Phenotypic and transcriptional changes in peripheral blood mononuclear cells during alphavirus encephalitis in mice.

Sindbis virus (SINV) infection of mice provides a model system for studying the pathogenesis of alphaviruses that infect the central nervous system (CNS) to cause encephalomyelitis. While studies of human viral infections typically focus on accessible cells from the blood, this compartment is rarely evaluated in mice. To bridge this gap, single-cell RNA sequencing (scRNAseq) was combined with flow cytometry to characterize the transcriptional and phenotypic changes of peripheral blood mononuclear cells (PBMCs) from SINV-infected mice. Twenty-one clusters were identified by scRNAseq at 7 days after infection, with a unique cluster and overall increase in naive B cells for infected mice. Uninfected mice had fewer immature T cells and CCR9+ CD4 T cells and a unique immature T cell cluster. Gene expression was most altered in the Ki67+ CD8 T cell cluster, with chemotaxis and proliferation-related genes upregulated. Global analysis indicated metabolic changes in myeloid cells and increased expression of Ccl5 by NK cells. Phenotypes of PBMCs and cells infiltrating the CNS were analyzed by flow cytometry over 14 days after infection. In PBMCs, CD8 and Th1 CD4 T cells increased in representation, while B cells showed a transient decrease at day 5 in total, Ly6a+, and naive cells, and an increase in activated B cells. In the brain, CD8 T cells increased for the first 7 days, while Th1 CD4 T cells and naive and Ly6a+ B cells continued to accumulate for 14 days. Therefore, dynamic immune cell changes can be identified in the blood as well as the CNS during viral encephalomyelitis. IMPORTANCE: The outcome of viral encephalomyelitis is dependent on the host immune response, with clearance and resolution of infection mediated by the adaptive immune response. These processes are frequently studied in mouse models of infection, where infected tissues are examined to understand the mechanisms of clearance and recovery. However, studies of human infection typically focus on the analysis of cells from the blood, a compartment rarely examined in mice, rather than inaccessible tissue. To close this gap, we used single-cell RNA sequencing and flow cytometry to profile the transcriptomic and phenotypic changes of peripheral blood mononuclear cells (PBMCs) before and after central nervous system (CNS) infection in mice. Changes to T and B cell gene expression and cell composition occurred in PBMC and during entry into the CNS, with CCL5 being a differentially expressed chemokine. Therefore, dynamic changes occur in the blood as well as the CNS during the response of mice to virus infection, which will inform the analysis of human studies.

Hereditary Connective Tissue Diseases and Risk of Post-Acute SARS-CoV-2.

We completed a retrospective review of data collected by the JH-CROWN consortium based on ICD10 codes for a hospitalized cohort. The severity and prevalence of COVID-19 and development of PASC within heritable connective tissue diseases were unknown; however, clinical observation suggested a thorough examination was necessary. We compared rates of disease severity, death, and PASC in connective tissue diseases versus the entire cohort as well as in diabetes and hypertension to determine if connective tissue disease was a risk factor. Of the 15,676 patients in the database, 63 (0.40%) had a connective tissue disease, which is elevated relative to the distribution in the population, suggesting a higher risk of severe disease. Within these 63 patients, 9.52% developed PASC compared to 2.54% in the entire cohort (p < 0.005). Elucidation of populations at high risk for severe disease and development of PASC is integral to improving treatment approaches. Further, no other study to date has examined the risk in those with connective tissue diseases and these data support a need for enhanced awareness among physicians, patients, and the community.

Development of automated microfluidic immunoassays for the detection of SARS-CoV-2 antibodies and antigen.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) global pandemic. Rapid and sensitive detection of the virus soon after infection is important for the treatment and prevention of transmission of COVID-19, and detection of antibodies is important for epidemiology, assessment of vaccine immunogenicity, and identification of the natural reservoir and intermediate host(s). Patient nasal or oropharyngeal swabs or saliva used in conjunction with polymerase chain reaction (PCR) detect SARS-CoV-2 RNA, whereas lateral flow immunoassays (LFI) detect SARS-CoV-2 proteins. Enzyme-linked immunosorbent assays (ELISA) detect anti-SARS-CoV-2 antibodies in blood. Although effective, these assays have poor sensitivity (e.g., LFI) or are labor intensive and time consuming (PCR and ELISA). Here we describe the development of rapid, automated ELISA-based immunoassays to detect SARS-CoV-2 antigens and antibodies against the virus. The Simple Plex™ platform uses rapid microfluidic reaction kinetics for sensitive analyte detection with small sample volumes. We developed three sensitive <90-min Simple Plex immunoassays that measure either the SARS-CoV-2 antigens or the immune response to SARS-CoV-2, including neutralizing antibodies, in serum from COVID-19 patients.

LIC in the time of COVID: experiences of LIC tutors during the COVID-19 pandemic in Scotland.

In March 2020, due to the escalating global coronavirus (COVID-19) pandemic, clinical placements for most medical students in the UK were suspended. A phased resumption of clinical placements started at the beginning of academic year 2020/2021. For the Scottish Graduate Entry Medicine programme (ScotGEM), 2020/21 was the first year that Dundee School of Medicine's comprehensive LIC was extended to all 54 students in the penultimate year of the ScotGEM programme. This cross-sectional qualitative study explored aspects of tutors' experiences of supporting LIC students in their practices. Thematic analysis of the data identified significant themes relating to the effects of the coronavirus pandemic on the organisation of the LIC placements and the experiences of the tutors, and the ways in which they adapted placements to the rapidly changing clinical and social landscapes. The changes necessitated by the pandemic posed significant challenges for practice-based tutors in ensuring that students had valuable educational experiences despite the constraints of social distancing requirements and the reduction in face-to-face consultations. However, tutors also identified several positive aspects of the changes which will be of interest to those involved in the organisation and delivery of both LIC and shorter General Practice based clinical attachments. Positive relationships between LIC students and practices enhanced the success of LIC placements. We will discuss how lessons learned from the experience of tutors in the pandemic could be used in the longer term to enrich the LIC experience and General Practice placements more generally.

Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs.

Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species of viral RNA were produced: genomic, subgenomic, and defective viral genomes (DVGs) encoding the RNA capping protein nsP1. Antibody treatment resulted in overall lower production of SINV RNA, decreased synthesis of subgenomic RNA relative to genomic RNA, and suppressed production of the nsP1 DVG. The nsP1 DVG was packaged into virus particles and could be translated. Because antibody-treated cells released a higher proportion of virions with noncapped genomes and transient transfection to express the nsP1 DVG improved viral RNA capping in antibody-treated cells, we postulate that one mechanism by which antibody inhibits SINV replication in neurons is to suppress DVG synthesis and thus decrease production of infectious virions containing capped genomes. IMPORTANCE Alphaviruses are important causes of viral encephalomyelitis without approved treatments or vaccines. Antibody to the Sindbis virus (SINV) E2 glycoprotein is required for immune-mediated noncytolytic virus clearance from neurons. We used direct RNA nanopore sequencing to evaluate how anti-E2 antibody affects SINV replication at the RNA level. Antibody altered the viral RNAs produced by decreasing the proportion of subgenomic relative to genomic RNA and suppressing production of a previously unrecognized defective viral genome (DVG) encoding nsP1, the viral RNA capping enzyme. Antibody-treated neurons released a lower proportion of SINV particles with capped genomes necessary for translation and infection. Decreased nsP1 DVG production in antibody-treated neurons led to lower expression of nsP1 protein, decreased genome capping efficiency, and release of fewer infectious virus particles. Capping was increased with exogenous expression of the nsP1 DVG. These studies identify a novel alphavirus DVG function and new mechanism for antibody-mediated control of virus replication.

Continued Virus-Specific Antibody-Secreting Cell Production, Avidity Maturation and B Cell Evolution in Patients Hospitalized with COVID-19.

Understanding the development and sustainability of the virus-specific protective immune response to infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains incomplete with respect to the appearance and disappearance of virus-specific antibody-secreting cells (ASCs) in circulation. Therefore, we performed cross-sectional and longitudinal analyses of peripheral blood mononuclear cells and plasma collected from 55 hospitalized patients up to 4 months after onset of COVID-19 symptoms. Spike (S)- and nucleocapsid (N)-specific IgM and IgG ASCs appeared within 2 weeks accompanied by flow cytometry increases in double negative plasmablasts consistent with a rapid extrafollicular B cell response. Total and virus-specific IgM and IgG ASCs peaked at 3-4 weeks and were still being produced at 3-4 months accompanied by increasing antibody avidity consistent with a slower germinal center B cell response. N-specific ASCs were produced for longer than S-specific ASCs and avidity maturation was greater for antibody to N than S. Patients with more severe disease produced more S-specific IgM and IgG ASCs than those with mild disease and had higher levels of N- and S-specific antibody. Women had more B cells in circulation than men and produced more S-specific IgA and IgG and N-specific IgG ASCs. Flow cytometry analysis of B cell phenotypes showed an increase in circulating B cells at 4-6 weeks with decreased percentages of switched and unswitched memory B cells. These data indicate ongoing antigen-specific stimulation, maturation, and production of ASCs for several months after onset of symptoms in patients hospitalized with COVID-19.

Disrupting the consultation: students empowering patients in a longitudinal clerkship.

Although well-established worldwide as a method of clinical medical education, Longitudinal Integrated Clerkships (LICs) are green shoots in the UK medical education landscape. The first comprehensive LIC in the UK was introduced in Dundee, Scotland in 2016. Substantial work has been carried out to evaluate the experiences of students and primary care tutors involved in the Dundee LIC, but the experiences of the patients LIC students cared for had not been evaluated. The purpose of this study was to explore the experiences of these patients, particularly the impact the involvement of a LIC student might have on their experience of healthcare. The study is a cross-sectional qualitative study involving semi-structured interviews with five patients who had experienced several contacts with LIC students. An interpretive phenomenological approach was taken. We describe the presence of the student as a disruptive force leading to the empowerment of patients. Students disrupted the status quo in the consultation by altering both the structure of the interaction and the doctor-patient relationship. The student-patient relationship was a powerful enabler of patient empowerment through the provision of education and information to the patient and through increasing patient centredness in the consultation. The positive social interaction provided by the student-patient relationship led to a reframing of patients' perceptions of the medical profession, challenging their perceptions of occupational hierarchy and power of the medical profession.

Genomic features of humoral immunity support tolerance model in Egyptian rousette bats.

Bats asymptomatically harbor many viruses that can cause severe human diseases. The Egyptian rousette bat (ERB) is the only known reservoir for Marburgviruses and Sosuga virus, making it an exceptional animal model to study antiviral mechanisms in an asymptomatic host. With this goal in mind, we constructed and annotated the immunoglobulin heavy chain locus, finding an expansion on immunoglobulin variable genes associated with protective human antibodies to different viruses. We also annotated two functional and distinct immunoglobulin epsilon genes and four distinctive functional immunoglobulin gamma genes. We described the Fc receptor repertoire in ERBs, including features that may affect activation potential, and discovered the lack of evolutionary conserved short pentraxins. These findings reinforce the hypothesis that a differential threshold of regulation and/or absence of key immune mediators may promote tolerance and decrease inflammation in ERBs.

Consensus statement on the content of clinical reasoning curricula in undergraduate medical education.

INTRODUCTION: Effective clinical reasoning is required for safe patient care. Students and postgraduate trainees largely learn the knowledge, skills and behaviours required for effective clinical reasoning implicitly, through experience and apprenticeship. There is a growing consensus that medical schools should teach clinical reasoning in a way that is explicitly integrated into courses throughout each year, adopting a systematic approach consistent with current evidence. However, the clinical reasoning literature is 'fragmented' and can be difficult for medical educators to access. The purpose of this paper is to provide practical recommendations that will be of use to all medical schools. METHODS: Members of the UK Clinical Reasoning in Medical Education group (CReME) met to discuss what clinical reasoning-specific teaching should be delivered by medical schools (what to teach). A literature review was conducted to identify what teaching strategies are successful in improving clinical reasoning ability among medical students (how to teach). A consensus statement was then produced based on the agreed ideas and the literature review, discussed by members of the consensus statement group, then edited and agreed by the authors. RESULTS: The group identified 30 consensus ideas that were grouped into five domains: (1) clinical reasoning concepts, (2) history and physical examination, (3) choosing and interpreting diagnostic tests, (4) problem identification and management, and (5) shared decision making. The literature review demonstrated a lack of effectiveness for teaching the general thinking processes involved in clinical reasoning, whereas specific teaching strategies aimed at building knowledge and understanding led to improvements. These strategies are synthesised and described. CONCLUSION: What is taught, how it is taught, and when it is taught can facilitate clinical reasoning development more effectively through purposeful curriculum design and medical schools should consider implementing a formal clinical reasoning curriculum that is horizontally and vertically integrated throughout the programme.

Acute RNA Viral Encephalomyelitis and the Role of Antibodies in the Central Nervous System.

Acute RNA viral encephalomyelitis is a serious complication of numerous virus infections. Antibodies in the cerebral spinal fluid (CSF) are correlated to better outcomes, and there is substantive evidence of antibody secreting cells (ASCs) entering the central nervous system (CNS) and contributing to resolution of infection. Here, we review the RNA viruses known to cause acute viral encephalomyelitis with mechanisms of control that require antibody or ASCs. We compile the cytokines, chemokines, and surface receptors associated with ASC recruitment to the CNS after infection and compare known antibody-mediated mechanisms as well as potential noncytolytic mechanisms for virus control. These non-canonical functions of antibodies may be employed in the CNS to protect precious non-renewable neurons. Understanding the immune-specialized zone of the CNS is essential for the development of effective treatments for acute encephalomyelitis caused by RNA viruses.

Correction to: The do's, don'ts and don't knows of establishing a sustainable longitudinal integrated clerkship.

Unfortunately information regarding the disclaimer of Paul Worley's affiliation is missing from the original article. Please find the information here:Paul Worley is affiliated to the Prideaux Centre for Research in Health Professions Education, Flinders University, Adelaide, Australia. He is the ….

The do's, don'ts and don't knows of establishing a sustainable longitudinal integrated clerkship.

INTRODUCTION: The longitudinal integrated clerkship is a model of clinical medical education that is increasingly employed by medical schools around the world. These guidelines are a result of a narrative review of the literature which considered the question of how to maximize the sustainability of a new longitudinal integrated clerkship program. METHOD: All four authors have practical experience of establishing longitudinal integrated clerkship programs. Each author individually constructed their Do's, Don'ts and Don't Knows and the literature that underpinned them. The lists were compiled and revised in discussion and a final set of guidelines was agreed. A statement of the strength of the evidence is included for each guideline. RESULTS: The final set of 18 Do's, Don'ts and Don't Knows is presented with an appraisal of the evidence for each one. CONCLUSION: Implementing a longitudinal integrated clerkship is a complex process requiring the involvement of a wide group of stakeholders in both hospitals and communities. The complexity of the change management processes requires careful and sustained attention, with a particular focus on the outcomes of the programs for students and the communities in which they learn. Effective and consistent leadership and adequate resourcing are important. There is a need to select teaching sites carefully, involve students and faculty in allocation of students to sites and support students and faculty though the implementation phase and beyond. Work is needed to address the Don't Knows, in particular the question of how cost-effectiveness is best measured.

First Evidence of Antibodies Against Lloviu Virus in Schreiber's Bent-Winged Insectivorous Bats Demonstrate a Wide Circulation of the Virus in Spain.

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber's Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber's Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber's Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber's Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment.

BACKGROUND: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. METHODS: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. FINDINGS: The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Équateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. INTERPRETATION: The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

2018 Ebola virus disease outbreak in Équateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation.

BACKGROUND: The 2018 Ebola virus disease (EVD) outbreak in Équateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). METHODS: We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Équateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. FINDINGS: A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name "Tumba". This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10-3 substitutions per site per year with "Tumba" vs 1·06 × 10-3 substitutions per site per year without "Tumba"). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. INTERPRETATION: Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

Dundee's Longitudinal Integrated Clerkship: drivers, implementation and early evaluation.

Dundee University School of Medicine established a pilot for a 40 week long comprehensive Longitudinal Integrated Clerkship (LIC) in 2016. Ten places for year 4 students are available which are shared between two regions of Scotland which are largely rural areas by UK definitions. This paper describes the drivers for the pilot, its implementation and early evaluation. For the evaluation, data were collected using focus groups and semi-structured interviews from the first cohort of seven students, four health service employed staff (two with leadership roles and two with regional student facing roles), 21 General Practitioner tutors, and from reflective audio-diaries kept by all students. Analysis was thematic, the themes being identified from the data. Summative assessment data were collated. Students reported positive learning experiences though access to secondary care learning linked to their patients was sometimes problematic. GP tutors were positive and enthusiastic about the programme and could see the potential benefits on recruitment to GP careers. Pre-existing workload pressures were a challenge. Summative assessment results were encouraging. The Dundee LIC is successful in delivering Dundee's year 4 curriculum. Ongoing development has been focused on improving awareness of the programme in secondary care services.

The do's, don't and don't knows of supporting transition to more independent practice.

INTRODUCTION: Transitions are traditionally viewed as challenging for clinicians. Throughout medical career pathways, clinicians need to successfully navigate successive transitions as they become progressively more independent practitioners. In these guidelines, we aim to synthesize the evidence from the literature to provide guidance for supporting clinicians in their development of independence, and highlight areas for further research. METHODS: Drawing upon D3 method guidance, four key themes universal to medical career transitions and progressive independence were identified by all authors through discussion and consensus from our own experience and expertise: workplace learning, independence and responsibility, mentoring and coaching, and patient perspectives. A scoping review of the literature was conducted using Medline database searches in addition to the authors' personal archives and reference snowballing searches. RESULTS: 387 articles were identified and screened. 210 were excluded as not relevant to medical transitions (50 at title screen; 160 at abstract screen). 177 full-text articles were assessed for eligibility; a further 107 were rejected (97 did not include career transitions in their study design; 10 were review articles; the primary references of these were screened for inclusion). 70 articles were included of which 60 provided extractable data for the final qualitative synthesis. Across the four key themes, seven do's, two don'ts and seven don't knows were identified, and the strength of evidence was graded for each of these recommendations. CONCLUSION: The two strongest messages arising from current literature are first, transitions should not be viewed as one moment in time: career trajectories are a continuum with valuable opportunities for personal and professional development throughout. Second, learning needs to be embedded in practice and learners provided with authentic and meaningful learning opportunities. In this paper, we propose evidence-based guidelines aimed at facilitating such transitions through the fostering of progressive independence.