Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

Exploring linkages: addressing the relationship between the climate crisis and HIV prevention with recommendations for emerging pre-exposure prophylaxis programs.

Short- and long-term impacts of the climate crisis continue to be felt across the public health landscape. Many individuals marginalized by the climate crisis also navigate a higher likelihood of exposure to HIV. By understanding this relationship, we can better position HIV prevention, and pre-exposure prophylaxis (PrEP) programs specifically, to meet user needs in communities experiencing the effects of the climate crisis. In support, we propose four recommendations for mitigating the impact of the climate crisis on those who may benefit from PrEP: (1) leverage existing and emerging research and lived experience to intentionally target and appropriately reach individuals affected by the climate crisis who may need or want PrEP; (2) emphasize the need for more climate-resilient PrEP products within the research and development pipeline; (3) build a continued understanding of the role of the climate crisis-HIV relationship in product introduction through national collaboration; and (4) strengthen the integration of PrEP service delivery and response to intimate partner violence. The PrEP market is set for rapid expansion with the introduction of new prevention methods to enable choice. To be comprehensively responsive to potential PrEP users, we must consider and address how the climate crisis changes not only the environmental landscape, but the prevention ecosystem.

Evaluation of a novel triple-action adulticide containing a pyrethroid, macrocyclic lactone, and fatty acid against pyrethroid-resistant Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae).

Insecticide resistance in mosquito populations has long been recognized as a significant global public health challenge, motivating the development of new control chemistries. ReMoa Tri is a novel triple-action space spray that employs a different mode of action than traditional adult mosquito control formulations. It combines 3 components: fenpropathrin, a mixed-type I/II pyrethroid; abamectin, a macrocyclic lactone; and C8910, a patented fatty acid chain. As an ultra-low volume adulticide, ReMoa Tri has the potential to target mosquito species that are resistant to pyrethroid and organophosphate-based control materials. To determine whether ReMoa Tri effectively targets resistant mosquito species in Florida's Collier County, United States, we conducted ground-based field cage trials using field-caught pyrethroid-resistant Culex quinquefasciatus (Say) and Aedes aegypti (L.), of which the latter also displayed developing resistance to organophosphates. Trials were also conducted against the same mosquito populations with Merus 3.0, a pyrethrin-based adulticide used by the Collier Mosquito Control District. ReMoa Tri was effective against Collier's pyrethroid-resistant Cx. quinquefasciatus, resulting in more than 95% mortality in semifield cage trials by 24 h postapplication. Similarly, ReMoa Tri applications against Collier's pyrethroid-resistant Ae. aegypti resulted in 72%-89% mortality at 24 h postapplication and 74%-97% mortality at 48 h postapplication. This study represents the first field data on this novel space spray, and its findings shed light on the performance of ReMoa Tri against local mosquito populations that have developed resistance to currently available adulticides.

A comparative analysis of nutritional quality, amino acid profile, and nutritional supplementations in plant-based products and their animal-based counterparts in the UK.

Plant-based (PB) food products have surged in popularity over the past decade. Available PB products in the UK market were extracted from NielsenIQ Brandbank and compared with animal-based (AB) counterparts in their nutrient contents and calculated Nutri-Scores. The amino acid contents of four beef products and their PB alternatives were analysed by LC-MS/MS. PB products consistently exhibited significantly higher fibre content across all food groups. Protein was significantly higher in AB products from all food groups except beef and ready meals. PB products were more likely to have higher Nutri-Scores compared to AB counterparts, albeit with greater score variability within each food group. Nutrient fortifications were primarily focused on dairy and ready meals; the most supplemented nutrient was vitamin B12 (found in 15% of all products). A higher proportion of EAAs in relation to total protein content was observed in all beef products.

Temporal-to-Nasal Macular Ganglion Cell and Inner Plexiform Layer Ratios in a Large Adult Twin Cohort: Correlations With Age and Heritability.

Purpose: Temporal-to-nasal macular ganglion cell layer thickness ratios are reduced in albinism. We explored similar ratios in a large twin cohort to investigate ranges in healthy adults, correlations with age, and heritability. Methods: More than 1000 twin pairs from TwinsUK underwent macular optical coherence tomography (OCT) scans. Automated segmentation yielded thicknesses for the combined ganglion cell and inner plexiform layer (GCIPL) in Early Treatment of Diabetic Retinopathy Study subfields. Participants with diseases likely to affect these layers or segmentation accuracy were excluded. Inner and outer ratios were defined as the ratio of temporal-to-nasal GCIPL thickness for inner and outer subfields respectively. Corresponding ratios were obtained from a smaller cohort undergoing OCTs with a different device (three-dimensional (3D)-OCT, Topcon, Japan). Results: Scans from 2300 twins (1150 pairs) were included (mean [SD] age, 53.9 (16.5) years). Mean (SD) inner and outer ratios were 0.89 (0.09) and 0.84 (0.11), correlating negatively with age (coefficients, -0.17 and -0.21, respectively). In males (150 pairs) ratios were higher and did not correlate significantly with age. Intrapair correlation coefficients were higher in monozygotic than dizygotic pairs; age-adjusted heritability estimates were 0.20 and 0.23 for inner and outer ratios, respectively. For the second cohort (n = 166), mean (SD) ratios were 0.93 (0.08) and 0.91 (0.09), significantly greater than for the larger cohort. Conclusions: Our study gives reference values for temporal-to-nasal macular GCIPL subfield ratios. Weak negative correlations with age emerged. Genetic factors may contribute to ∼20% to 23% of the variance in healthy individuals. The ratios differ according to the OCT platform used.

Development and assessment of educational materials for spinal muscular atrophy carrier screening in the Plain community.

Spinal muscular atrophy (SMA) has been reported in both Amish and Mennonite (Plain) communities, and a higher incidence has been observed in certain Mennonite communities compared to the general population. There are several therapies for SMA, but all are most effective in pre-symptomatic newborns. To identify couples from the Wisconsin Plain community who are most likely to have a child with SMA, carrier screening is offered via mailed kits with at-home specimen collection. Our survey data about Plain families' perspectives on genetic testing suggest educational materials are needed for individuals providing informed consent with at-home specimen collection. We therefore developed a Plain population-specific educational trifold brochure about SMA carrier screening by incorporating existing medical education strategies and feedback from Plain community members and their health care providers. Along with the brochure, surveys were included in the kits to assess baseline knowledge about SMA carrier screening ("pre-education") as well as improvement in knowledge after reviewing the brochure and cultural appropriateness of the brochure ("post-education"). Fifty-five testing kits were distributed, and 26 survey pairs (pre- and post-education) were returned and analyzed (response rate 47%). Respondents had high baseline knowledge with an average of 5 of 7 questions (71%) answered correctly on the pre-education survey. Knowledge improved after reviewing the brochure as the average score increased to 6.5 of 7 questions (93%) answered correctly. Questions about risks of having an affected child after positive or negative carrier screening showed the most improvement from the pre-education to post-education surveys. Most respondents indicated the brochure was helpful, was easy to understand, and contained the right amount of information. Overall, incorporating elements of existing medical education strategies with feedback from the target population and stakeholders about appropriate language seems to be an effective method for creating beneficial, culturally responsive educational materials for the Plain population.

Neural cell engraftment therapy for sporadic Creutzfeldt-Jakob disease restores neuroelectrophysiological parameters in a cerebral organoid model.

BACKGROUND: Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is a fatal neurodegenerative disease with currently no treatment options. Stem cell therapy for neurodegenerative diseases is emerging as a possible treatment option. However, while there are a few clinical trials for other neurodegenerative disorders such as Parkinson's disease, prion disease cell therapy research has so far been confined to animal models. METHODS: Here, we use a novel approach to study cell therapies in sCJD using a human cerebral organoid model. Cerebral organoids can be infected with sCJD prions allowing us to assess how neural precursor cell (NPC) therapy impacts the progression of sCJD. After 90 days of sCJD or mock infection, organoids were either seeded with NPCs or left unseeded and monitored for cellular composition changes, prion infection parameters and neuroelectrophysiological function at 180 days post-infection. RESULTS: Our results showed NPCs integrated into organoids leading to an increase in neuronal markers and changes in cell signaling irrespective of sCJD infection. Although a small, but significant, decrease in protease-resistant PrP deposition was observed in the CJD-infected organoids that received the NPCs, other disease-associated parameters showed minimal changes. However, the NPCs had a beneficial impact on organoid function following infection. sCJD infection caused reduction in neuronal spike rate and mean burst spike rate, indicative of reduced action potentials. NPC seeding restored these electrophysiological parameters to the uninfected control level. CONCLUSIONS: Together with the previous animal studies, our results support that cell therapy may have some functional benefit for the treatment of human prion diseases.

Demystifying human research ethics committee applications.

BACKGROUND: The National Health and Medical Research Council's National Statement on Ethical Conduct in Human Research and updated Guidelines for Ethical Conduct in Aboriginal and Torres Strait Islander Health Research provide guidance for primary care researchers. OBJECTIVE: This paper describes a step-by-step approach to ethics applications for research projects in primary care for new or inexperienced researchers, or those new to primary care research. DISCUSSION: Domains that may enhance ethics applications include increased consumer involvement; comprehensive literature reviews; evidence of researcher training in ethical research and clinical trials; the use of online platforms for participant information, consent processes and surveys; and consideration of the risks of genomic research or research in subpopulations. This paper discusses steps required when preparing ethics applications to ensure the community, clinicians and researchers are protected.

Prevalence, Concordance, and Heritability of Vitreomacular Interface Abnormalities in a Twin Study.

Purpose: The relative importance of genetic factors in common vitreomacular interface (VMI) abnormalities is unknown. The aim of this classical twin study is to determine the prevalence case wise concordance between monozygotic and dizygotic twin pairs, and heritability of common VMI abnormalities, including epiretinal membrane (ERM), posterior vitreous detachment (PVD), vitreomacular adhesion (VMA), vitreomacular traction (VMT), lamellar macular holes (LMHs), and full-thickness macular holes (FTMHs). Methods: This is a single-center, cross-sectional classical twin study of 3406 TwinsUK participants over the age of 40 years who underwent spectral domain macular optical coherence tomography (SD-OCT) scans which were graded for signs of VMI abnormalities. Case wise concordance was calculated and the heritability of each VMI abnormality was estimated using OpenMx structural equation modeling. Results: In this population (mean age = 62.0 years [SD = 10.4 years], range = 40-89 years) the overall prevalence of ERM was 15.6% (95% confidence interval [CI] = 14.4-16.9) and increased with age, posterior vitreous detachment affected 21.3% (20.0-22.7), and VMA was diagnosed in 11.8% (10.8-13.0). Monozygotic twins were more concordant for all traits than dizygotic twins, and age, spherical equivalent refraction (SER), and lens status-adjusted heritability was estimated at 38.9% (95% CI = 33.6-52.8) for ERM, 53.2% (95% CI = 41.8-63.2) for PVD, and 48.1% (95% CI = 33.6-58) for VMA. Conclusions: Common VMI abnormalities are heritable and therefore have an underlying genetic component. Given the sight-threatening potential of VMI abnormalities, further genetic studies, such as genomewide association studies, would be useful to identify genes and pathways implicated in their pathogenesis.

Temporary alteration of neuronal network communication is a protective response to redox imbalance that requires GPI-anchored prion protein.

Cellular prion protein (PrPC) protects neurons against oxidative stress damage. This role is lost upon its misfolding into insoluble prions in prion diseases, and correlated with cytoskeletal breakdown and neurophysiological deficits. Here we used mouse neuronal models to assess how PrPC protects the neuronal cytoskeleton, and its role in network communication, from oxidative stress damage. Oxidative stress was induced extrinsically by potassium superoxide (KO2) or intrinsically by Mito-Paraquat (MtPQ), targeting the mitochondria. In mouse neural lineage cells, KO2 was damaging to the cytoskeleton, with cells lacking PrPC (PrP-/-) damaged more than wild-type (WT) cells. In hippocampal slices, KO2 acutely inhibited neuronal communication in WT controls without damaging the cytoskeleton. This inhibition was not observed in PrP-/- slices. Neuronal communication and the cytoskeleton of PrP-/- slices became progressively disrupted and degenerated post-recovery, whereas the dysfunction in WT slices recovered in 5 days. This suggests that the acute inhibition of neuronal activity in WT slices in response to KO2 was a neuroprotective role of PrPC, which PrP-/- slices lacked. Heterozygous expression of PrPC was sufficient for this neuroprotection. Further, hippocampal slices from mice expressing PrPC without its GPI anchor (PrPGPI-/-) displayed acute inhibition of neuronal activity by KO2. However, they failed to restore normal activity and cytoskeletal formation post-recovery. This suggests that PrPC facilitates the depressive response to KO2 and its GPI anchoring is required to restore KO2-induced damages. Immuno spin-trapping showed increased radicals formed on the filamentous actin of PrP-/- and PrPGPI-/- slices, but not WT and PrP+/- slices, post-recovery suggesting ongoing dysregulation of redox balance in the slices lacking GPI-anchored PrPC. The MtPQ treatment of hippocampal slices temporarily inhibited neuronal communication independent of PrPC expression. Overall, GPI-anchored PrPC alters synapses and neurotransmission to protect and repair the neuronal cytoskeleton, and neuronal communication, from extrinsically induced oxidative stress damages.

Defining principles for a choice-based approach to HIV prevention.

The HIV prevention landscape is on the cusp of an unprecedented era of multiple biomedical prevention products available for distribution. Several HIV prevention options, such as oral pre-exposure prophylaxis (PrEP), dapivirine vaginal rings, and injectable cabotegravir for PrEP, are becoming more widely available. Although the future HIV prevention market promises to be rich in options, it would benefit from a core set of principles that uphold choice in all phases of product development, assessment, and introduction. These principles, as presented in this Viewpoint, show the applicability, opportunities, and challenges of choice in different contexts of HIV prevention and provide checkpoints of accountability. By committing to these principles, stakeholders at national and global levels can advance choice across all phases of the HIV prevention market, thereby ensuring that individuals can realise their right to choose when and how to prevent HIV in their own lives.

Evolution of a Mosquito's Hatching Behavior to Match Its Human-Provided Habitat.

AbstractA subspecies of the yellow fever mosquito, Aedes aegypti, has recently evolved to specialize in biting and living alongside humans. It prefers human odor over the odor of nonhuman animals and breeds in human-provided artificial containers rather than the forest tree holes of its ancestors. Here, we report one way this human specialist has adapted to the distinct ecology of human environments. While eggs of the ancestral subspecies rarely hatch in pure water, those of the derived human specialist do so readily. We trace this novel behavior to a shift in how eggs respond to dissolved oxygen, low levels of which may signal food abundance. Moreover, we show that while tree holes are consistently low in dissolved oxygen, artificial containers often have much higher levels. There is thus a concordance between the hatching behavior of each subspecies and the aquatic habitat it uses in the wild. We find this behavioral variation is heritable, with both maternal and zygotic effects. The zygotic effect depends on dissolved oxygen concentration (i.e., a genotype-environment interaction, or G×E), pointing to potential changes in oxygen-sensitive circuits. Together, our results suggest that a shift in hatching response contributed to the pernicious success of this human-specialist mosquito and illustrate how animals may rapidly adapt to human-driven changes in the environment.

Sporadic Creutzfeldt-Jakob disease infected human cerebral organoids retain the original human brain subtype features following transmission to humanized transgenic mice.

Human cerebral organoids (COs) are three-dimensional self-organizing cultures of cerebral brain tissue differentiated from induced pluripotent stem cells. We have recently shown that COs are susceptible to infection with different subtypes of Creutzfeldt-Jakob disease (CJD) prions, which in humans cause different manifestations of the disease. The ability to study live human brain tissue infected with different CJD subtypes opens a wide array of possibilities from differentiating mechanisms of cell death and identifying neuronal selective vulnerabilities to testing therapeutics. However, the question remained as to whether the prions generated in the CO model truly represent those in the infecting inoculum. Mouse models expressing human prion protein are commonly used to characterize human prion disease as they reproduce many of the molecular and clinical phenotypes associated with CJD subtypes. We therefore inoculated these mice with COs that had been infected with two CJD subtypes (MV1 and MV2) to see if the original subtype characteristics (referred to as strains once transmitted into a model organism) of the infecting prions were maintained in the COs when compared with the original human brain inocula. We found that disease characteristics caused by the molecular subtype of the disease associated prion protein were similar in mice inoculated with either CO derived material or human brain material, demonstrating that the disease associated prions generated in COs shared strain characteristics with those in humans. As the first and only in vitro model of human neurodegenerative disease that can faithfully reproduce different subtypes of prion disease, these findings support the use of the CO model for investigating human prion diseases and their subtypes.

Dysregulation of PRMT5 in chronic lymphocytic leukemia promotes progression with high risk of Richter's transformation.

Richter's Transformation (RT) is a poorly understood and fatal progression of chronic lymphocytic leukemia (CLL) manifesting histologically as diffuse large B-cell lymphoma. Protein arginine methyltransferase 5 (PRMT5) is implicated in lymphomagenesis, but its role in CLL or RT progression is unknown. We demonstrate herein that tumors uniformly overexpress PRMT5 in patients with progression to RT. Furthermore, mice with B-specific overexpression of hPRMT5 develop a B-lymphoid expansion with increased risk of death, and Eµ-PRMT5/TCL1 double transgenic mice develop a highly aggressive disease with transformation that histologically resembles RT; where large-scale transcriptional profiling identifies oncogenic pathways mediating PRMT5-driven disease progression. Lastly, we report the development of a SAM-competitive PRMT5 inhibitor, PRT382, with exclusive selectivity and optimal in vitro and in vivo activity compared to available PRMT5 inhibitors. Taken together, the discovery that PRMT5 drives oncogenic pathways promoting RT provides a compelling rationale for clinical investigation of PRMT5 inhibitors such as PRT382 in aggressive CLL/RT cases.

Altered energy metabolism in Fatal Familial Insomnia cerebral organoids is associated with astrogliosis and neuronal dysfunction.

Fatal familial insomnia (FFI) is a rare neurodegenerative disease caused by a dominantly inherited single amino acid substitution (D178N) within the prion protein (PrP). No in vitro human brain tissue model for this disease has previously been available. Consequently, how this mutation exerts its damaging effect on brain cells is still unknown. Using CRISPR-Cas9 engineered induced pluripotent stem cells, we made D178N cerebral organoids and compared these with isotype control organoids. We found that, in the absence of other hallmarks of FFI, the D178N organoids exhibited astrogliosis with cellular oxidative stress. Abnormal post-translational processing of PrP was evident but no tissue deposition or propagation of mis-folded PrP isoforms were observed. Neuronal electrophysiological function was compromised and levels of neurotransmitters, particularly acetylcholine and GABA, altered. Underlying these dysfunctions were changes in cellular energy homeostasis, with substantially increased glycolytic and Krebs cycle intermediates, and greater mitochondrial activity. This increased energy demand in D178N organoids was associated with increased mitophagy and depletion of lipid droplets, in turn resulting in shifts of cellular lipid composition. Using a double mutation (178NN) we could confirm that most changes were caused by the presence of the mutation rather than interaction with PrP molecules lacking the mutation. Our data strongly suggests that shifting biosynthetic intermediates and oxidative stress, caused by an imbalance of energy supply and demand, results in astrogliosis with compromised neuronal activity in FFI organoids. They further support that many of the disease associated changes are due to a corruption of PrP function and do not require propagation of PrP mis-folding.

Practical Mouse Model to Investigate Therapeutics for Staphylococcusaureus Contaminated Surgical Mesh Implants.

INTRODUCTION: The use of prosthetic mesh in hernia repair provides a powerful tool to increase repair longevity, decrease recurrence rates, and facilitate complex abdominal wall reconstruction. Overall infection rates with mesh are low, but for those affected there is high morbidity and economic cost. The availability of a practicable small animal model would be advantageous for the preclinical testing of prophylactics, therapeutics, and new biomaterials. To this end, we have developed a novel mouse model for implantation of methicillin-resistant Staphylococcus aureus-infected surgical mesh and provide results from antibiotic and immunotherapeutic testing. MATERIALS AND METHODS: Implantation of surgical mesh between fascial planes of the mouse hind limb was used to approximate hernia repair in humans. Surgical mesh was inoculated with methicillin-resistant Staphylococcus aureus to test the efficacy of antibiotic therapy with daptomycin and/or immunotherapy to induce macrophage phagocytosis using antibody blockade of the CD47 "don't eat me" molecule. Clinical outcomes were assessed by daily ambulation scores of the animals and by enumeration of mesh-associated bacteria at predetermined end points. RESULTS: A single prophylactic treatment with daptomycin at the time of surgery led to improved ambulation scores and undetectable levels of bacteria in seven of eight mice by 21 days postinfection. Anti-CD47, an activator of macrophage phagocytosis, was ineffective when administered alone or in combination with daptomycin treatment. Ten days of daily antibiotic therapy begun 3 days after infection was ineffective at clearing infection. CONCLUSIONS: This fast and simple model allows rapid in vivo testing of novel antimicrobials and immunomodulators to treat surgical implant infections.

Cross-sectional survey on genetic testing utilization and perceptions in Wisconsin Amish and Mennonite communities.

Amish and Mennonite (Plain) communities have increased prevalence of many recessively inherited disorders due to founder variants that can be identified using next-generation sequencing (NGS). We assessed newborn screening (NBS) utilization, prior genetic testing, and perceptions of genetic testing among Wisconsin Plain communities to guide implementation and utilization of a population-specific NGS gene panel testing. A mailed paper survey (N = 959) of demographics, NBS utilization, prior genetic testing, and preferences for categorical genetic disorder and defined clinical context testing was developed. Overall response rate was 39% (N = 378; 183 Amish, 193 Mennonite; 2 not Amish/Mennonite). Mennonites were more likely to respond in favor of carrier screening for metabolic disorders and other surgical conditions and less likely to respond in favor of asymptomatic testing for neurologic disorders and lethal disorders compared to Amish. Reported utilization of NBS was positively associated with stated interest in genetic testing for an asymptomatic child. Reported prior genetic testing was positively associated with stated interest in carrier screening and negatively associated with testing a symptomatic child. Although Plain community members share many common outward characteristics, our survey responses suggest diversity in their views of genetic testing and support laboratory methods that can be flexible to varied needs of individuals.

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Second passage experiments of chronic wasting disease in transgenic mice overexpressing human prion protein.

Chronic wasting disease (CWD) is a prion disease of cervids including deer, elk, reindeer, and moose. Human consumption of cervids is common, therefore assessing the risk potential of CWD transmission to humans is critical. In a previous study, we tested CWD transmission via intracerebral inoculation into transgenic mice (tg66 and tgRM) that over-expressed human prion protein. Mice screened by traditional prion detection assays were negative. However, in a group of 88 mice screened by the ultrasensitive RT-QuIC assay, we identified 4 tg66 mice that produced inconsistent positive RT-QuIC reactions. These data could be false positive reactions, residual input inoculum or indicative of subclinical infections suggestive of cross species transmission of CWD to humans. Additional experiments were required to understand the nature of the prion seeding activity in this model. In this manuscript, second passage experiments using brains from mice with weak prion seeding activity showed they were not infectious to additional recipient tg66 mice. Clearance experiments showed that input CWD prion seeding activity was eliminated by 180 days in tg66 mice and PrPKO mice, which are unable to replicate prion protein, indicating that the weak positive levels of seeding activity detected at later time points was not likely residual inoculum. The failure of CWD prions to cause disease in tg66 after two sequential passages suggested that a strong species barrier prevented CWD infection of mice expressing human prion protein.