the initially deceased sister, compatible with a diagnosis of iatrogenic CJD. Clinical and familial histories provided no evidence for potential horizontal transmission. This observation of 2 siblings suffering from CJD without mutations in the PRNP gene suggests potential involvement of non-PRNP genes in prion disease etiology.