TRIM7 restricts Coxsackievirus and norovirus infection by detecting the C-terminalglutamine generated by 3C protease processing

TRIM7 catalyses the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7s extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The helix-{Phi}Q degron motif recognised by TRIM7 is reminiscent of the N-end degron system and is found in [~] 1% of cellular proteins. These features, together with TRIM7s restricted tissue expression and lack of immune regulation suggest that viral restriction may not be its physiological function.

SARS-CoV-2 Omicron spike mediated immune escape, infectivity and cell-cell fusion

The SARS-CoV-2 Omicron BA.1 variant emerged in late 2021 and is characterised by multiple spike mutations across all spike domains. Here we show that Omicron BA.1 has higher affinity for ACE2 compared to Delta, and confers very significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesevir and molnupiravir retain efficacy against Omicron BA.1. We found that in human nasal epithelial 3D cultures replication was similar for both Omicron and Delta. However, in lower airway organoids, Calu-3 lung cells and gut adenocarcinoma cell lines live Omicron virus demonstrated significantly lower replication in comparison to Delta. We noted that despite presence of mutations predicted to favour spike S1/S2 cleavage, the spike protein is less efficiently cleaved in live Omicron virions compared to Delta virions. We mapped the replication differences between the variants to entry efficiency using spike pseudotyped virus (PV) entry assays. The defect for Omicron PV in specific cell types correlated with higher cellular RNA expression of TMPRSS2, and accordingly knock down of TMPRSS2 impacted Delta entry to a greater extent as compared to Omicron. Furthermore, drug inhibitors targeting specific entry pathways demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that mediates cell entry via plasma membrane fusion. Instead, we demonstrate that Omicron spike has greater dependency on cell entry via the endocytic pathway requiring the activity of endosomal cathepsins to cleave spike. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was dramatically impaired compared to the Delta spike. Overall, Omicron appears to have gained significant evasion from neutralising antibodies whilst maintaining sensitivity to antiviral drugs targeting the polymerase. Omicron has shifted cellular tropism away from TMPRSS2 expressing cells that are enriched in cells found in the lower respiratory and GI tracts, with implications for altered pathogenesis.

Towards Internationally standardised humoral Immune Correlates of Protection from SARS CoV 2 infection and COVID-19 disease

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

SARS-CoV-2 B.1.617 emergence and sensitivity to vaccine-elicited antibodies

The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

Protected code blue: using in situ simulation to develop a protected code blue and modify staff training protocol-experience in a large community teaching hospital during the COVID-19 pandemic.

The surge in clinical demand, shortage in personal protective equipment and high-exposure risk for healthcare workers during the COVID-19 pandemic has challenged hospital common practices and forced a reassessment of care delivery models. Code blue teams are highly specialised units that partake in life-saving situations that can jeopardise the safety of team members. There is a paucity of guidance in regards to proper infection control measures to protect the responders.This study describes a methodical approach to assessing vulnerabilities to transmission of SARS-CoV-2 within existing code blue practices, modalities to limit the number of code blue team responders and modifications to the protocol at a large community teaching hospital. The effort undertaken faced challenges due to the nature of the pandemic and the increased demand on healthcare workers. Quality improvement methods facilitated our protocol design and implementation. To this date, there has been no identified COVID-19 disease in any protected code blue (PCB) team members. We recommend that similar practices be considered and adopted widely and practised periodically.

Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion ΔH69/V70

SARS-CoV-2 amino acid replacements in the receptor binding domain (RBD) occur relatively frequently and some have a consequence for immune recognition. Here we report recurrent emergence and significant onward transmission of a six-nucleotide out of frame deletion in the S gene, which results in loss of two amino acids: H69 and V70. We report that in human infections {Delta}H69/V70 often co-occurs with the receptor binding motif amino acid replacements N501Y, N439K and Y453F, and in the latter two cases has followed the RBD mutation. One of the {Delta}H69/V70+ N501Y lineages, now known as B.1.1.7, has undergone rapid expansion and includes eight S gene mutations: RBD (N501Y and A570D), S1 ({Delta}H69/V70 and {Delta}144) and S2 (P681H, T716I, S982A and D1118H). In vitro, we show that {Delta}H69/V70 does not reduce serum neutralisation across multiple convalescent sera. However, {Delta}H69/V70 increases infectivity and is associated with increased incorporation of cleaved spike into virions. {Delta}H69/V70 is able to compensate for small infectivity defects induced by RBD mutations N501Y, N439K and Y453F. In addition, replacement of H69 and V70 residues in the B.1.1.7 spike reduces its infectivity and spike mediated cell-cell fusion. Based on our data {Delta}H69/V70 likely acts as a permissive mutation that allows acquisition of otherwise deleterious immune escape mutations. Enhanced surveillance for the {Delta}H69/V70 deletion with and without RBD mutations should be considered as a global priority not only as a marker for the B.1.1.7 variant, but potentially also for other emerging variants of concern. Vaccines designed to target the deleted spike protein could mitigate against its emergence as increased selective forces from immunity and vaccines increase globally. HighlightsO_LI{Delta}H69/V70 is present in at least 28 SARS-CoV-2 lineages C_LIO_LI{Delta}H69/V70 does not confer escape from convalescent sera C_LIO_LI{Delta}H69/V70 increases spike infectivity and compensates for RBD mutations C_LIO_LI{Delta}H69/V70 is associated with greater spike cleavage C_LIO_LIB.1.1.7 requires {Delta}H69/V70 for optimal spike cleavage and infectivity C_LI

Critical care workers have lower seroprevalence of SARS-CoV-2 IgG compared with non-patient facing staff in first wave of COVID19.

With the first 2020 surge of the COVID-19 pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams to look after high numbers of patients with severe COVID-19. There was considerable anxiety of increased risk of COVID19 for staff working in these environments. Using a multiplex platform to assess serum IgG responses to SARS-CoV-2 N, S and RBD proteins, and detailed symptom reporting, we screened over 500 HCW (25% of the total workforce) in a quaternary level hospital to explore the relationship between workplace and evidence of exposure to SARS-CoV-2. Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, overall seroprevalence was 14% with anosmia and fever being the most discriminating symptoms for seropositive status. There was a significant difference in seropositive status between staff working in clinical and non-clinical roles (9% patient facing critical care, 15% patient facing non-critical care, 22% nonpatient facing). In the seropositive cohort, symptom severity increased with age for men and not for women. In contrast, there was no relationship between symptom severity and age or sex in the seronegative cohort reporting possible COVID-19 symptoms. Of the 12 staff screened PCR positive (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. ConclusionThe current approach to Personal Protective Equipment (PPE) appears highly effective in protecting staff from patient acquired infection in the critical care environment including protecting staff managing interhospital transfers of COVID-19 patients. The relationship between seroconversion and disease severity in different demographics warrants further investigation. Longitudinally paired virological and serological surveillance, with symptom reporting are urgently required to better understand the role of antibody in the outcome of HCW exposure during subsequent waves of COVID-19 in health care environments.

The Impact of Implementing an Intensivist Model With Nighttime In-Hospital Nocturnist and Effect on ICU Outcomes.

BACKGROUND: Various intensivist staffing models have been suggested, but the long-term sustainability and outcomes vary and may not be sustained. We examined the impact of implementing a high-intensity intensivist coverage model with a nighttime in-house nocturnist (non-intensivist) and its effect on intensive care unit (ICU) outcomes. METHODS: We obtained historical control baseline data from 2007 to 2011 and compared the same data from 2011 to 2015. The Acute Physiological and Chronic Health Evaluation outcomes system was utilized to collect clinical, physiological, and outcome data on all adult patients in the medical ICU and to provide severity-adjusted outcome predictions. The model consists of a mandatory in-house daytime intensivist service that leads multidisciplinary rounds, and an in-house nighttime coverage is provided by nocturnist (nonintensivists) with current procedural skills in airways management, vascular access, and commitment to supervise house staff as needed. The intensivist continues to be available remotely at nighttime for house staff and consultation with the nocturnist. A backup intensivist is available for surge management. RESULTS: First year yielded improved throughput (2428 patients/year to 2627 then 2724 at fifth year). Case mix stable at 53.7 versus 55.2. The ICU length of stay decreased from 4.7 days (predicted 4.25 days) to 3.8 days (4.15) in first year; second year: 3.63 days (4.29 days); third year: 3.24 days (4.37), fourth year: 3.34 days (4.45), and fifth year: 3.61 days (4.42). Intensive care unit <24 hours readmission remained at 1%; >24 hours increased from 4% to 6%. Low-risk monitoring admissions remained at an average 17% (benchmark 17.18%). Intensive care unit mortality improved with standardized mortality ration averaging at 0.84. Resident satisfaction surveys improved. CONCLUSIONS: Implementing an intensivist service with nighttime nocturnist staffing in a high-intensity large teaching hospital is feasible and improved ICU outcomes in a sustained manner that persisted after the initial implementation phase. The model resulted in reduced and sustained observed-to-predicted length of ICU stay.

Analgesia-first sedation in critically ill adults: A U.S. pilot, randomized controlled trial.

PURPOSE: To determine the feasibility of conducting a multicenter ICU RCT of AFS compared to either protocol-directed sedation (PDS) or both PDS and daily sedation interruption (DSI) in North America. MATERIALS AND METHODS: This single-center RCT compared AFS [fentanyl (bolus ± infusions) to reach CPOT ≤2; if RASS ≥1, CPOT ≤2 and additional fentanyl failed to reach RASS goal (-2 to 0), low-dose propofol (up to 6 h) was given] with either PDS or both PDS and DSI daily in adults mechanically ventilated (MV) ≥48 h. Relevant feasibility, safety, and clinical outcomes were defined and evaluated. RESULTS: 90 of 160 eligible patients were enrolled [AFS = 27; PDS = 28; PDS + DSI = 31]; rate = 3/month. Time from intubation to randomization was 17.5 ±â€¯11.6 h. Study days fully adherent to the study intervention [AFS = 95%; PDS = 99%; PDS + DSI = 96%] and time spent in the first 48 h after randomization without pain (CPOT ≤2)[AFS = 82%; PDS = 78%; PDS + DSI = 77%] and at goal RASS[AFS = 88%; PDS = 83%; PDS + DSI = 95%] were high and similar. Nurse-perceived [median (IQR)] study workload (10-point VAS) was higher with AFS [4(2-6)] than PDS [1(1-3)] or PDS + DSI [2(1-5)]; p = .002). Unplanned extubation was rare (AFS = 1; PDS = 0; PDS + DSI = 1). Days [median (IQR)] free of MV in the 28d after intubation [AFS 24(23,26); PDS 24(20,26); PDS + DSI 24(21,26)] was not different (p = .62). CONCLUSION: A multicenter RCT evaluating AFS is feasible to conduct in North America.

Decolonization to Reduce Postdischarge Infection Risk among MRSA Carriers.

BACKGROUND: Hospitalized patients who are colonized with methicillin-resistant Staphylococcus aureus (MRSA) are at high risk for infection after discharge. METHODS: We conducted a multicenter, randomized, controlled trial of postdischarge hygiene education, as compared with education plus decolonization, in patients colonized with MRSA (carriers). Decolonization involved chlorhexidine mouthwash, baths or showers with chlorhexidine, and nasal mupirocin for 5 days twice per month for 6 months. Participants were followed for 1 year. The primary outcome was MRSA infection as defined according to Centers for Disease Control and Prevention (CDC) criteria. Secondary outcomes included MRSA infection determined on the basis of clinical judgment, infection from any cause, and infection-related hospitalization. All analyses were performed with the use of proportional-hazards models in the per-protocol population (all participants who underwent randomization, met the inclusion criteria, and survived beyond the recruitment hospitalization) and as-treated population (participants stratified according to adherence). RESULTS: In the per-protocol population, MRSA infection occurred in 98 of 1063 participants (9.2%) in the education group and in 67 of 1058 (6.3%) in the decolonization group; 84.8% of the MRSA infections led to hospitalization. Infection from any cause occurred in 23.7% of the participants in the education group and 19.6% of those in the decolonization group; 85.8% of the infections led to hospitalization. The hazard of MRSA infection was significantly lower in the decolonization group than in the education group (hazard ratio, 0.70; 95% confidence interval [CI], 0.52 to 0.96; P=0.03; number needed to treat to prevent one infection, 30; 95% CI, 18 to 230); this lower hazard led to a lower risk of hospitalization due to MRSA infection (hazard ratio, 0.71; 95% CI, 0.51 to 0.99). The decolonization group had lower likelihoods of clinically judged infection from any cause (hazard ratio, 0.83; 95% CI, 0.70 to 0.99) and infection-related hospitalization (hazard ratio, 0.76; 95% CI, 0.62 to 0.93); treatment effects for secondary outcomes should be interpreted with caution owing to a lack of prespecified adjustment for multiple comparisons. In as-treated analyses, participants in the decolonization group who adhered fully to the regimen had 44% fewer MRSA infections than the education group (hazard ratio, 0.56; 95% CI, 0.36 to 0.86) and had 40% fewer infections from any cause (hazard ratio, 0.60; 95% CI, 0.46 to 0.78). Side effects (all mild) occurred in 4.2% of the participants. CONCLUSIONS: Postdischarge MRSA decolonization with chlorhexidine and mupirocin led to a 30% lower risk of MRSA infection than education alone. (Funded by the AHRQ Healthcare-Associated Infections Program and others; ClinicalTrials.gov number, NCT01209234 .).

Influence of sedation strategies on unplanned extubation in a mixed intensive care unit.

BACKGROUND: Identifying risk factors for unplanned extubation in patients receiving mechanical ventilation can help guide prevention strategies. OBJECTIVE: To assess the risk of unplanned extubation with different sedation strategies. METHODS: A 36-month quality improvement study in a 33-bed intensive care unit at a tertiary-care center. RESULTS: A total of 92 unplanned extubations occurred (7.5 events/1000 days of mechanical ventilation): patients who were receiving continuous sedation protocol with daily interruption of sedatives had 1.5 events/1000 ventilator days, patients receiving the intermittent sedation protocol had 5.0 events/1000 days, and patients with no sedation protocol had 16 events/1000 days (P < .05). Median duration of mechanical ventilation before unplanned extubation was 2 days. Most unplanned extubations (94%) were deliberate, and 53% occurred in patients scheduled for weaning. Most unplanned extubations in the continuous sedation protocol group (71%) occurred during weaning, in comparison to the intermittent sedation protocol (54%) and no sedation protocol groups (48%, P< .05). The highest incidences of agitation were in patients receiving the intermittent sedation protocol as compared with the other 2 groups (77% vs 50% vs 49%, P < .05). Overall, 73% of patients who had an unplanned extubation did not require reintubation; those who did were older (mean age: 68 vs 53 years, P = .01) and were male (80% vs 20%, P= .02). Reintubation was unrelated to the time of unplanned extubation. CONCLUSION: Strategies of no sedation or intermittent sedation are both associated with higher rates of unplanned extubation when compared to a strategy of continuous sedation with daily interruption of sedatives. Sedation strategies that allow agitation may increase the risk of unplanned extubation.

A unique hospital physician disaster response system for a nonemployed medical staff.

Private hospitals with nonemployed, volunteer medical staffs face a special challenge in meeting the patient-care needs posed by a mass casualty incident (MCI). Although most disaster response systems focus on emergency department and trauma management, such systems often do not provide for the need to triage existing inpatients to create room for incoming casualties, for continuity of physician care for those patients, as well as for MCI victims in case of major disaster. Such systems must also provide a mechanism for ethical and appropriate rationing of limited resources during a MCI. Community hospitals without 24/7 in-house physicians must provide a mechanism for physician care for patients in situations in which access to the hospital may be limited by the disaster (eg, major earthquake or flood). This article describes a system established at Long Beach Memorial Medical Center, a 740-bed not-for-profit hospital with a volunteer medical staff to ensure continuity of physician care in a major disaster. To our knowledge, this is the first published report of such a system.