Physical Therapy versus Glucocorticoid Injection for Osteoarthritis of the Knee.

BACKGROUND: Both physical therapy and intraarticular injections of glucocorticoids have been shown to confer clinical benefit with respect to osteoarthritis of the knee. Whether the short-term and long-term effectiveness for relieving pain and improving physical function differ between these two therapies is uncertain. METHODS: We conducted a randomized trial to compare physical therapy with glucocorticoid injection in the primary care setting in the U.S. Military Health System. Patients with osteoarthritis in one or both knees were randomly assigned in a 1:1 ratio to receive a glucocorticoid injection or to undergo physical therapy. The primary outcome was the total score on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) at 1 year (scores range from 0 to 240, with higher scores indicating worse pain, function, and stiffness). The secondary outcomes were the time needed to complete the Alternate Step Test, the time needed to complete the Timed Up and Go test, and the score on the Global Rating of Change scale, all assessed at 1 year. RESULTS: We enrolled 156 patients with a mean age of 56 years; 78 patients were assigned to each group. Baseline characteristics, including severity of pain and level of disability, were similar in the two groups. The mean (±SD) baseline WOMAC scores were 108.8±47.1 in the glucocorticoid injection group and 107.1±42.4 in the physical therapy group. At 1 year, the mean scores were 55.8±53.8 and 37.0±30.7, respectively (mean between-group difference, 18.8 points; 95% confidence interval, 5.0 to 32.6), a finding favoring physical therapy. Changes in secondary outcomes were in the same direction as those of the primary outcome. One patient fainted while receiving a glucocorticoid injection. CONCLUSIONS: Patients with osteoarthritis of the knee who underwent physical therapy had less pain and functional disability at 1 year than patients who received an intraarticular glucocorticoid injection. (ClinicalTrials.gov number, NCT01427153.).

The lived experience of remembering a 'good' interview: Micro-phenomenology applied to itself.

Micro-phenomenology is an interview and analysis method for investigating subjective experience. As a research tool, it provides detailed descriptions of brief moments of any type of subjective experience and offers techniques for systematically comparing them. In this article, we use an auto-ethnographic approach to present and explore the method. The reader is invited to observe a dialogue between two authors that illustrates and comments on the planning, conducting and analysis of a pilot series of five micro-phenomenological interviews. All these interviews asked experienced researchers of micro-phenomenology to browse their memories to identify one successful and one challenging instance of working with micro-phenomenology. The interview then focused on this reflective task to investigate whether applying the method to itself might reveal quality criteria. The article starts by presenting a shortened and edited version of the first of these interviews. Keeping the dialogue format, we then outline the micro-phenomenological analysis procedure by demonstrating its application to part of this data and corresponding passages of other interviews. We focus on one unexpected finding: interviewed researchers judge the quality of an interview in part based on a connection or contact between interviewer and interviewee. We discuss these results in the context of the means and intentions of the method and suggest avenues for future research.

Volatile Chemical Product Enhancements to Criteria Pollutants in the United States.

Volatile chemical products (VCPs) are a significant source of reactive organic carbon emissions in the United States with a substantial fraction (>20% by mass) serving as secondary organic aerosol (SOA) precursors. Here, we incorporate a new nationwide VCP inventory into the Community Multiscale Air Quality (CMAQ) model with VCP-specific updates to better model air quality impacts. Model results indicate that VCPs mostly enhance anthropogenic SOA in densely populated areas with population-weighted annual average SOA increasing 15-30% in Southern California and New York City due to VCP emissions (contribution of 0.2-0.5 µg m-3). Annually, VCP emissions enhance total population-weighted PM2.5 by ∼5% in California, ∼3% in New York, New Jersey, and Connecticut, and 1-2% in most other states. While the maximum daily 8 h ozone enhancements from VCP emissions are more modest, their influence can cause a several ppb increase on select days in major cities. Printing Inks, Cleaning Products, and Paints and Coatings product use categories contribute ∼75% to the modeled VCP-derived SOA and Cleaning Products, Paints and Coatings, and Personal Care Products contribute ∼81% to the modeled VCP-derived ozone. Overall, VCPs enhance multiple criteria pollutants throughout the United States with the largest impacts in urban cores.

National Exposure Models for Source-Specific Primary Particulate Matter Concentrations Using Aerosol Mass Spectrometry Data.

This paper investigates the feasibility of developing national empirical models to predict ambient concentrations of sparsely monitored air pollutants at high spatial resolution. We used a data set of cooking organic aerosol (COA) and hydrocarbon-like organic aerosol (HOA; traffic primary organic PM) measured using aerosol mass spectrometry across the continental United States. The monitoring locations were selected to span the national distribution of land-use and source-activity variables commonly used for land-use regression modeling (e.g., road length, restaurant count, etc.). The models explain about 60% of the spatial variability of the measured data (R2 0.63 for the COA model and 0.62 for the HOA model). Extensive cross-validation suggests that the models are robust with reasonable transferability. The models predict large urban-rural and intra-urban variability with hotspots in urban areas and along the road corridors. The predicted national concentration surfaces show reasonable spatial correlation with source-specific national chemical transport model (CTM) simulations (R2: 0.45 for COA, 0.4 for HOA). Our measured data, empirical models, and CTM predictions all show that COA concentrations are about two times higher than HOA. Since COA and HOA are important contributors to the intra-urban spatial variability of the total PM2.5, our results highlight the potential importance of controlling commercial cooking emissions for air quality management in the United States.

Characterizing the Air Emissions, Transport, and Deposition of Per- and Polyfluoroalkyl Substances from a Fluoropolymer Manufacturing Facility.

Per- and polyfluoroalkyl substances (PFASs) have been released into the environment for decades, yet contributions of air emissions to total human exposure, from inhalation and drinking water contamination via deposition, are poorly constrained. The atmospheric transport and fate of a PFAS mixture from a fluoropolymer manufacturing facility in North Carolina were investigated with the Community Multiscale Air Quality (CMAQ) model applied at high resolution (1 km) and extending ∼150 km from the facility. Twenty-six explicit PFAS compounds, including GenX, were added to CMAQ using current best estimates of air emissions and relevant physicochemical properties. The new model, CMAQ-PFAS, predicts that 5% by mass of total emitted PFAS and 2.5% of total GenX are deposited within ∼150 km of the facility, with the remainder transported out. Modeled air concentrations of total GenX and total PFAS around the facility can reach 24.6 and 8500 ng m-3 but decrease to ∼0.1 and ∼10 ng m-3 at 35 km downwind, respectively. We find that compounds with acid functionality have higher deposition due to enhanced water solubility and pH-driven partitioning to aqueous media. To our knowledge, this is the first modeling study of the fate of a comprehensive, chemically resolved suite of PFAS air emissions from a major manufacturing source.

Does the nursing curriculum influence feelings of gender-role conflict in a cohort of nursing degree male students?

It is estimated that more than 9% of the global nursing workforce is male and that this share will gradually rise over the next decade. Although there are some positive aspects of having a male nursing workforce, men in the profession still experience discriminatory behaviours and practices. Fortunately, this does not deter a number of men entering undergraduate degree programmes. The aim of this study was to understand the experiences of 14 male nursing students in their first year of the adult Bachelor of Nursing programme. Using the Inventory of Male Friendliness in Nursing Programs and the Gender Role Conflict Scale, this study found that the male students felt welcomed, supported and included into the nursing programme. In addition, they felt no overall gender-role conflict, although feelings of success and achievement caused some challenges. The results of this study suggest that the male students did not necessarily experience those inequitable behaviours and practices reported in the literature. It has been suggested that perhaps the reality of clinical practice may change the perception of nursing for male students. Therefore, implications for further research could include a longitudinal study to ascertain where the perceptions of the nursing programme change for the male nursing students over time.

Transitioning Transdiagnostic CBT from Face-to-Face to Telephone Delivery During the Coronavirus Pandemic: A Case Study.

The coronavirus pandemic led to worldwide disruption in the delivery of face-to-face mental health services. This impact was marked for individuals with long-term health conditions and comorbid depression and anxiety. Many face-to-face mental health services switched to remote delivery or paused therapeutic input entirely, despite the lack of research on the efficacy of switching between modalities mid-therapy or having breaks in therapy. This paper presents the case of a patient with long-term health conditions who experienced both breaks in therapy and a switch in modalities from face-to-face to telephone delivery. The intervention used was based on transdiagnostic cognitive behavioral therapy and self-report measures were completed at the beginning and end of the twelve sessions. Despite the shift in modalities, the patient experienced clinically significant recovery on all measures, indicating the efficacy of therapy was not greatly affected by the shift in modalities. Long breaks in therapy were linked to deterioration in mental health, although this could be due to the deterioration in physical health that necessitated these breaks. This case highlights the benefits and challenges of a shifting modality of therapy during treatment and in response to a pandemic for a shielding population. From the work presented here, it seems beneficial for services to be able to work across multiple modalities to suit the needs of the patients and ensure continuity of treatment. It also indicates that pauses in therapy may risk deterioration. Further work is needed to prevent digital exclusion of patients.

Impact of a care delivery redesign initiative for vascular surgery.

OBJECTIVE: A hospital-wide quality improvement process through a care delivery redesign (CDR) was initiated to improve patient care efficiency, clinical documentation, and length of stay (LOS). The impact of CDR was assessed through LOS, unplanned readmission rates, and hospital financial metrics. METHODS: The CDR team consisted of the Chief of Vascular Surgery, inpatient nurse practitioner, dedicated case manager, clinical documentation improvement specialist, and vascular surgery residents and faculty. The nurse practitioner facilitated patient care coordination, resident system-based education, and multidisciplinary collaboration. Tools created to track performance and to ensure sustainability included daily discussions of patient care barriers and solutions; standardized order sets; a mobile app for residents containing resident service expectations, disease-specific resources, and vascular surgery journal links; and a weekly inpatient tracker showing real-time patient care data. Outcome measures included LOS, case mix index, contribution margin, and unplanned readmissions. Each outcome was determined for all inpatient admissions the year before and the 12 months after CDR was initiated. Outcomes were compared between the two groups. RESULTS: Implementation of CDR resulted in a 23% decrease in LOS (P = .003), reducing the gap to the Centers for Medicare and Medicaid Services geometric mean LOS from 2.1 days to 0.5 day (P < .001). Clinical documentation resulted in an increase in case mix index of 10% (P = .011). The 30-day unplanned readmission rates did not change in the 12 months after CDR was initiated compared with the year before (P = .92). Financial data demonstrated decreased variable cost and increased revenue resulting in a $1.89 million increase in contribution margin. CONCLUSIONS: A CDR predicated on a dedicated service line advanced practitioner, clinical documentation education, weekly service tracker review, and real-time management of system-related barriers to patient care is described. Implementation of the CDR reduced hospital LOS with no change in unplanned readmissions and provided significant financial benefit to the hospital by increasing revenue and decreasing variable cost.

The work and relatedness of ties mediated online in supporting long-term condition self-management.

The 'care transition' is characterised by reduced state involvement in chronic illness management in response to socio-political movements aimed at meeting the challenges presented by an increased prevalence of chronic illness. Amongst these changes has been online communities' rising importance in everyday interactions and attention is being increasingly paid towards the ways online contacts might contribute to self-management. Whilst research has illuminated the relevance of personal networks in long-term condition management, it is relevant to extend this work to consider the place of ties mediated online in this bricolage of support, including better understanding the work drawn from them and the strategies involved in eliciting it. This study examined the work and relatedness of 30 participants, who used online communities. Participants were asked about the role of on and offline ties and ego network mapping was used to frame conversations about the nature of this support. The context of engagement followed three main themes. Participants drew from online communities in response to deficits in offline support, they used online ties to leverage support or action from offline ties and they used online ties to substitute offline support, with less intimate online ties.

Pharmacokinetics of Benzylpenicillin (Penicillin G) during Prolonged Intermittent Renal Replacement Therapy.

Prolonged intermittent renal replacement therapy (PIRRT) is an increasingly adopted method of renal replacement in critically ill patients. Like continuous renal replacement therapy, PIRRT can alter the pharmacokinetics (PK) of many drugs. In this setting, dosing data for antibiotics like benzylpenicillin are lacking. In order to enable clinicians to prescribe benzylpenicillin safely and effectively, knowledge of the effects of PIRRT on the plasma PK of benzylpenicillin is required. Herein, we describe the PK of benzylpenicillin in 2 critically ill patients on PIRRT for the treatment of penicillin-susceptible Staphylococcus aureus bacteremia complicated by infective endocarditis. Blood samples were taken for each patient taken over dosing periods during PIRRT and off PIRRT. Two-compartment PK models described significant differences in the mean clearance of benzylpenicillin with and without PIRRT (6.61 vs. 3.04 L/h respectively). We would suggest a benzylpenicillin dose of 1,800 mg (3 million units) every 6-h during PIRRT therapy as sufficient to attain PK/pharmacodynamic target.

Long-Term Condition Self-Management Support in Online Communities: A Meta-Synthesis of Qualitative Papers.

BACKGROUND: Recent years have seen an exponential increase in people with long-term conditions using the Internet for information and support. Prior research has examined support for long-term condition self-management through the provision of illness, everyday, and emotional work in the context of traditional offline communities. However, less is known about how communities hosted in digital spaces contribute through the creation of social ties and the mobilization of an online illness "workforce." OBJECTIVE: The aim was to understand the negotiation of long-term condition illness work in patient online communities and how such work may assist the self-management of long-term conditions in daily life. METHODS: A systematic search of qualitative papers was undertaken using various online databases for articles published since 2004. A total of 21 papers met the inclusion criteria of using qualitative methods and examined the use of peer-led online communities for those with a long-term condition. A qualitative meta-synthesis was undertaken and the review followed a line of argument synthesis. RESULTS: The main themes identified in relation to the negotiation of self-management support were (1) redressing offline experiential information and knowledge deficits, (2) the influence of modeling and learning behaviors from others on self-management, (3) engagement that validates illness and negates offline frustrations, (4) tie formation and community building, (5) narrative expression and cathartic release, and (6) dissociative anonymity and invisibility. These translated into a line of argument synthesis in which four network mechanisms for self-management support in patient online communities were identified. These were (1) collective knowledge and identification through lived experience; (2) support, information, and engagement through readily accessible gifting relationships; (3) sociability that extends beyond illness; and (4) online disinhibition as a facilitator in the negotiation of self-management support. CONCLUSIONS: Social ties forged in online spaces provide the basis for performing relevant self-management work that can improve an individual's illness experience, tackling aspects of self-management that are particularly difficult to meet offline. Membership in online groups can provide those living with a long-term condition with ready access to a self-management support illness workforce and illness and emotional support. The substitutability of offline illness work may be particularly important to those whose access to support offline is either limited or absent. Furthermore, such resources require little negotiation online because information and support is seemingly gifted to the community by its members.

A randomized trial of tenecteplase versus alteplase for acute ischemic stroke.

BACKGROUND: Intravenous alteplase is the only approved treatment for acute ischemic stroke. Tenecteplase, a genetically engineered mutant tissue plasminogen activator, is an alternative thrombolytic agent. METHODS: In this phase 2B trial, we randomly assigned 75 patients to receive alteplase (0.9 mg per kilogram of body weight) or tenecteplase (0.1 mg per kilogram or 0.25 mg per kilogram) less than 6 hours after the onset of ischemic stroke. To favor the selection of patients most likely to benefit from thrombolytic therapy, the eligibility criteria were a perfusion lesion at least 20% greater than the infarct core on computed tomographic (CT) perfusion imaging at baseline and an associated vessel occlusion on CT angiography. The coprimary end points were the proportion of the perfusion lesion that was reperfused at 24 hours on perfusion-weighted magnetic resonance imaging and the extent of clinical improvement at 24 hours as assessed on the National Institutes of Health Stroke Scale (NIHSS, a 42-point scale on which higher scores indicate more severe neurologic deficits). RESULTS: The three treatment groups each comprised 25 patients. The mean (±SD) NIHSS score at baseline for all patients was 14.4±2.6, and the time to treatment was 2.9±0.8 hours. Together, the two tenecteplase groups had greater reperfusion (P=0.004) and clinical improvement (P<0.001) at 24 hours than the alteplase group. There were no significant between-group differences in intracranial bleeding or other serious adverse events. The higher dose of tenecteplase (0.25 mg per kilogram) was superior to the lower dose and to alteplase for all efficacy outcomes, including absence of serious disability at 90 days (in 72% of patients, vs. 40% with alteplase; P=0.02). CONCLUSIONS: Tenecteplase was associated with significantly better reperfusion and clinical outcomes than alteplase in patients with stroke who were selected on the basis of CT perfusion imaging. (Funded by the Australian National Health and Medical Research Council; Australia New Zealand Clinical Trials Registry number, ACTRN12608000466347.).

Could artificial intelligence write mental health nursing care plans?

WHAT IS KNOWN ON THE SUBJECT?: Artificial intelligence (AI) is freely available, responds to very basic text input (such as a question) and can now create a wide range of outputs, communicating in many languages or art forms. AI platforms like OpenAI's ChatGPT can now create passages of text that could be used to create plans of care for people with mental health needs. As such, AI output can be difficult to distinguish from human-output, and there is a risk that its use could go unnoticed. WHAT THIS PAPER ADDS TO EXISTING KNOWLEDGE?: Whilst it is known that AI can produce text or pass pre-registration health-profession exams, it is not known if AI can produce meaningful results for care delivery. We asked ChatGPT basic questions about a fictitious person who presents with self-harm and then evaluated the quality of the output. We found that the output could look reasonable to laypersons but there were significant errors and ethical issues. There are potential harms to people in care if AI is used without an expert correcting or removing these errors. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: We suggest that there is a risk that AI use could cause harm if it was used in direct care delivery. There is a lack of policy and research to safeguard people receiving care - and this needs to be in place before AI should be used in this way. Key aspects of the role of a mental health nurse are relational and AI use may diminish mental health nurses' ability to provide safe care in its current form. Many aspects of mental health recovery are linked to relationships and social engagement, however AI is not able to provide this and may push the people who are in most need of help further away from services that assist recovery. ABSTRACT: Background Artificial intelligence (AI) is being increasingly used and discussed in care contexts. ChatGPT has gained significant attention in popular and scientific literature although how ChatGPT can be used in care-delivery is not yet known. Aims To use artificial intelligence (ChatGPT) to create a mental health nursing care plan and evaluate the quality of the output against the authors' clinical experience and existing guidance. Materials & Methods Basic text commands were input into ChatGPT about a fictitious person called 'Emily' who presents with self-injurious behaviour. The output from ChatGPT was then evaluated against the authors' clinical experience and current (national) care guidance. Results ChatGPT was able to provide a care plan that incorporated some principles of dialectical behaviour therapy, but the output had significant errors and limitations and thus there is a reasonable likelihood of harm if used in this way. Discussion AI use is increasing in direct-care contexts through the use of chatbots or other means. However, AI can inhibit clinician to care-recipient engagement, 'recycle' existing stigma, and introduce error, which may thus diminish the ability for care to uphold personhood and therefore lead to significant avoidable harms. Conclusion Use of AI in this context should be avoided until a point where policy and guidance can safeguard the wellbeing of care recipients and the sophistication of AI output has increased. Given ChatGPT's ability to provide superficially reasonable outputs there is a risk that errors may go unnoticed and thus increase the likelihood of patient harms. Further research evaluating AI output is needed to consider how AI may be used safely in care delivery.

Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease.

Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies-outlined in a validated pathogenicity scoring system--is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt--tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as "definitely pathogenic" mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as 'possibly pathogenic' (m.4289T>C, m.7554G>A, and m.8304G>A).

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 µM) and thonzonium bromide (EC(50) = 69 µM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 µM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

E-cigarette puffing topography: The importance of assessing user behaviour to inform emissions testing.

Analysis of the chemical composition of e-cigarette emissions is an important step in determining whether e-cigarettes offer both individual and population-level harm reduction potential. Commonly, e-cigarette emissions for chemical analysis are collected when using e-cigarettes according to standardised puffing regimens, such as those recommended by the International Organization for Standardization (ISO) or the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA). While the use of such standard puffing regimens affords a degree of uniformity between studies and are also recommended by regulatory authorities who require the submission of e-cigarette emissions data to make decisions regarding allowing a product to be commercially marketed, the standardised regimens do not necessarily reflect human puffing behaviour. This can lead to under- or over-estimating real-world emissions from e-cigarettes and inaccuracy in determining their harm reduction potential. In this review, we describe how human puffing behaviour (topography) information can be collected both in the clinical laboratory and in the real world using a variety of different methodologies. We further discuss how this information can be used to dictate e-cigarette puffing regimens for collecting emissions for chemical analyses and how this may lead to better predictions both of human yields of e-cigarette emissions constituents and of risk assessments to predict e-cigarette tobacco harm reduction potential.

Intracellular diversity of WNV within circulating avian peripheral blood mononuclear cells reveals host-dependent patterns of polyinfection.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author Summary: WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.