The unidirectional flagellum of R. sphaeroides : cloning and analysis of genes encoding regulatory, structural and motor components

In this study several components responsible for the formation and function of the unidirectional flagellum of R. sphaeroides WS8 were identified via the characterisation of motility impaired TnphoA mutants. The role of the alternative sigma factor sigma 54 in flagellar gene regulation was also examined. Mutant M18 was defective in a fliI homologue, characterisation of this mutant revealed that FliI is not essential for flagellar formation in R. sphaeroides. This differs from that reported in the literature for S. typhimurium and so highlights the importance of studying R. sphaeroides as a model for flagellar motility. Analysis of another mutant Nm7 revealed that it was defective in FliF, a rotor component around which other flagellar components assemble. Overexpression of a FliF fusion protein allowed the production of anti FliF antiserum. DNA sequencing upstream and downstream of the fliF gene, revealed several other genes encoding flagellar components and a potential flagellar gene regulator (Torf). fliE, encoding a component of the basal body of unknown function, was identified upstream of fliF, an interposon mutant was created and was unable to be complemented by the wild type gene in trans suggesting a dominant effect. This is the first dominant mutation to be isolated in any fliE . The gene encoding the motor component FliG was also identified downstream of fliF and its C-terminal motility domain was found to contain regions that are conserved between FliG proteins from unidirectional and bidirectional motors, these may play a role in motor rotation and not switching. An overexpressed poly histidine FliG fusion protein was found to form a complex with the FliF-GST fusion protein ill vitro. The torf gene encodes a protein with homology to sigma 54 enhancer binding proteins. The Torf protein lacks any obvious DNA binding motif and may represent a novel member of the sigma 54 enhancer binding protein family

The unidirectional flagellum of R. sphaeroides : cloning and analysis of genes encoding regulatory, structural and motor components

In this study several components responsible for the formation and function of the unidirectional flagellum of R. sphaeroides WS8 were identified via the characterisation of motility impaired TnphoA mutants. The role of the alternative sigma factor sigma 54 in flagellar gene regulation was also examined. Mutant M18 was defective in a fliI homologue, characterisation of this mutant revealed that FliI is not essential for flagellar formation in R. sphaeroides. This differs from that reported in the literature for S. typhimurium and so highlights the importance of studying R. sphaeroides as a model for flagellar motility. Analysis of another mutant Nm7 revealed that it was defective in FliF, a rotor component around which other flagellar components assemble. Overexpression of a FliF fusion protein allowed the production of anti FliF antiserum. DNA sequencing upstream and downstream of the fliF gene, revealed several other genes encoding flagellar components and a potential flagellar gene regulator (Torf). fliE, encoding a component of the basal body of unknown function, was identified upstream of fliF, an interposon mutant was created and was unable to be complemented by the wild type gene in trans suggesting a dominant effect. This is the first dominant mutation to be isolated in any fliE . The gene encoding the motor component FliG was also identified downstream of fliF and its C-terminal motility domain was found to contain regions that are conserved between FliG proteins from unidirectional and bidirectional motors, these may play a role in motor rotation and not switching. An overexpressed poly histidine FliG fusion protein was found to form a complex with the FliF-GST fusion protein ill vitro. The torf gene encodes a protein with homology to sigma 54 enhancer binding proteins. The Torf protein lacks any obvious DNA binding motif and may represent a novel member of the sigma 54 enhancer binding protein family

Screening of healthcare workers for SARS-CoV-2 highlights the role of asymptomatic carriage in COVID-19 transmission.

Significant differences exist in the availability of healthcare worker (HCW) SARS-CoV-2 testing between countries, and existing programmes focus on screening symptomatic rather than asymptomatic staff. Over a 3 week period (April 2020), 1032 asymptomatic HCWs were screened for SARS-CoV-2 in a large UK teaching hospital. Symptomatic staff and symptomatic household contacts were additionally tested. Real-time RT-PCR was used to detect viral RNA from a throat+nose self-swab. 3% of HCWs in the asymptomatic screening group tested positive for SARS-CoV-2. 17/30 (57%) were truly asymptomatic/pauci-symptomatic. 12/30 (40%) had experienced symptoms compatible with coronavirus disease 2019 (COVID-19)>7 days prior to testing, most self-isolating, returning well. Clusters of HCW infection were discovered on two independent wards. Viral genome sequencing showed that the majority of HCWs had the dominant lineage B∙1. Our data demonstrates the utility of comprehensive screening of HCWs with minimal or no symptoms. This approach will be critical for protecting patients and hospital staff.This work was supported by the Wellcome Trust Senior Research Fellowships 108070/Z/15/Z to MPW, 215515/Z/19/Z to SGB and 207498/Z/17/Z to IGG; Collaborative award 206298/B/17/Z to IGG; Principal Research Fellowship 210688/Z/18/Z to PJL; Investigator Award 200871/Z/16/Z to KGCS; Addenbrooke’s Charitable Trust (to MPW, SGB, IGG and PJL); the Medical Research Council (CSF MR/P008801/1 to NJM); NHS Blood and Transfusion (WPA15-02 to NJM); National Institute for Health Research (Cambridge Biomedical Research Centre at CUHNFT), to JRB, MET, AC and GD, Academy of Medical Sciences and the Health Foundation (Clinician Scientist Fellowship to MET), Engineering and Physical Sciences Research Council (EP/P031447/1 and EP/N031938/1 to RS),Cancer Research UK (PRECISION Grand Challenge C38317/A24043 award to JY). Components of this work were supported by the COVID-19 Genomics UK Consortium, (COG-UK), which is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institut

Effective control of SARS-CoV-2 transmission between healthcare workers during a period of diminished community prevalence of COVID-19

Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; FundRef: http://dx.doi.org/10.13039/501100002927Funder: National Institute for Health Research; FundRef: http://dx.doi.org/10.13039/501100000272Previously, we showed that 3% (31/1032)of asymptomatic healthcare workers (HCWs) from a large teaching hospital in Cambridge, UK, tested positive for SARS-CoV-2 in April 2020. About 15% (26/169) HCWs with symptoms of coronavirus disease 2019 (COVID-19) also tested positive for SARS-CoV-2 (Rivett et al., 2020). Here, we show that the proportion of both asymptomatic and symptomatic HCWs testing positive for SARS-CoV-2 rapidly declined to near-zero between 25th April and 24th May 2020, corresponding to a decline in patient admissions with COVID-19 during the ongoing UK ‘lockdown’. These data demonstrate how infection prevention and control measures including staff testing may help prevent hospitals from becoming independent ‘hubs’ of SARS-CoV-2 transmission, and illustrate how, with appropriate precautions, organizations in other sectors may be able to resume on-site work safely

Combined point of care nucleic acid and antibody testing for SARS-CoV-2 following emergence of D614G Spike Variant

Rapid COVID-19 diagnosis in hospital is essential, though complicated by 30-50% of nose/throat swabs being negative by SARS-CoV-2 nucleic acid amplification testing (NAAT). Furthermore, the D614G spike mutant now dominates the pandemic and it is unclear how serological tests designed to detect anti-Spike antibodies perform against this variant. We assess the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease due to either wild type or the D614G spike mutant SARS-CoV-2. The overall detection rate for COVID-19 is 79.2% (95CI 57.8-92.9%) by rapid NAAT alone. Combined point of care antibody test and rapid NAAT is not impacted by D614G and results in very high sensitivity for COVID-19 diagnosis with very high specificity

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

Understanding the drivers for spread of SARS-CoV-2 in higher education settings is important to limit transmission between students, and onward spread into at-risk populations. In this study, we prospectively sequenced 482 SARS-CoV-2 isolates derived from asymptomatic student screening and symptomatic testing of students and staff at the University of Cambridge from 5 October to 6 December 2020. We performed a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. After a limited number of viral introductions into the university, the majority of student cases were linked to a single genetic cluster, likely dispersed across the university following social gatherings at a venue outside the university. We identified considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and dramatically reduced following a national lockdown. We observed that transmission clusters were largely segregated within the university or within the community. This study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics

Genomic epidemiology of COVID-19 in care homes in the east of England

Funder: National Institute for Health Research; FundRef: http://dx.doi.org/10.13039/501100000272COVID-19 poses a major challenge to care homes, as SARS-CoV-2 is readily transmitted and causes disproportionately severe disease in older people. Here, 1167 residents from 337 care homes were identified from a dataset of 6600 COVID-19 cases from the East of England. Older age and being a care home resident were associated with increased mortality. SARS-CoV-2 genomes were available for 700 residents from 292 care homes. By integrating genomic and temporal data, 409 viral clusters within the 292 homes were identified, indicating two different patterns – outbreaks among care home residents and independent introductions with limited onward transmission. Approximately 70% of residents in the genomic analysis were admitted to hospital during the study, providing extensive opportunities for transmission between care homes and hospitals. Limiting viral transmission within care homes should be a key target for infection control to reduce COVID-19 mortality in this population

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

Understanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response, but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)- 6. These late immunometabolic and immune defects may have clinical implication

Single-dose BNT162b2 vaccine protects against asymptomatic SARS-CoV-2 infection

The BNT162b2 mRNA COVID-19 vaccine (Pfizer-BioNTech) is being utilised internationally for mass COVID-19 vaccination. Evidence of single-dose protection against symptomatic disease has encouraged some countries to opt for delayed booster doses of BNT162b2, but the effect of this strategy on rates of asymptomatic SARS-CoV-2 infection remains unknown. We previously demonstrated frequent pauci- and asymptomatic SARS-CoV-2 infection amongst healthcare workers (HCWs) during the UK’s first wave of the COVID-19 pandemic, using a comprehensive PCR-based HCW screening programme (Rivett et al., 2020; Jones et al., 2020). Here, we evaluate the effect of first-dose BNT162b2 vaccination on test positivity rates and find a fourfold reduction in asymptomatic infection amongst HCWs ≥12 days post-vaccination. These data provide real-world evidence of short-term protection against asymptomatic SARS-CoV-2 infection following a single dose of BNT162b2 vaccine, suggesting that mass first-dose vaccination will reduce SARS-CoV-2 transmission, as well as the burden of COVID-19 disease