Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein ...(PrP
res) that replicates in the body by inducing the misfolding of the cellular prion protein (PrP
C). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrP
C → PrP
res conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrP
res. The in vitro-generated forms of PrP
res share similar biochemical and structural properties with PrP
res derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrP
res led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.
Respiratory viruses such as influenza do not typically cause viremia; however, SARS-CoV-2 has been detected in the blood of COVID-19 patients with mild and severe symptoms. Detection of SARS-CoV-2 in ...blood raises questions about its role in pathogenesis as well as transfusion safety concerns. Blood donor reports of symptoms or a diagnosis of COVID-19 after donation (post-donation information, PDI) preceded or coincided with increased general population COVID-19 mortality. Plasma samples from 2,250 blood donors who reported possible COVID-19-related PDI were tested for the presence of SARSCoV-2 RNA. Detection of RNAemia peaked at 9%-15% of PDI donors in late 2020 to early 2021 and fell to approximately 4% after implementation of widespread vaccination in the population. RNAemic donors were 1.2- to 1.4-fold more likely to report cough or shortness of breath and 1.8-fold more likely to report change in taste or smell compared with infected donors without detectable RNAemia. No infectious virus was detected in plasma from RNAemic donors; inoculation of permissive cell lines produced less than 0.7-7 plaque-forming units (PFU)/mL and in susceptible mice less than 100 PFU/mL in RNA-positive plasma based on limits of detection in these models. These findings suggest that blood transfusions are highly unlikely to transmit SARS-CoV-2 infection.
Background
SARS‐CoV‐2 RNA prevalence in blood donors from large geographic areas of high community transmission is limited. We tested residual donor plasma minipools (MPs) to determine SARS‐CoV‐2 ...RNAemia prevalence in six United States areas.
Study Design/Methods
Blood donations collected from 7 March 2020 to 25 September 2020 were tested for SARS‐CoV‐2 RNA (vRNA) in MP of 6 or 16 donations using the Grifols Procleix SARS‐CoV‐2 research‐use only (RUO) transcription‐mediated amplification (TMA) assay. Reactive results were confirmed using an alternate target region TMA assay. Reactive MPs were tested by TMA after serial dilution to estimate viral load. Testing for anti‐SARS‐CoV‐2 antibodies and infectivity was performed.
Results
A total of 17,995 MPs corresponding to approximately 258,000 donations were tested for vRNA. Three confirmed reactive MP16 were identified. The estimated prevalence of vRNA reactive donations was 1.16/100,000 (95% CI 0.40, 3.42). The vRNA‐reactive samples were non‐reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP ranged from <1000 to <4000 copies/ml. When tested, no infectivity was observed in inoculated permissive cell cultures.
Discussion
Blood donation MP‐nucleic acid testing (NAT) indicated that SARS‐CoV‐2 RNAemia is infrequent and, when detected, the vRNA was at low concentrations. Only one RNA‐reactive MP could be tested for infectivity for operational reasons and was not infectious in cell culture. These findings support current recommendations from international and national regulatory agencies to not screen donors by NAT.
Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under ...different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.
► The presence of P. putida quickened biofilm formation and increased BAC resistance of L. monocytogenes. ► The association between P. putida and L. monocytogenes varies depending of the involved strains. ► The correct evaluation of biofilm formation should consider by indirect variables such as resistance to disinfectants.
Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). ...Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrPSc starting from a 1 × 10-12 dilution of scrapie hamster brain, which contains the equivalent of ∼26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as one oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrPSc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.
Presymptomatic Detection of Prions in Blood Saá, Paula; Castilla, Joaquín; Soto, Claudio
Science (American Association for the Advancement of Science),
07/2006, Letnik:
313, Številka:
5783
Journal Article
Recenzirano
Prions are thought to be the proteinaceous infectious agents responsible for transmissible spongiform encephalopathies (TSEs). PrPSc, the main component of the infectious agent, is also the only ...validated surrogate marker for the disease, and its sensitive detection is critical for minimizing the spread of the disease. We detected PrPSc biochemically in the blood of hamsters infected with scrapie during most of the presymptomatic phase of the disease. At early stages of the incubation period, PrPSc detected in blood was likely to be from the peripheral replication of prions, whereas at the symptomatic phase, PrPSc in blood was more likely to have leaked from the brain. The ability to detect prions biochemically in the blood of infected but not clinically sick animals offers a great promise for the noninvasive early diagnosis of TSEs.
BACKGROUND
Characteristics of US blood donors with recent (RBI) or occult (OBI) hepatitis B virus (HBV) infection are not well defined.
METHODS
Donors with RBI and OBI were identified by nucleic acid ...and serologic testing among 34.4 million donations during 2009–2015. Consenting donors were interviewed and their HBV S‐gene sequenced.
RESULTS
The overall rate of HBV‐infected donors was 7.95 per 100,000; of these, 0.35 per 100,000 and 1.70 per 100,000 were RBI and OBI, respectively. RBI (n = 120) and OBI (n = 583) donors constituted 26% of all HBV‐infected (n = 2735) donors. Detection of HBV DNA in 92% of OBI donors required individual donation nucleic acid testing. Donors with OBI compared to RBI were older (mean age, 48 vs 39 years; p < 0.0001) with lower median viral loads (9 vs. 529 IU/mL; p < 0.0001). A higher proportion of OBI than RBI donors were born or resided in an endemic country (39% vs. 5%; p = 0.0078). Seventy‐seven percent of all RBI and OBI donors had multiple sex partners, an HBV‐risk factor. Of 40 RBI and 10 OBI donors whose S gene was sequenced, 33 (83%) and 6 (60%), respectively, carried HBV subgenotype A2; 18 (55%) and 2 (33%), respectively, shared an identical sequence. Infection with 1 or more putative HBV‐immune‐escape mutants was identified in 5 (50%) of OBI but no RBI donors.
CONCLUSION
RBI and OBI continue to be identified at low rates, confirming the importance of comprehensive HBV DNA screening of US blood donations. HBV‐infected donors require referral for care and evaluation and contact tracing; their HBV strains may provide important information on emergent genotypes.
Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrPSc) binds to the cellular form (PrPC), generating additional molecules of PrPSc. While several ...regions of the PrPC molecule have been suggested to play a role in PrPSc formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrPSc. These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrPC that binds to PrPSc and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to beta -sheet-rich oligomers that bind to PrPC.
•PMCA is essential for studying biological and molecular aspects of TSE pathogenesis.•Its sensitivity allows preclinical PrPTSE detection in biological fluids and tissues.•It provides an invaluable ...matrix for testing potential therapeutic molecules.•PMCA also facilitates risk assessment studies to minimize iatrogenic TSE transmission.•It can be applied to investigate protein conformational disorders such as AD and PD.
Transmissible spongiform encephalopathies (TSEs) most commonly known as prion diseases are invariably fatal neurological disorders that affect humans and animals. These disorders differ from other neurodegenerative conformational diseases caused by the accumulation in the brain of misfolded proteins, sometimes with amyloid properties, in their ability to infect susceptible species by various routes. While the infectious properties of amyloidogenic proteins, other than misfolded prion protein (PrPTSE), are currently under scrutiny, their potential to transmit from cell to cell, one of the intrinsic properties of the prion, has been recently shown in vitro and in vivo. Over the decades, various cell culture and laboratory animal models have been developed to study TSEs. These assays have been widely used in a variety of applications but showed to be time consuming and entailed elevated costs. Novel economic and fast alternatives became available with the development of in vitro assays that are based on the property of conformationally abnormal PrPTSE to recruit normal cellular PrPC to misfold. These include the cell-free conversion assay, protein misfolding cyclic amplification (PMCA) and quaking induced conversion assay (QuIC), of which the PMCA has been the only technology shown to generate infectious prions. Moreover, it allows indefinite amplification of PrPTSE with strain-specific biochemical and biological properties of the original molecules and under certain conditions may give rise to new spontaneously generated prions. The method also allows addressing the species barrier phenomena and assessing possible risks of animal-to-animal and animal-to-human transmission. Additionally, its unprecedented sensitivity has made possible the detection of as little as one infectious dose of PrPTSE and the biochemical identification of this protein in different tissues and biological fluids, including blood, cerebral spinal fluid (CSF), semen, milk, urine and saliva during the pre-clinical and clinical phases of the disease. The mechanistic similarities between TSEs and other conformational disorders have resulted in the adaptation of the PMCA to the amplification and detection of various amyloidogenic proteins. Here we provide a compelling discussion of the different applications of this technology to the study of TSEs and other neurodegenerative diseases.
PDF
