Patient and immunological factors associated with delayed clearance of mucosal SARS-CoV-2 RNA and symptom persistence.

Serial blood and mucosal samples were characterized for 102 participants enrolled a median of 7.0 days post-COVID-19 diagnosis. Mucosal RNA was detectable a median 31.5 (95% CI 20.5 - 63.5) days, with persistence ≥1 month associated with obesity (BMI ≥30, OR 3.9, 95% CI 1.2 - 13.8) but not age, sex, or chronic conditions. Fifteen participants had likely reinfection; lower serum anti-S IgG levels were associated with reinfection risk. Nearly half of participants (47%) reported symptoms lasting ≥2-3 months; persistence ≥3 months was associated with BMI ≥30 (OR = 4.2 95% CI 1.1 - 12.8) and peak anti-S and anti-NC antibody levels.

Estimates of SARS-CoV-2 Seroprevalence and Incidence of Primary SARS-CoV-2 Infections Among Blood Donors, by COVID-19 Vaccination Status - United States, April 2021-September 2022.

Changes in testing behaviors and reporting requirements have hampered the ability to estimate the U.S. SARS-CoV-2 incidence (1). Hybrid immunity (immunity derived from both previous infection and vaccination) has been reported to provide better protection than that from infection or vaccination alone (2). To estimate the incidence of infection and the prevalence of infection- or vaccination-induced antibodies (or both), data from a nationwide, longitudinal cohort of blood donors were analyzed. During the second quarter of 2021 (April-June), an estimated 68.4% of persons aged ≥16 years had infection- or vaccination-induced SARS-CoV-2 antibodies, including 47.5% from vaccination alone, 12.0% from infection alone, and 8.9% from both. By the third quarter of 2022 (July-September), 96.4% had SARS-CoV-2 antibodies from previous infection or vaccination, including 22.6% from infection alone and 26.1% from vaccination alone; 47.7% had hybrid immunity. Prevalence of hybrid immunity was lowest among persons aged ≥65 years (36.9%), the group with the highest risk for severe disease if infected, and was highest among those aged 16-29 years (59.6%). Low prevalence of infection-induced and hybrid immunity among older adults reflects the success of public health infection prevention efforts while also highlighting the importance of older adults staying up to date with recommended COVID-19 vaccination, including at least 1 bivalent dose.*,†.

Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions.

Prions are unconventional infectious agents composed exclusively of misfolded prion protein (PrP(Sc)), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrP(C)). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here, we report the generation of infectious prions by interspecies transmission of PrP(Sc) misfolding by in vitro PMCA amplification. Hamster PrP(C) misfolded by mixing with mouse PrP(Sc) generated unique prions that were infectious to wild-type hamsters, and similar results were obtained in the opposite direction. Successive rounds of PMCA amplification result in adaptation of the in vitro-produced prions, in a process reminiscent of strain stabilization observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool for the investigation of cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.

Patterns of Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 Among 1.6 Million Blood Donors: Impact of Vaccination, United States, December 2020-June 2021.

From December 2020 to June 2021, 1654487 blood donors were tested for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1 protein, and 1028547 (62.17%) were reactive. A rapid increase in prevalence was due to vaccination. Among a subset of 1567446 donors, 729771 (46.56%) reported SARS-CoV-2 vaccination, of whom 633769 (86.84%) were S1-antibody reactive only in response to vaccination and 68269 (9.35%) were reactive to both S1 and nucleocapsid in response to prior infection; the remainder were not reactive to either antibody. Among the 837675 (53.44%) donors who did not report vaccination, 210022 (25.07%) had reactivity to both antibodies and 29446 (3.52%) to S1 only.

Use of US Blood Donors for National Serosurveillance of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies: Basis for an Expanded National Donor Serosurveillance Program.

BACKGROUND: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) Epidemiology, Surveillance and Preparedness of the Novel SARS-CoV-2 Epidemic (RESPONSE) seroprevalence study conducted monthly cross-sectional testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood donors in 6 US metropolitan regions to estimate the extent of SARS-CoV-2 infections over time. METHODS: During March-August 2020, approximately ≥1000 serum specimens were collected monthly from each region and tested for SARS-CoV-2 antibodies using a well-validated algorithm. Regional seroprevalence estimates were weighted based on demographic differences compared with the general population. Seroprevalence was compared with reported coronavirus disease 2019 (COVID-19) case rates over time. RESULTS: For all regions, seroprevalence was <1.0% in March 2020. New York, New York, experienced the biggest increase (peak seroprevalence, 15.8% in May). All other regions experienced modest increases in seroprevalence (1%-2% in May-June to 2%-4% in July-August). Seroprevalence was higher in younger, non-Hispanic black, and Hispanic donors. Temporal increases in donor seroprevalence correlated with reported case rates in each region. In August, 1.3-5.6 estimated cumulative infections (based on seroprevalence data) per COVID-19 case were reported to the Centers for Disease Control and Prevention. CONCLUSIONS: Increases in seroprevalence were found in all regions, with the largest increase in New York. Seroprevalence was higher in non-Hispanic black and Hispanic than in non-Hispanic white blood donors. SARS-CoV-2 antibody testing of blood donor samples can be used to estimate the seroprevalence in the general population by region and demographic group. The methods derived from the RESPONSE seroprevalence study served as the basis for expanding SARS-CoV-2 seroprevalence surveillance to all 50 states and Puerto Rico.

Population-Weighted Seroprevalence From Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection, Vaccination, and Hybrid Immunity Among US Blood Donations From January to December 2021.

BACKGROUND: Previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination, independently and combined ("hybrid immunity"), result in partial protection from subsequent infection and strong protection from severe disease. Proportions of the US population who have been infected, vaccinated, or have hybrid immunity remain unclear, posing a challenge for assessing effective pandemic mitigation strategies. METHODS: In this serial cross-sectional study, nationwide blood donor specimens collected during January-December 2021 were tested for anti-spike and anti-nucleocapsid antibodies, and donor COVID-19 vaccination history of ≥1 dose was collected. Monthly seroprevalence induced from SARS-CoV-2 infection, COVID-19 vaccination, or both, were estimated. Estimates were weighted to account for demographic differences from the general population and were compared temporally and by demographic factors. RESULTS: Overall, 1 123 855 blood samples were assayed. From January to December 2021, the weighted percentage of donations with seropositivity changed as follows: seropositivity due to vaccination without previous infection, increase from 3.5% (95% confidence interval, 3.4%-3.7%) to 64.0%, (63.5%-64.5%); seropositivity due to previous infection without vaccination, decrease from 15.6% (15.2%-16.0%) to 11.7% (11.4%-12.0%); and seropositivity due to hybrid immunity, increase from 0.7% (0.6%-0.7%) to 18.9% (18.5%-19.3%). Combined seroprevalence from infection, vaccination, or both increased from 19.8% (19.3%-20.2%) to 94.5% (93.5%-94.0%). Infection- and vaccination-induced antibody responses varied significantly by age, race-ethnicity, and region, but not by sex. CONCLUSIONS: Our results indicate substantial increases in population humoral immunity from SARS-CoV-2 infection, COVID-19 vaccination, and hybrid immunity during 2021. These findings are important to consider in future COVID-19 studies and long-term pandemic mitigation efforts.

How do we form and coordinate a national serosurvey of SARS-CoV-2 within the blood collection industry?

BACKGROUND: A national serosurvey of U.S. blood donors conducted in partnership with the Centers for Disease Control and Prevention (CDC) was initiated to estimate the prevalence of SARS-CoV-2 infections and vaccinations. METHODS: Beginning in July 2020, the Nationwide Blood Donor Seroprevalence Study collaborated with multiple blood collection organizations, testing labs, and leadership from government partners to capture, test, and analyze approximately 150,000 blood donation specimens per month in a repeated, cross-sectional seroprevalence survey. RESULTS: A CDC website (https://covid.cdc.gov/covid-data-tracker/#nationwide-blood-donor-seroprevalence) provided stratified, population-level results to public health professionals and the general public. DISCUSSION: The study adapted operations as the pandemic evolved, changing specimen flow and testing algorithms, and collecting additional data elements in response to changing policies on universal blood donation screening and administration of SARS-CoV-2 spike-based vaccines. The national serosurvey demonstrated the utility of serosurveillance testing of residual blood donations and highlighted the role of the blood collection industry in public-private partnerships during a public health emergency.

Investigational Testing for Zika Virus among U.S. Blood Donors.

BACKGROUND: Because of the potential severe clinical consequences of Zika virus (ZIKV) infection, the large numbers of asymptomatic travelers returning from ZIKV-active areas, the detection of ZIKV nucleic acid in blood, and reports of transmission of ZIKV through transfusion, in 2016 the Food and Drug Administration released recommendations for individual-unit nucleic acid testing to minimize the risk of transmission of ZIKV through blood transfusions. METHODS: The American Red Cross implemented investigational screening of donated blood for ZIKV RNA by means of transcription-mediated amplification (TMA). Confirmatory testing of reactive donations involved repeat TMA, TMA testing in exploratory minipools, real-time reverse-transcriptase polymerase chain reaction, IgM serologic testing, and red-cell TMA. Viral loads in plasma and red cells were estimated by means of end-point TMA. The costs of interdicting a donation that was confirmed to be positive were calculated for the 15-month period between June 2016 and September 2017. RESULTS: Of the 4,325,889 donations that were screened, 393,713 (9%) were initially tested in 24,611 minipools, and no reactive donations were found. Of the 3,932,176 donations that were subsequently tested individually, 160 were initially reactive and 9 were confirmed positive (a 1:480,654 confirmed-positive rate overall; positive predictive value, 5.6%; specificity, 99.997%). Six (67%) of the confirmed-positive donations were reactive on repeat TMA, of which 4 were IgM-negative; of these 4, all 3 that could be tested were reactive on minipool TMA. Two confirmed-positive donors had infections that had been transmitted locally (in Florida), 6 had traveled to ZIKV-active areas, and 1 had received an experimental ZIKV vaccine. ZIKV RNA levels in red cells ranged from 40 to 800,000 copies per milliliter and were detected up to 154 days after donation, as compared with 80 days of detection in plasma at levels of 12 to 20,000 copies per milliliter. On the basis of industry-reported costs of testing and the yield of the tests in our study, the cost of identifying 8 mosquito-borne ZIKV infections through individual-unit nucleic acid testing was $5.3 million per ZIKV RNA-positive donation. CONCLUSIONS: Screening of U.S. blood donations for ZIKV by individual-donation TMA was costly and had a low yield. Among the 9 confirmed ZIKV-positive donations, only 4 were IgM-negative; of these donations, all 3 that were tested were reactive on minipool TMA. (Funded by the American Red Cross and Grifols Diagnostic Solutions.).

Minipool testing for SARS-CoV-2 RNA in United States blood donors.

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.

Estimated US Infection- and Vaccine-Induced SARS-CoV-2 Seroprevalence Based on Blood Donations, July 2020-May 2021.

Importance: People who have been infected with or vaccinated against SARS-CoV-2 have reduced risk of subsequent infection, but the proportion of people in the US with SARS-CoV-2 antibodies from infection or vaccination is uncertain. Objective: To estimate trends in SARS-CoV-2 seroprevalence related to infection and vaccination in the US population. Design, Setting, and Participants: In a repeated cross-sectional study conducted each month during July 2020 through May 2021, 17 blood collection organizations with blood donations from all 50 US states; Washington, DC; and Puerto Rico were organized into 66 study-specific regions, representing a catchment of 74% of the US population. For each study region, specimens from a median of approximately 2000 blood donors were selected and tested each month; a total of 1 594 363 specimens were initially selected and tested. The final date of blood donation collection was May 31, 2021. Exposure: Calendar time. Main Outcomes and Measures: Proportion of persons with detectable SARS-CoV-2 spike and nucleocapsid antibodies. Seroprevalence was weighted for demographic differences between the blood donor sample and general population. Infection-induced seroprevalence was defined as the prevalence of the population with both spike and nucleocapsid antibodies. Combined infection- and vaccination-induced seroprevalence was defined as the prevalence of the population with spike antibodies. The seroprevalence estimates were compared with cumulative COVID-19 case report incidence rates. Results: Among 1 443 519 specimens included, 733 052 (50.8%) were from women, 174 842 (12.1%) were from persons aged 16 to 29 years, 292 258 (20.2%) were from persons aged 65 years and older, 36 654 (2.5%) were from non-Hispanic Black persons, and 88 773 (6.1%) were from Hispanic persons. The overall infection-induced SARS-CoV-2 seroprevalence estimate increased from 3.5% (95% CI, 3.2%-3.8%) in July 2020 to 20.2% (95% CI, 19.9%-20.6%) in May 2021; the combined infection- and vaccination-induced seroprevalence estimate in May 2021 was 83.3% (95% CI, 82.9%-83.7%). By May 2021, 2.1 SARS-CoV-2 infections (95% CI, 2.0-2.1) per reported COVID-19 case were estimated to have occurred. Conclusions and Relevance: Based on a sample of blood donations in the US from July 2020 through May 2021, vaccine- and infection-induced SARS-CoV-2 seroprevalence increased over time and varied by age, race and ethnicity, and geographic region. Despite weighting to adjust for demographic differences, these findings from a national sample of blood donors may not be representative of the entire US population.

Qualification of the Geenius HIV 1/2 supplemental assay for use in the HIV blood donation screening algorithm.

BACKGROUND: A single, simplified approach for human immunodeficiency virus (HIV)-1/HIV-2 antibody confirmation/differentiation is needed for the HIV blood donation supplemental algorithm used in the United States. A clinical evaluation of the Geenius assay was performed-the same assay used for HIV diagnostic confirmation/differentiation in the United States since 2014. STUDY DESIGN AND METHODS: Well-characterized unlinked donation samples classified as HIV negative, false positive, or confirmed positive were included in the study: 200 antibody-nonreactive, 200 HIV-1 immunofluorescence assay (IFA) confirmed-positive, and 100 antibody-screen false-positive donations, equally divided between serum and plasma. Samples were retrieved from a repository, relabeled, and tested by an immunochromatographic test (Geenius HIV 1/2 Supplemental Assay, Bio-Rad). Comparator testing involved parallel US Food and Drug Administration (FDA)-licensed HIV-1 IFA or HIV-2 enzyme immunoassay (EIA) supplemental testing for any sample missing those results as part of routine testing (otherwise test-of-record results were used). Samples with discordant results were further tested with a rapid test (Multispot HIV-1/HIV-2 Rapid Test, Bio-Rad) to provide final sample interpretations. Testing volume reductions with the Geenius were estimated from screening performed by the American Red Cross from September 2016 to April 2019. RESULTS: Clinical results were 100% sensitivity and specificity with an indeterminate rate of 4.0% to 5.0%. From 2016 to 2019, sole use of the Geenius would reduce testing complexity for 5265 antibody repeat-reactive donations including 95.7% (5028) false positives, eliminating approximately 5000 unnecessary tests. CONCLUSION: Geenius FDA licensure (August 26, 2019) adding the HIV-1/HIV-2 differentiation/confirmation donation supplemental claim will enable replacement of previously used FDA-licensed supplemental assays while maintaining comparable sensitivity, avoiding thousands of unnecessary HIV-1-IFA, western blot, and HIV-2-EIA tests.

Acute Zika virus infection in an asymptomatic blood donor at the onset of the Puerto Rico epidemic.

BACKGROUND: Zika virus (ZIKV) spread to Puerto Rico likely originated from southeastern Brazil approximately 8.5 months earlier than blood donation screening for ZIKV was initiated, but the time of ZIKV introduction in the blood donor population remains unknown. METHODS: To better understand when arboviral infections first appeared in the blood donor pool in Puerto Rico, we retrospectively screened for ZIKV RNA (as well as chikungunya [CHIKV] and dengue [DENV] viral RNA) a repository of 1186 linked blood donor and recipient samples collected from February 2015 to May 2016 as an endpoint efficacy measure following the introduction of platelet pathogen reduction (PR). Phylogenetic analysis identified relatedness of donor strain to other circulating strains, and molecular clock analysis identified the estimated time of introduction. RESULTS: An asymptomatic donor collected in December 2015 was ZIKV RNA confirmed positive, 4 months BEFORE investigational nucleic acid testing (NAT) implementation in April 2016, coincident and related to the first reported autochthonous cases. No CHIKV RNA or DENV RNA reactives were identified in donors or recipients, and no adverse events were reported from PR use in recipients. Phylogenetic analysis confirmed the molecular relatedness of the donor ZIKV strain to the Puerto Rico lineage likely introduced approximately 4.5 months earlier. CONCLUSION: This study identified an asymptomatic ZIKV infection in a blood donor occurring before those previously recognized by blood donation screening. NAT and PR continue to be used as acceptable strategies to prevent transfusion-transmitted arboviral infections worldwide; however, repeated arboviral outbreaks warrant consideration of PR as a more proactive approach.

Recent and occult hepatitis B virus infections among blood donors in the United States.

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.

Redefining Blood Donation-Path to Inclusivity and Safety.

This Viewpoint reviews the vicissitudes of establishing fair and equitable blood donation policies based on research and improved serological testing and promotes expanding the pool of potential donors.

Mechanisms of prion-induced neurodegeneration.

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders characterised by long incubation period, short clinical duration, and transmissibility to susceptible species. Neuronal loss, spongiform changes, gliosis and the accumulation in the brain of the misfolded version of a membrane-bound cellular prion protein (PrP(C)), termed PrP(TSE), are diagnostic markers of these diseases. Compelling evidence links protein misfolding and its accumulation with neurodegenerative changes. Accordingly, several mechanisms of prion-mediated neurotoxicity have been proposed. In this paper, we provide an overview of the recent knowledge on the mechanisms of neuropathogenesis, the neurotoxic PrP species and the possible therapeutic approaches to treat these devastating disorders.

Are prions transported by plasma exosomes?

Blood has been shown to contain disease-associated misfolded prion protein (PrP(TSE)) in animals naturally and experimentally infected with various transmissible spongiform encephalopathy (TSE) agents, and in humans infected with variant Creutzfeldt-Jakob disease (vCJD). Recently, we have demonstrated PrP(TSE) in extracellular vesicle preparations (EVs) containing exosomes from plasma of mice infected with mouse-adapted vCJD by Protein Misfolding Cyclic Amplification (PMCA). Here we report the detection of PrP(TSE) by PMCA in EVs from plasma of mice infected with Fukuoka-1 (FU), an isolate from a Gerstmann-Sträussler-Scheinker disease patient. We used Tga20 transgenic mice that over-express mouse cellular prion protein, to assay by intracranial injections the level of infectivity in a FU-infected brain homogenate from wild-type mice (FU-BH), and in blood cellular components (BCC), consisting of red blood cells, white blood cells and platelets, plasma EVs, and plasma EVs subjected to multiple rounds of PMCA. Only FU-BH and plasma EVs from FU-infected mice subjected to PMCA that contained PrP(TSE) transmitted disease to Tga20 mice. Plasma EVs not subjected to PMCA and BCC from FU-infected mice failed to transmit disease. These findings confirm the high sensitivity of PMCA for PrP(TSE) detection in plasma EVs and the efficiency of this in vitro method to produce highly infectious prions. The results of our study encourage further research to define the role of EVs and, more specifically exosomes, as blood-borne carriers of PrP(TSE).

First demonstration of transmissible spongiform encephalopathy-associated prion protein (PrPTSE) in extracellular vesicles from plasma of mice infected with mouse-adapted variant Creutzfeldt-Jakob disease by in vitro amplification.

The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP(TSE)), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP(C)) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP(TSE) in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations.

The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc).

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) 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 PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) 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 ß-sheet-rich oligomers that bind to PrP(C).

Strain-specific role of RNAs in prion replication.

Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.