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Objective To determine whether or not radiofrequency ablation (RFA) for persistent atrial fibrillation in patients with advanced heart failure leads to improvements in cardiac function. Setting Patients were recruited from heart failure outpatient clinics in Scotland. Design and intervention Patients with advanced heart failure and severe left ventricular dysfunction were randomised to RFA (rhythm control) or continued medical treatment (rate control). Patients were followed up for a minimum of 6months. Main outcome measure Change in left ventricular ejection fraction (LVEF) measured by cardiovascular MRI. Results 22 patients were randomised to RFA and 19 to medical treatment. In the RFA group, 50% of patients were in sinus rhythm at the end of the study (compared with none in the medical treatment group). The increase in cardiovascular magnetic resonance (CMR) LVEF in the RFA group was 4.5±11.1% compared with 2.8±6.7% in the medical treatment group (p=0.6). The RFA group had a greater increase in radionuclide LVEF (a prespecified secondary end point) than patients in the medical treatment group (+8.2±12.0% vs +1.4±5.9%; p=0.032). RFA did not improve N-terminal pro-B-type natriuretic peptide, 6min walk distance or quality of life. The rate of serious complications related to RFA was 15%. Conclusions RFA resulted in long-term restoration of sinus rhythm in only 50% of patients. RFA did not improve CMR LVEF compared with a strategy of rate control. RFA did improve radionuclide LVEF but did not improve other secondary outcomes and was associated with a significant rate of serious complications. Clinical trials registration number NCT00292162. [ABSTRACT FROM AUTHOR]

Background: Pericardial effusions frequently present challenging clinical dilemmas. Whether or not to drain an effusion, and if so by what method, are two common decisions facing cardiologists. We performed a survey to evaluate pericardiocentesis practice in the United Kingdom (UK). Methods: A total of 640 questionnaires were sent to all cardiologists in the UK Directory of Cardiology in March 2003. Results: A total of 274 (43%) completed questionnaires were returned, 88% from consultants, equally distributed between tertiary referral centres and district general hospitals. More than 1500 procedures were performed, largely using a paraxiphoid approach (89%). Clinical tamponade was the commonest indication for pericardiocentesis (83%). However, the majority of respondents (69%) considered echocardiographic features alone an indication for pericardiocentesis, even in the absence of clinical tamponade. The commonest perceived indications for drainage were right ventricular diastolic collapse and right atrial collapse (69% and 33% of respondents respectively). For guidance, 82% use echocardiography, either alone or with fluoroscopy or the electrocardiogram (ECG) injury trace. 11% employ fluoroscopy alone or with the ECG injury trace. The remaining 11% stated that they would use the ECG injury trace alone or use no guidance. Using the ECG injury trace alone is said by the European Society of Cardiology (ESC) guidelines to offer an inadequate safeguard. Reported complications included ventricular puncture (n = 12, 0.8%) and hepatic damage (n = 4, 0.3%). Conclusion: Pericardiocentesis practice varies substantially in the UK. Many cardiologists would perform pericardiocentesis based on echocardiographic features alone. 11% of cardiologists use guidance that is considered inadequate by the ESC guidelines. [ABSTRACT FROM AUTHOR]

Objective: To examine the prevalence and incidence of unrecognized myocardial infarction in a contemporary population with type 2 diabetes.Research Design and Methods: We performed a retrospective analysis of the electrocardiograms (ECGs) recorded at baseline and after 2 years for the first 1,004 type 2 diabetic individuals to be randomized in the Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) study.Results: ECGs suitable for analysis were obtained from 669 participants. The prevalence of unrecognized Q-wave myocardial infarction at baseline was 1.9% (n = 13). The incidence of unrecognized Q-wave myocardial infarction at the end of 2 years of follow-up was 1.5/1,000-person-years (n = 2). One-third (13 of 39) of prevalent and one-quarter (2 of 8) of incident myocardial infarctions were unrecognized.Conclusions: Although the prevalence and incidence of myocardial infarction was low, unrecognized Q-wave myocardial infarctions made up a substantial proportion of all events. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis (TBE) virus is the most prevalent tick-transmitted orthoflavivirus in Europe. Due to the nonspecific nature of its symptoms, TBE is primarily diagnosed by ELISA-based detection of specific antibodies in the patient serum. However, cross-reactivity between orthoflaviviruses complicates the diagnosis. Specificity issues may be mitigated by serum neutralization assays (SNT), although the handling of clinically relevant orthoflaviviruses requires biosafety level (BSL) 3 conditions and they have highly divergent viral kinetics and cell tropisms. In the present study, we established a reporter virus particle (RVP)-based SNT in which the infectivity is measured by luminescence and that can be performed under BSL-2 conditions. The RVP-based SNT for TBEV exhibited a highly significant correlation with the traditional virus-based SNT (R 2  = 0.8637, p < 0.0001). The RVP-based assay demonstrated a sensitivity of 92.3% (95% CI: 79.7%-97.4%) and specificity of 100% (95% CI: 81.6%-100%). We also tested the cross-reactivity of serum samples in RVP-based assays against other orthoflaviviruses (yellow fever virus, dengue virus type 2, Zika virus, West Nile virus and Japanese encephalitis virus). Interestingly, all serum samples which had tested TBEV-positive by ELISA but negative by RVP-based SNT were reactive for antibodies against other orthoflaviviruses. Thus, the RVP-based seroneutralization assay provides an added value in clinical diagnostics as well as in epidemiological studies., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Lyme disease (LD) affects ∼476,000 people each year in the United States. Symptoms are variable and include rash and flu-like symptoms. Reasons for the wide variation in disease outcomes are unknown. Powassan virus (POWV) is a tick-borne flavivirus that causes disease ranging from asymptomatic infection to encephalitis, neurologic damage, and death. POWV and LD geographic case distributions overlap, with Ixodes species ticks as the common vectors. Clinical ramifications of coinfection or sequential infection are unknown. Objectives: This study's primary objective was to determine the prevalence of POWV-reactive antibodies in sera samples collected from previously studied cohorts of individuals with self-reported LD history residing in the Northeastern United States. As a secondary objective, we studied clinical differences between people with self-reported LD history and low versus high POWV antibody levels. Methods: We used an enzyme-linked immunosorbent assay (ELISA) to quantify IgG directed at the POWV envelope (E) protein domain III in 538 samples from individuals with self-reported LD history and 16 community controls. The samples were also tested with an ELISA assay to quantify IgG directed at the POWV NS1 protein. Results: The percentage of individuals with LD history and possible evidence of POWV exposure varied depending on the assay utilized. We found no significant difference in clinical symptoms between those with low or high POWV IgG levels in the in-house assay. Congruence of the EDIII and NS1 assays was low with only 12% of those positive in the in-house EDIII ELISA testing positive in the POWV NS1 ELISA. Conclusions: The results highlight the difficulty in flavivirus diagnostic testing, particularly in the retrospective detection of flavivirus exposure. The findings suggest that a prospective study with symptomatic patients using approved clinical testing is necessary to address the incidence and clinical implications of LD and POWV co-infection or sequential infection.

Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components., Competing Interests: Competing interests statement:The authors declare no competing interest.

Covering: up to fall 2022.Nonribosomal peptide synthetases (NRPSs) are a family of modular, multidomain enzymes that catalyze the biosynthesis of important peptide natural products, including antibiotics, siderophores, and molecules with other biological activity. The NRPS architecture involves an assembly line strategy that tethers amino acid building blocks and the growing peptides to integrated carrier protein domains that migrate between different catalytic domains for peptide bond formation and other chemical modifications. Examination of the structures of individual domains and larger multidomain proteins has identified conserved conformational states within a single module that are adopted by NRPS modules to carry out a coordinated biosynthetic strategy that is shared by diverse systems. In contrast, interactions between modules are much more dynamic and do not yet suggest conserved conformational states between modules. Here we describe the structures of NRPS protein domains and modules and discuss the implications for future natural product discovery.

Mosquito-borne West Nile virus (WNV) infection is benign in most individuals but can cause encephalitis in <1% of infected individuals. We show that ∼35% of patients hospitalized for WNV disease (WNVD) in six independent cohorts from the EU and USA carry auto-Abs neutralizing IFN-α and/or -ω. The prevalence of these antibodies is highest in patients with encephalitis (∼40%), and that in individuals with silent WNV infection is as low as that in the general population. The odds ratios for WNVD in individuals with these auto-Abs relative to those without them in the general population range from 19.0 (95% CI 15.0-24.0, P value <10-15) for auto-Abs neutralizing only 100 pg/ml IFN-α and/or IFN-ω to 127.4 (CI 87.1-186.4, P value <10-15) for auto-Abs neutralizing both IFN-α and IFN-ω at a concentration of 10 ng/ml. These antibodies block the protective effect of IFN-α in Vero cells infected with WNV in vitro. Auto-Abs neutralizing IFN-α and/or IFN-ω underlie ∼40% of cases of WNV encephalitis., (© 2023 Gervais et al.)

Although dengue virus (DENV) infection typically causes asymptomatic disease, DENV-infected patients can experience severe complications. A risk factor for symptomatic disease is pre-existing anti-DENV IgG antibodies. Cellular assays suggested that these antibodies can enhance viral infection of Fcγ receptor (FcγR)-expressing myeloid cells. Recent studies, however, revealed more complex interactions between anti-DENV antibodies and specific FcγRs by demonstrating that modulation of the IgG Fc glycan correlates with disease severity. To investigate the in vivo mechanisms of antibody-mediated dengue pathogenesis, we developed a mouse model for dengue disease that recapitulates the unique complexity of human FcγRs. In in vivo mouse models of dengue disease, we discovered that the pathogenic activity of anti-DENV antibodies is exclusively mediated through engagement of FcγRIIIa on splenic macrophages, resulting in inflammatory sequelae and mortality. These findings highlight the importance of IgG-FcγRIIIa interactions in dengue, with important implications for the design of safer vaccination approaches and effective therapeutic strategies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Aims: To investigate the effect of high-dose iron vs. low-dose intravenous (IV) iron on myocardial infarction (MI) in patients on maintenance haemodialysis., Methods and Results: This was a pre-specified analysis of secondary endpoints of the Proactive IV Iron Therapy in Hemodialysis Patients trial (PIVOTAL) randomized, controlled clinical trial. Adults who had started haemodialysis within the previous year, who had a ferritin concentration <400 μg per litre and a transferrin saturation <30% were randomized to high-dose or low-dose IV iron. The main outcome measure for this analysis was fatal or non-fatal MI. Over a median of 2.1 years of follow-up, 8.4% experienced a MI. Rates of type 1 MIs (3.2/100 patient-years) were 2.5 times higher than type 2 MIs (1.3/100 patient-years). Non-ST-elevation MIs (3.3/100 patient-years) were 6 times more common than ST-elevation MIs (0.5/100 patient-years). Mortality was high after non-fatal MI (1- and 2-year mortality of 40% and 60%, respectively). In time-to-first event analyses, proactive high-dose IV iron reduced the composite endpoint of non-fatal and fatal MI [hazard ratio (HR) 0.69, 95% confidence interval (CI) 0.52-0.93, P = 0.01] and non-fatal MI (HR 0.69, 95% CI 0.51-0.93; P = 0.01) when compared with reactive low-dose IV iron. There was less effect of high-dose IV iron on recurrent MI events than on the time-to-first event analysis., Conclusion: In total, 8.4% of patients on maintenance haemodialysis had an MI over 2 years. High-dose compared to low-dose IV iron reduced MI in patients receiving haemodialysis., Eudract Registration Number: 2013-002267-25., Competing Interests: Conflict of interest: M.C.P. reported receiving lecture fees from AstraZeneca, Novartis, and Eli Lilly, grant support, advisory board fees, and fees for serving on an endpoint committee from Boehringer Ingelheim, advisory board fees, lecture fees, and fees for serving on an endpoint committee from Novo Nordisk, advisory board fees from Napp Pharmaceuticals, and fees for serving on an endpoint committee from Takeda Pharmaceutical and Bayer. P.S.J. receiving consulting fees, advisory board fees, and lecture fees from Novartis, advisory board fees from Cytokinetics, and grant support from Boehringer Ingelheim. S.D.A. reported receiving fees for serving on a steering committee for Vifor, Bayer, Boehringer Ingelheim, Novartis, Servier, and grant support from Abbott Vascular. S.B. reported receiving lecture fees from Vifor Pharma and Pharmacosmos. P.A.K. reported receiving grants, personal fees and non-financial support from Vifor Fresenius, personal fees and non-financial support from Pharmacosmos. D.C.W. reported receiving personal fees from AstraZeneca, Akebia, Boehringer Ingelheim, Jannsen, Napp, Vifor Fresenius, and Amgen. C.R.V.T. has nothing to disclose. I.F. reported receiving grant support from Kidney Research UK, Vifor Pharma, and Pharmacosmos. J.J.V.Mc.M. reports receiving fees (all fees listed paid to Glasgow University) for serving on a steering committee from Bayer, fees for serving on a steering committee, fees for serving on an endpoint committee, and travel support from Cardiorentis, fees for serving on a steering committee and travel support from Amgen, fees for serving on a steering committee and travel support from Oxford University–Bayer, fees for serving as principal investigator of a trial and travel support from Theracos, fees for serving on a steering committee and travel support from AbbVie, fees for serving on a steering committee from DalCor Pharmaceuticals, fees for serving on a data and safety monitoring committee from Pfizer, fees for serving on a data and safety monitoring committee from Merck, fees for serving on an executive committee, fees for serving as co-principal investigator of a trial, fees for serving on a steering committee, fees for serving on an executive committee, travel support, and advisory board fees from Novartis, fees for serving as co-principal investigator for a trial, fees for serving on a steering committee, and travel support from GlaxoSmithKline, fees for serving on a steering committee from Bristol-Myers Squibb, and fees for serving on a steering committee, fees for serving on an endpoint adjudication committee, and travel support from Vifor Pharma–Fresenius. I.C.M. reported receiving grants from Kidney Research UK, Akebia, Bayer, and Astellas, personal fees from AMAG, FibroGen, Pharmacosmos and Vifor Pharma and grants and personal fees from GlaxoSmithKline., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity., Competing Interests: Declaration of interests J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

The non-ribosomal peptide synthetases (NRPSs) are a family of modular enzymes involved in the production of peptide natural products. Not restricted by the constraints of ribosomal peptide and protein production, the NRPSs are able to incorporate unusual amino acids and other suitable building blocks into the final product. The NRPSs operate with an assembly line strategy in which peptide intermediates are covalently tethered to a peptidyl carrier protein and transported to different catalytic domains for the multiple steps in the biosynthesis. Often the carrier and catalytic domains are joined into a single large multidomain protein. This chapter serves to introduce the NRPS enzymes, using the nocardicin NRPS system as an example that highlights many common features to NRPS biochemistry. We then describe recent advances in the structural biology of NRPSs focusing on large multidomain structures that have been determined., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V ( RhoV ) and WW domain-containing transcription regulator 1 ( WWTR1 ) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Objectives: This study sought to examine the effect of intravenous iron on heart failure events in hemodialysis patients., Background: Heart failure is a common and deadly complication in patients receiving hemodialysis and is difficult to diagnose and treat., Methods: The study analyzed heart failure events in the PIVOTAL (Proactive IV Iron Therapy in Hemodialysis Patients) trial, which compared intravenous iron administered proactively in a high-dose regimen with a low-dose regimen administered reactively. Heart failure hospitalization was an adjudicated outcome, a component of the primary composite outcome, and a prespecified secondary endpoint in the trial., Results: Overall, 2,141 participants were followed for a median of 2.1 years. A first fatal or nonfatal heart failure event occurred in 51 (4.7%) of 1,093 patients in the high-dose iron group and in 70 (6.7%) of 1,048 patients in the low-dose group (HR: 0.66; 95% CI: 0.46-0.94; P = 0.023). There was a total of 63 heart failure events (including first and recurrent events) in the high-dose iron group and 98 in the low-dose group, giving a rate ratio of 0.59 (95% CI: 0.40-0.87; P = 0.0084). Most patients presented with pulmonary edema and were mainly treated by mechanical removal of fluid. History of heart failure and diabetes were independent predictors of a heart failure event., Conclusions: Compared with a lower-dose regimen, high-dose intravenous iron decreased the occurrence of first and recurrent heart failure events in patients undergoing hemodialysis, with large relative and absolute risk reductions. (UK Multicentre Open-label Randomised Controlled Trial Of IV Iron Therapy In Incident Haemodialysis Patients; 2013-002267-25)., Competing Interests: Funding Support and Author Disclosures This work was supported by Kidney Research UK, which was supported by an unrestricted grant from Vifor Fresenius Medical Care Renal Pharma. Drs. Petrie and McMurray are supported by British Heart Foundation Centre of Research Excellence Grant RE/18/6/34217. The supporter/ funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication. Dr Jhund has received consulting fees from Novartis; advisory board fees from Novartis and Cytokinetics; lecture fees from Novartis; and grant support from Boehringer Ingelheim. Dr Petrie has received lecture fees from AstraZeneca, Novartis, Eli Lilly, and Novo Nordisk; grant support from Boehringer Ingelheim; advisory board fees from Boehringer Ingelheim, Novo Nordisk, and Napp Pharmaceuticals; and fees for serving on an endpoint committee from Boehringer Ingelheim, Novo Nordisk, Takeda Pharmaceutical, and Bayer. Dr Anker has received receiving fees for serving on steering committees for Vifor, Bayer, Boehringer Ingelheim, Novartis, and Servier; and grant support from Abbott Vascular. Dr Bhandari has received lecture fees from Vifor Pharma and Pharmacosmos. Dr Kalra has received grants from Vifor Fresenius; and personal fees and nonfinancial support from Vifor Fresenius and Pharmacosmos. Dr Wheeler has received personal fees from AstraZeneca, Akebia, Boehringer Ingelheim, Janssen, Napp, Vifor Fresenius, and Amgen. Dr Ford has received grant support from Kidney Research UK, Vifor Pharma and Pharmacosmos. Dr McMurray has received fees (all fees listed paid to Glasgow University) for serving on a steering committee from Bayer, Cardiorentis, Amgen, Oxford University–Bayer, AbbVie, DalCor Pharmaceuticals, Novartis, Bristol Myers Squibb, Vifor Pharma–Fresenius; fees for serving on an endpoint committee from Cardiorentis; travel support from Cardiorentis, Amgen, Oxford University–Bayer, Theracos, AbbVie, Novartis, GlaxoSmithKline, Vifor Pharma–Fresenius; fees for serving as principal investigator of a trial from Theracos; fees for serving on a data and safety monitoring committee from Pfizer and Merck; fees for serving on an executive committee from Novartis; fees for serving as co-principal investigator of a trial from Novartis and GlaxoSmithKline; advisory board fees from Novartis; and fees for serving on an endpoint adjudication committee from Vifor Pharma–Fresenius. Dr Macdougall has received grants from Kidney Research UK, Akebia, Bayer, and Astellas; personal fees from AMAG, FibroGen, Pharmacosmos, and Vifor Pharma; and grants and personal fees from GlaxoSmithKline. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021. Published by Elsevier Inc.)

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV., Competing Interests: Disclosures: M. Agudelo, D.F. Robbiani, and M.C. Nussenzweig reported a patent to Broadly Neutralizing Antibodies to Tick-Borne Encephalitis and Related Viruses (US 63/118,461) pending. M.C. Nussenzweig reported personal fees from Celldex outside the submitted work. Additionally, M.C. Nussenzweig is a Frontier Bioscience SAB member. No other disclosures were reported., (© 2021 Agudelo et al.)

Yellow fever virus (YFV) live attenuated vaccine can, in rare cases, cause life-threatening disease, typically in patients with no previous history of severe viral illness. Autosomal recessive (AR) complete IFNAR1 deficiency was reported in one 12-yr-old patient. Here, we studied seven other previously healthy patients aged 13 to 80 yr with unexplained life-threatening YFV vaccine-associated disease. One 13-yr-old patient had AR complete IFNAR2 deficiency. Three other patients vaccinated at the ages of 47, 57, and 64 yr had high titers of circulating auto-Abs against at least 14 of the 17 individual type I IFNs. These antibodies were recently shown to underlie at least 10% of cases of life-threatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination., Competing Interests: Disclosures: A. Homma reported, "Our institution is a non-profit producer of the yellow fever vaccine. We are public institution, part of our Ministry of Health, and provide vaccine only for National Immunization Program and UNICEF, PAHO Revolving Fund, GAVI, and WHO. We are very much interested to know all relevant scientific issues involved with our vaccine." J.L. Casanova reported a patent to application number 63/055,155, filed July 22, 2020 pending. No other disclosures were reported., (© 2021 Bastard et al.)

Background: Little is known regarding the impact of socioeconomic factors on the use of evidence-based therapies and outcomes in patients with heart failure with reduced ejection fraction across Asia., Methods: We investigated the association of both patient-level (household income, education levels) and country-level (regional income level by World Bank classification, income disparity by Gini index) socioeconomic indicators on use of guideline-directed therapy and clinical outcomes (composite of 1-year mortality or HF hospitalization, quality of life) in the prospective multinational ASIAN-HF study (Asian Sudden Cardiac Death in Heart Failure)., Results: Among 4540 patients (mean age: 60±13 years, 23% women) with heart failure with reduced ejection fraction, 39% lived in low-income regions; 34% in regions with high-income disparity (Gini ≥42.8%); 64.4% had low monthly household income (interaction <0.001 for both), where the association of low household income and low education status with poor outcomes was more pronounced in high-income compared with lower income regions., Conclusions: These findings highlight the importance of socioeconomic determinants among patients with heart failure in Asia and suggest that attention should be paid to address disparities in access to care among the poor and less educated, including those from wealthy regions. Registration: URL: https://clinicaltrials.gov; Unique Identifier: NCT01633398.

Background QRS duration (QRSd) is a marker of electrical remodeling in heart failure. Anthropometrics and left ventricular size may influence QRSd and, in turn, may influence the association between QRSd and heart failure outcomes. Methods and Results Using the prospective, multicenter, multinational ASIAN-HF (Asian Sudden Cardiac Death in Heart Failure) registry, this study evaluated whether electroanatomic ratios (QRSd indexed for height or left ventricular end-diastole volume) are associated with 1-year mortality in individuals with heart failure with reduced ejection fraction. The study included 4899 individuals (aged 60±19 years, 78% male, mean left ventricular ejection fraction: 27.3±7.1%). In the overall cohort, QRSd was not associated with all-cause mortality (hazard ratio [HR], 1.003; 95% CI, 0.999-1.006, P =0.142) or sudden cardiac death (HR, 1.006; 95% CI, 1.000-1.013, P =0.059). QRS/height was associated with all-cause mortality (HR, 1.165; 95% CI, 1.046-1.296, P =0.005 with interaction by sex p interaction =0.020) and sudden cardiac death (HR, 1.270; 95% CI, 1.021-1.580, P =0.032). QRS/left ventricular end-diastole volume was associated with all-cause mortality (HR, 1.22; 95% CI, 1.05-1.43, P =0.011) and sudden cardiac death (HR, 1.461; 95% CI, 1.090-1.957, P =0.011) in patients with nonischemic cardiomyopathy but not in patients with ischemic cardiomyopathy (all-cause mortality: HR, 0.94; 95% CI, 0.79-1.11, P =0.467; sudden cardiac death: HR, 0.734; 95% CI, 0.477-1.132, P =0.162). Conclusions Electroanatomic ratios of QRSd indexed for body size or left ventricular size are associated with mortality in individuals with heart failure with reduced ejection fraction. In particular, increased QRS/height may be a marker of high risk in individuals with heart failure with reduced ejection fraction, and QRS/left ventricular end-diastole volume may further risk stratify individuals with nonischemic heart failure with reduced ejection fraction. Registration URL: https://Clinicaltrials.gov. Unique identifier: NCT01633398.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks., Competing Interests: Declaration of interests C.D.A. is a co-founder of Chroma Therapeutics and Constellation Pharmaceuticals and a Scientific Advisory Board member of EpiCypher. S.W.L. is an advisor for and has equity in the following biotechnology companies: ORIC Pharmaceuticals, Faeth Therapeutics, Blueprint Medicines, Geras Bio, Mirimus Inc., PMV Pharmaceuticals, and Constellation Pharmaceuticals. C.M.R. is a founder of Apath LLC, a Scientific Advisory Board member of Imvaq Therapeutics, Vir Biotechnology, and Arbutus Biopharma and is an advisor for Regulus Therapeutics and Pfizer. The remaining authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics., Competing Interests: Declaration of Interests S.W.L. is an advisor for and has equity in the following biotechnology companies: ORIC Pharmaceuticals, Faeth Therapeutics, Blueprint Medicines, Geras Bio, Mirimus Inc., PMV Pharmaceuticals, and Constellation Pharmaceuticals. C.M.R. is a founder of Apath LLC; a Scientific Advisory Board member of Imvaq Therapeutics; Vir Biotechnology, and Arbutus Biopharma; and an advisor for Regulus Therapeutics and Pfizer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication., Competing Interests: Declaration of Interests C.M. Rice is a founder of Apath LLC; a Scientific Advisory Board member of Imvaq Therapeutics, Vir Biotechnology, and Arbutus Biopharma; and an advisor for Regulus Therapeutics and Pfizer. The remaining authors declare no competing interests. C.M. Rudin serves on the Scientific Advisory Boards of Bridge Medicines, Earli, and Harpoon Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Screening for genes differentially expressed in placental tissues, aiming to identify transcriptional signatures that may be involved in ZIKV congenital pathogenesis., Methods: Transcriptome data from placental tissues of pregnant women naturally infected with Zika virus during the third trimester were compared to those from women who tested negative for Zika infection. The findings were validated using both a cell culture model and an immunohistochemistry/morphological analysis of naturally infected placental tissues., Results: Transcriptome analysis revealed that Zika virus infection induces downregulation of insulin-like growth factor II (IGF2) gene, an essential factor for fetal development. The Caco-2 cell culture model that constitutively expresses IGF2 was used for the transcriptome validation. Asiatic and African Zika virus strains infection caused downregulated IGF2 gene expression in Caco-2 cells, whereas other flaviviruses, such as dengue serotype 1, West Nile and wild-type yellow fever viruses, had no effect on this gene expression. Immunohistochemical assays on decidual tissues corroborated our transcriptome analysis, showing that IGF2 is reduced in the decidua of Zika virus-infected women., Conclusions: Our results draw attention to IGF2 modulation in uterine tissues, and this finding is expected to support future studies on strategies to ameliorate the harmful effects of Zika virus infection during pregnancy., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Yellow fever virus (YFV), a member of the Flaviviridae family, is an arthropod-borne virus that can cause severe disease in humans with a lethality rate of up to 60%. Since 2017, increases in YFV activity in areas of South America and Africa have been described. Although a vaccine is available, named strain 17D (Theiler and Smith, 1937), it is contraindicated for use in the elderly, expectant mothers, immunocompromised people, among others. To this day there is no antiviral treatment against YFV to reduce the severity of viral infection. Here, we used a circular polymerase extension reaction (CPER)-based reverse genetics approach to generate a full-length reporter virus (YFVhb) by introducing a small HiBit tag in the NS1 protein. The reporter virus replicates at a similar rate to the parental YFV in HuH-7 cells. Using YFVhb, we designed a high throughput antiviral screening luciferase-based assay to identify inhibitors that target any step of the viral replication cycle. We validated our assay by using a range of inhibitors including drugs, immune sera and neutralizing single chain variable fragments (scFv). In light of the recent upsurge in YFV and a potential spread of the virus, this assay is a further tool in the development of antiviral therapy against YFV., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Free clinics manage a diversity of diseases among the uninsured. We sought to assess the medical management of stroke in a population of uninsured patients., Methods: A retrospective chart review was conducted to collect chronic disease statistics from 6558 electronic medical records and paper charts at nine free clinics in Tampa, Florida, from January 2016 to December 2017. Demographics and risk factors were compared between stroke patients and non-stroke patients. Medication rates for several comorbidities were also assessed., Results: Two percent (107) of patients had been diagnosed with a stroke. Stroke patients were older (mean (M) = 56.0, standard deviation (SD) = 11.2) than the rest of the sample (M = 43.3, SD = 15.4), p < 0.001 and a majority were men (n = 62, 58%). Of the stroke patients with hypertension (n = 79), 81% (n = 64) were receiving anti-hypertensive medications. Of the stroke patients with diabetes (n = 43), 72% (n = 31) were receiving diabetes medications. Among all stroke patients, 44% were receiving aspirin therapy (n = 47). Similarly, 39% of all stroke patients (n = 42) were taking statins., Conclusions: Uninsured patients with a history of stroke may not be receiving adequate secondary prevention highlighting the risk and vulnerability of uninsured patients. This finding identifies an area for improvement in secondary stroke prevention in free clinics., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease., Competing Interests: The authors have declared that no competing interests exist.

Aims: We aimed to characterize ethnic differences in prevalence, clinical correlates, and outcomes of atrial fibrillation (AF) in heart failure (HF) with preserved and reduced ejection fraction (HFpEF and HFrEF) across Asia., Methods and Results: Among 5504 patients with HF prospectively recruited across 11 Asian regions using identical protocols in the Asian Sudden Cardiac Death in Heart Failure study (mean age 61 ± 13 years, 27% women, 83% HFrEF), 1383 (25%) had AF defined as a history of AF and/or AF/flutter on baseline electrocardiogram. Clinical correlates of AF were similar across ethnicities and included older age, prior stroke, higher NT-proBNP, and larger left atria. Diabetes was associated with lower odds of AF in HFrEF [adjusted odds ratio (AOR) 0.79, 95% CI 0.66-0.95] and HFpEF (AOR 0.58, 95% CI 0.39-0.84) regardless of ethnicity. Compared with Chinese ethnicity, Japanese/Koreans had higher odds of AF in HFrEF (AOR 1.76, 95% CI 1.40-2.21), while Indians had lower odds in HFrEF (AOR 0.18, 95% CI 0.13-0.24) and HFpEF (AOR 0.28, 95% CI 0.16-0.49) even after adjusting for clinical covariates. Interaction between ethnicity and region was observed among Indians, with Southeast Asian Indians having higher odds of AF (AOR 3.01, 95% CI 1.60-5.67) compared with South Asian Indians. AF was associated with poorer quality of life and increased risk of 1 year all-cause mortality or HF hospitalisation (adjusted hazard ratio 1.39, 95% CI 1.18-1.63) regardless of ethnicity., Conclusions: Among patients with HF across Asia, clinical correlates and adverse outcomes associated with AF are similar across ethnicities; however, there are striking ethnic variations in the prevalence of AF that are not accounted for by known risk factors., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Aims: Our study aimed to compare the effectiveness of telemonitoring over structured telephone support in reducing heart failure-related healthcare utilization., Methods: This was a non-randomised controlled study comparing 150 recently discharged heart failure patients enrolled into telemonitoring and 55 patients who only received structured telephone support after rejecting telemonitoring. Patient activation, knowledge and self-management levels were measured at baseline and the one year upon programme completion using the Patient Activation Measure, the Dutch Heart Failure Knowledge Scale and the Self-Care of Heart Failure Index respectively. Differences in heart failure-related and all-cause hospitalization rates, total bed days and mortality rates at 180 days and at one year, knowledge and self-management scores and total cost of care between groups at one year were analysed., Results: Average age of telemonitoring was 57.9 years and 63.9 years for structured telephone support. Significant difference in adjusted 180-day all-cause bed days (telemonitoring: five days versus structured telephone support: 9.8 days), heart failure-related bed days (telemonitoring: 1.2 days versus structured telephone support: six days) and adjusted one-year heart failure-related bed days (telemonitoring: 2.2 days versus structured telephone support: 6.6 days) were observed. Telemonitoring was associated with reduced all-cause one-year mortality (hazard ratio 0.32, p  = 0.02). Estimated mean maintenance and confidence scores were significantly higher in the telemonitoring group at one year. No differences in all-cause and HF-related readmission rates and knowledge levels were observed. The one-year total cost of care was predicted to be Singapore dollars (SG$) 2774.4 lower ( p  = 0.07) in telemonitoring., Conclusion: In conclusion, telemonitoring was associated with lower all-cause and heart failure-related total bed days at 180 days, lower heart failure-related total bed days and total cost of care at one year as compared with structured telephone support.

Recent epidemics demonstrate the global threat of Zika virus (ZIKV), a flavivirus transmitted by mosquitoes. Although infection is usually asymptomatic or mild, newborns of infected mothers can display severe symptoms, including neurodevelopmental abnormalities and microcephaly. Given the large-scale spread, symptom severity, and lack of treatment or prophylaxis, a safe and effective ZIKV vaccine is urgently needed. However, vaccine design is complicated by concern that elicited antibodies (Abs) may cross-react with other flaviviruses that share a similar envelope protein, such as dengue virus, West Nile virus, and yellow fever virus. This cross-reactivity may worsen symptoms of a subsequent infection through Ab-dependent enhancement. To better understand the neutralizing Ab response and risk of Ab-dependent enhancement, further information on germline Ab binding to ZIKV and the maturation process that gives rise to potently neutralizing Abs is needed. Here we use binding and structural studies to compare mature and inferred-germline Ab binding to envelope protein domain III of ZIKV and other flaviviruses. We show that affinity maturation of the light-chain variable domain is important for strong binding of the recurrent VH3-23/VK1-5 neutralizing Abs to ZIKV envelope protein domain III, and identify interacting residues that contribute to weak, cross-reactive binding to West Nile virus. These findings provide insight into the affinity maturation process and potential cross-reactivity of VH3-23/VK1-5 neutralizing Abs, informing precautions for protein-based vaccines designed to elicit germline versions of neutralizing Abs., Competing Interests: Competing interest statement: D.F.R., M.C.N., and The Rockefeller University have filed a patent application for antibody Z004.