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The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 47 fragments with diverse scaffolds were identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we performed deep mutational scanning of NS2B-NS3 protease. Merging fragment hits yields an extensive set of 'mergers', defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points that maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development., Competing Interests: Competing Interest Statement A.S.G consults for DNDi and MMV.

Venezuelan, western, and eastern equine encephalitis virus (VEEV, WEEV, and EEEV) cause a febrile illness that may result in fatal neurological disease in humans and equines. Human infections are typically from mosquito bites, although cases from respiratory exposure in laboratory accidents have been documented. In addition to natural mosquito-borne infection, the potential biothreat inherent in the ability to disseminate these viruses via the respiratory route has driven the development of antiviral drugs for this route of exposure. To address this gap, we tested the prophylactic administration of a novel brain-penetrant, antiviral, BDGR-49, against a lethal intranasal challenge of VEEV, WEEV, or EEEV in BALB/c mouse model. BDGR-49 conferred 100% protection with 6 mg kg -1 twice per day for 6 days for VEEV, but not EEEV or WEEV. By 8 days post-infection (dpi), infectious virus, viral RNA, and viral antigen in the brain of BDGR-49-treated mice were significantly reduced. Brains of VEEV TrD-infected, BDGR-49-treated mice showed a significant reduction in the expression of genes associated with inflammation ( IFNB1 , TNF , IL6 , and CCL5 ) and cell death ( CASP4 , GSDMD , PYCARD , and ZBP1 ). At dpi 14, histopathology showed that neuronal lesions and inflammatory cell infiltrates were essentially absent, and viral antigen was not detected in the brains of VEEV TrD-infected, BDGR-49-treated mice. In summary, although BDGR-49 treatment showed significant promise for the treatment of mice exposed intranasally to VEEV, the more rapid and efficient entry of EEEV and WEEV by this route into the central nervous system will require additional optimization of the dosing regimen.IMPORTANCEProphylactic and therapeutic treatment of viruses that cause encephalitis requires fast-acting drugs that rapidly penetrate the blood-brain barrier. Currently, clinicians have only a limited set of antivirals for the treatment of neurotropic infections such as herpesviruses or HIV-1, and none for alphaviruses, and treatment outcomes remain poor. New medical countermeasures will address the gap in treatment of viral encephalitis such as those caused by the neurotropic alphaviruses and others.

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5'-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca 2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.

Young adults from underserved racial/ethnic groups are critically needed as unrelated hematopoietic stem cell (HSC) donors, yet they are more likely than other groups to opt out of donation after having matched a patient. Understanding which factors are most strongly associated with opting out among young underserved racial/ ethnic registered donors compared with their White counterparts will provide the basis for specific interventions to improve donor retention. We sought to determine the key, modifiable psychosocial, registry-related, and donation-related characteristics that are uniquely associated with opting out across 5 key racial/ethnic groups of young HSC donor registry members who had been contacted as a potential match for a patient. This study examines data from a large cross-sectional survey of young (age 18 to 30) registry members shortly after they preliminarily matched a patient (CT-stage) and continued toward or opted out of donation (CT-C and CT-NI), stratified by racial/ethnic group and sex. We assessed psychosocial, registry-related, and donation-related characteristics for all participants. We used chi-squared and F tests to assess differences between racial/ethnic groups. A separate logistic regression analysis for each racial/ethnic group was conducted to quantify adjusted associations between each variable and opting out. Then, we compared these associations across the racial/ethnic groups by evaluating the interaction effect between each variable and racial/ethnic group, with the same outcome (CT-C versus CT-NI) in question. Nine hundred thirty-five participants were surveyed, including 284 White, 165 Hispanic, 191 Black, 192 Asian/Pacific Islander, and 103 Multiracial/multiethnic participants. There were significant differences across racial/ethnic groups in values/goals, religious objections to donation, HSC-related medical mistrust, and parental involvement in donation decisions. Adjusted logistic regression subgroup analyses indicated that ambivalence was strongly associated with opting out across all racial/ethnic groups. Greater focus on intrinsic life goals (e.g., raising a family, becoming a community leader, influencing social values) was associated with opting out in the Multiracial/multiethnic, Hispanic, and Asian/Pacific Islander groups. Healthcare mistrust and insufficient registry contact was a significant factor for Hispanic participants. Protective factors against opting out included remembering joining the registry (Black participants), and parental support for donation decision (Asian/Pacific Islander participants). The performance of each logistic regression model was strong, with area-under-the curve ≥.88, CT-stage outcome classification accuracy ≥89%, and good fit between expected and observed opt-out probabilities. In the analysis across different racial/ethnic groups, the only significant interaction was race/ethnicity by whether more contact with the registry would have changed the decision at CT-stage; this variable was significant only for the Hispanic group. In the within-group analysis for Hispanic participants, the "more registry contact" variable was strongly associated with opting out (odds ratio 5.8, P = .03). Consistent with a growing body of HSC donor research, ambivalence was a key factor associated with opting-out for all racial/ethnic groups. Other key variables were differentially associated with opting-out depending on racial/ethnic group. Our study highlights key variables that registries should focus on as they develop targeted and tailored strategies to enhance commitment and reduce attrition of potential donors., (Copyright © 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16 . Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance., Competing Interests: The authors declare no conflict of interest.

Several hantaviruses result in zoonotic infections of significant public health concern, causing hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in the Old and New World, respectively. Given a 35% case fatality rate, disease-causing New World hantaviruses require a greater understanding of their biology, genetic diversity, and geographical distribution. Juquitiba hantaviruses have been identified in Oligoryzomys nigripes in Brazil, Paraguay, and Uruguay. Brazil has reported the most HCPS cases associated with this virus. We used a multiplexed, amplicon-based PCR strategy to screen and deep-sequence the virus harbored within lung tissues collected from Oligoryzomys species during rodent field collections in southern (Itapúa) and western (Boquerón) Paraguay. No Juquitiba-like hantaviruses were identified in Boquerón. Herein, we report the full-length S and M segments of the Juquitiba hantaviruses identified in Paraguay from O. nigripes . We also report the phylogenetic relationships of the Juquitiba hantaviruses in rodents collected from Itapúa with those previously collected in Canindeyú. We showed, using the TN93 nucleotide substitution model, the coalescent (constant-size) population tree model, and Bayesian inference implemented in the Bayesian evolutionary analysis by sampling trees (BEAST) framework, that the Juquitiba virus lineage in Itapúa is distinct from that in Canindeyú. Our spatiotemporal analysis showed significantly different time to the most recent ancestor (TMRA) estimates between the M and S segments, but a common geographic origin. Our estimates suggest the additional geographic diversity of the Juquitiba virus within the Interior Atlantic Forest and highlight the need for more extensive sampling across this biome.

Hantaviruses, genus Orthohantavirus , family Hantaviridae , order Bunyavirales , are negative-sense, single-stranded, tri-segmented RNA viruses that persistently infect rodents, shrews, and moles. Of these, only certain virus species harbored by rodents are pathogenic to humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (LMVEC). The reason why certain rodent-borne hantavirus species are pathogenic has long been hypothesized to be related to their ability to downregulate and dysregulate the immune response as well as increase vascular permeability of infected endothelial cells. We set out to study the temporal dynamics of host immune response modulation in primary human LMVECs following infection by Prospect Hill (nonpathogenic), Andes (pathogenic), and Hantaan (pathogenic) viruses. We measured the level of RNA transcripts for genes representing antiviral, proinflammatory, anti-inflammatory, and metabolic pathways from 12 to 72 h with time points every 12 h. Gene expression analysis in conjunction with mathematical modeling revealed a similar profile for all three viruses in terms of upregulated genes that partake in interferon signaling ( TLR3 , IRF7 , IFNB1 ), host immune cell recruitment ( CXCL10 , CXCL11 , and CCL5 ), and host immune response modulation ( IDO1 ). We examined secreted protein levels of IFN-β, CXCL10, CXCL11, CCL5, and IDO in two male and two female primary HLMVEC donors at 48 and 60 h post infection. All three viruses induced similar levels of CCL5, CXCL10, and CXCL11 within a particular donor, and the levels were similar in three of the four donors. All three viruses induced different protein secretion levels for both IFN-β and IDO and secretion levels differed between donors. In conclusion, we show that there was no difference in the transcriptional profiles of key genes in primary HLMVECs following infection by pathogenic and nonpathogenic hantaviruses, with protein secretion levels being more donor-specific than virus-specific., Competing Interests: The authors declare no conflict of interest.

Objectives: To determine the prevalence of the utilization of primary intensivists and primary nurses for long-stay patients in large, academic PICU and ascertain how these practices are operationalized and perceived., Design: A cross-sectional survey., Setting: U.S. PICUs with accredited Pediatric Critical Care Medicine fellowships., Subjects: One senior physician and one senior nurse at each institution., Intervention: None., Measurements and Main Results: Separate but largely analogous questionnaires for intensivists and nurses were created using an iterative process to enhance content/face validity and readability. Sixty-seven intensivists (representing 93% of the 72 institutions with fellowship programs and their PICUs) and 59 nurses (representing 82%) responded. Twenty-four institutions utilize primary intensivists; 30 utilize primary nurses; and 13 utilize both. Most institutions use length of stay and/or other criteria (e.g., medical complexity) for eligibility. Commonly, not all patients that meet eligibility criteria receive primaries. Primary providers are overwhelmingly volunteers, and often only a fraction of providers participate. Primary intensivists at a large majority (>75%) of institutions facilitate information sharing and decision-making, attend family/team meetings, visit patients/families regularly, and are otherwise available upon request. Primary nurses at a similar majority of institutions provide consistent bedside care, facilitate information sharing, and attend family/team meetings. A large majority of respondents thought that primary intensivists increase patient/family satisfaction, reduce their stress, improve provider communication, and reduce conflict, whereas primary nurses similarly increase patient/family satisfaction. More than half of respondents shared that these practices can sometimes require effort (e.g., time and emotion), complicate decision-making, and/or reduce staffing flexibility., Conclusions: Primary practices are potential strategies to augment rotating PICU care models and better serve the needs of long-stay and other patients. These practices are being utilized to varying extents and with some operationalization uniformity at large, academic PICUs., Competing Interests: Dr. Aponte-Patel received funding from the Columbia University Department of Psychiatry and NYP Weill Cornell. Dr. Baird received funding from I-PASS Patient Safety Institute. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2023 by the Society of Critical Care Medicine and the World Federation of Pediatric Intensive and Critical Care Societies.)

The evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of several variants of concern (VOC) with increased immune evasion and transmissibility. This has motivated studies to assess protection conferred by earlier strains following infection or vaccination to each new VOC. We hypothesized that while NAbs play a major role in protection against infection and disease, a heterologous reinfection or challenge may gain a foothold in the upper respiratory tract (URT) and result in a self-limited viral infection accompanied by an inflammatory response. To test this hypothesis, we infected K18-hACE2 mice with SARS-CoV-2 USA-WA1/2020 (WA1) and, after 24 days, challenged with WA1, Alpha, or Delta. While NAb titers against each virus were similar across all cohorts prior to challenge, the mice challenged with Alpha and Delta showed weight loss and upregulation of proinflammatory cytokines in the URT and lower RT (LRT). Mice challenged with WA1 showed complete protection. We noted increased levels of viral RNA transcripts only in the URT of mice challenged with Alpha and Delta. In conclusion, our results suggested self-limiting breakthrough infections of Alpha or Delta in the URT, which correlated with clinical signs and a significant inflammatory response in mice.

Young adults enrolled in hematopoietic stem cell (HSC) donation registries, including the Be The Match registry in the United States, often opt out of the registry when a potential recipient is identified. This results in a limited supply from the most desirable allogeneic source of HSCs used in transplantation to treat serious health conditions. The differences in demographic, psychosocial, registry-related, and donation-related characteristics between those who continue to donation and those who opt out may elucidate the modifiable risk factors for attrition, but these characteristics have not been extensively studied in young donors up to age 30 years. Our goal was to study demographic, psychosocial, registry-related, and donation-related characteristics in a group of young HSC donor registry members who had recently been contacted about a potential recipient, to determine the key characteristics that differ between those who continued toward donation and those who opted out and to examine the extent of these differences. We conducted a cross-sectional survey in a random sample of young (age 18 to 30 years) current and former registry members, stratified by race/ethnicity and sex. Demographic, psychosocial (eg, life goals, HSC allocation mistrust), registry-related (eg context and motive for joining the registry), and donation-related (eg, ambivalence, religious objections to donation, knowledge about donation) characteristics were assessed. Chi-square and 2-sample t tests were used to examine differences between those who continued (CT-C group) and those who opted out (CT-NI group). Hierarchical logistic regression was used to estimate adjusted covariate effects on the odds of opting out. A total of 935 participants were surveyed. Donation-related knowledge was higher in the CT-C group than in the CT-NI group. HSC allocation mistrust, religious objections, and concerns about donation were higher in the CT-NI group. After adjusting for covariates in a logistic regression model, we found that having more intrinsic life goals, having more ambivalence, and talking with registry staff only once/twice were significantly associated with opting out of the registry. Ambivalence had the strongest association with opting out. In contrast, remembering joining the registry, believing that parents would support donation, and having medical concerns were significantly associated with continuing toward donation. This effect of medical concerns on donation was discovered only after adjusting for the related but distinct ambivalence variable, with the remaining effect of medical concerns relating to engagement with the donation process and information-seeking. The model had strong discriminative ability (area under the receiver operating characteristic curve = .92) and classification accuracy (86.6%). Our data indicate that among young adult members of a national HSC donor registry, ambivalence and limited contact with registry staff were more strongly associated with opting out of donation. Medical concerns were associated with continuing toward donation. Further studies are needed to confirm a causal link between medical concerns and continuing to donation among young donors. Our study suggests that these concerns might not be directly related to attrition, whereas other factors (eg, ambivalence, low donation-related knowledge) are associated with attrition and thus should be targeted for attrition reduction strategies., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium., (© 2023 New York Academy of Sciences.)

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Rationale: Primary continuity intensivists and nurses for long-stay patients (LSPs) in pediatric intensive care units (PICUs) are understudied strategies used to mitigate the fragmented care of typical rotating care models. Objectives: To investigate the advantages and disadvantages of primary continuity intensivists and nurses for LSPs as perceived by their parents and PICU providers. Methods: We conducted a prospective cross-sectional mixed-methods study of the perspectives of parents whose children were admitted to a PICU for >10 days and had one or more complex chronic conditions regarding the care provided by their PICU intensivists and nurses. As part of a trial, patients had been randomized to care provided by a rotating on-service intensivist who changed weekly and by PICU nurses who changed every 12 hours (usual care group) or to care provided by the same on-service intensivist plus a primary continuity intensivist and primary nurses (primary group). In addition, PICU providers (intensivists, fellows, and nurses) were queried for their perspectives on primary intensivists and nurses. Novel questionnaires, assessed for content and face validity and for readability, were used. The parental questionnaire involved indicating their degree of agreement with 16 statements about their children's care. The provider questionnaire involved rating potential advantages of primary continuity intensivists and nurses and estimating the frequency of disadvantages. Descriptive statistics and divergent stack bar charts were used; parents' and providers' responses were compared, stratified by their children's group (usual care or primary) and provider role, respectively. Results: The parental response rate was 71% (120 completed questionnaires). For 10 of 16 statements, parents whose children had primary continuity intensivists and nurses indicated significantly more positive perceptions of care (e.g., communication, listening, decision making, problems due to changing providers). The provider response rate was 61% (117 completed questionnaires); more than 80% believed that primary intensivists and nurses were highly or very highly beneficial for LSPs. Providers perceived more benefits for patients/families (e.g., informational continuity, facilitating and expediting decision making) than for staff/institutions (e.g., staff satisfaction). Providers reported associated stress, expenditure of time and effort, and decreased staffing flexibility with primary practices. Conclusions: Perceived benefits of primary continuity intensivists and nurses by both parents and providers support more widespread adoption and study of these continuity strategies.

Background: For long-stay patients (LSP) in pediatric intensive care units (PICU), frequently rotating providers can lead to ineffective information sharing and retention, varying goals and timelines, and delayed decisions, likely contributing to prolonged admissions. Primary intensivists (one physician serves as a consistent resource for the patient/family and PICU providers) and primary nurses (a small team of PICU nurses provide consistent bedside care) seek to augment usual transitory PICU care, by enhancing continuity and, potentially, decreasing length of stay (LOS). Methods: A single-centered, partially blinded randomized controlled trial of primary intensivists and nurses versus usual care. PICU patients admitted for or expected to be admitted for >10 days and who had ≥1 complex chronic condition were eligible. A block randomization with 1:1 allocation was used. The primary outcome was PICU LOS. Multiple secondary outcomes were explored. Results: Two hundred LSPs were randomized-half to receive primaries and half to usual care. The two groups were not significantly different in their baseline and admission characteristics. LSPs randomized to receive primaries had a shorter, but non-significant, mean LOS than those randomized to usual care (32.5 vs. 37.1 days, respectively, p  = .19). Compared to LSPs in the usual care group, LSPs in the primary group had fewer unplanned intubations. Among LSPs that died, DNR orders were more prevalent in the primary group. Other secondary outcome and balance metrics were not significantly different between the two groups. Conclusion: Primary intensivists and nurses may be an effective strategy to counteract transitory PICU care and serve the distinctive needs of LSPs. However, additional studies are needed to determine the ways and to what extent they may accomplish this.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The Hantaviridae constitute a family of viruses harbored by mice, rats, shrews, voles, moles and bats. Intriguingly, only viruses harbored by mice and rats may cause disease in humans with up to 40% case fatality rate in the Americas. Transmission of virus from rodents to humans occurs via the respiratory route and results in replication of the virus in the microvascular endothelial cells of the lung or kidney. Understanding the replication kinetics of these viruses in various cell types and how replication is abrogated by the host is critical to the development of effective therapeutics for treatment for which there are none. We formulate several new ordinary differential equation (ODE) models to examine the replication kinetics of Prospect Hill orthohantavirus (PHV). The models are distinguished by the distribution of the viral replication delay. A new threshold, R GE , the genome equivalent replication number, is defined in terms of the model parameters. New final density relations are derived that associate R GE to the asymptotic number of virions in each model. All models are fit to real time (qRT)-PCR data of genomic RNA from PHV released from Vero E6 cells over 192 h. A sensitivity analysis of the parameters is performed and models are tested for best fit. Our findings provide a basis for future research into formulating more complex mathematical models for evaluation of the replication of hantaviruses in various cell types and sources., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Attrition of young adult registry members is a significant issue impacting hematopoietic stem cell (HSC) donation registries, including the Be The Match registry in the US. The resulting limited supply of allogeneic HSCs, used to treat serious health conditions, has a stronger impact on racial/ethnic minority groups in the US. Compared with young white adults, young adults identifying with these minority groups are more likely to drop out of the donor registry when called to donate. However, the underlying psychosocial factors that differ between white and nonwhite registrants have not been fully investigated. The central goal of this study was to examine demographic, registry-related, and donation-related characteristics in a young, newly registered group of potential donors and to determine whether these characteristics differed by, or were distributed differently among, racial/ethnic groups. We conducted a cross-sectional survey in a random sample of young (age 18 to 30 years) newly registered members, stratified by racial/ethnic group and sex. Demographic, registry-related (eg, context and motive for joining the registry), and donation-related (eg, ambivalence, religious objections to donation, knowledge about donation) characteristics were assessed. The chi-square test and analysis of variance were used to examine differences among racial/ethnic groups. Discriminant function analysis was used to assess whether patterns of the 3 classes of characteristics were associated with membership in particular racial/ethnic groups. A total of 524 participants were surveyed. Joining online was most common among white individuals, whereas joining at college was most common among black and Hispanic individuals. Ambivalence toward donation was higher among Asian/Pacific Islanders compared with white or multiracial/multiethnic individuals. Discriminant function analysis revealed 4 psychosocial/attitudinal functions predicting membership in certain racial/ethnic groups. The function accounting for the most variance in responses included mistrust of HSC allocation, religious objections to donation, low parental support, and low knowledge level. This function discriminated significantly between the white and nonwhite groups. Another function also identified ambivalence as a discriminating factor, which was most strongly associated with Asian/Pacific Islanders. Among young adult members of an HSC donor registry, such factors as ambivalence, family concerns about donation, mistrust of HSC allocation, religious objections, and less knowledge about donation were more strongly associated with membership in the nonwhite groups compared with the white group. These factors are known to be associated with a higher risk of opting out after having been preliminarily matched with a patient. The finding that these characteristics are associated with racial/ethnic minority group membership provides targets for recruitment strategies aimed at improving retention of young registry members., Competing Interests: Conflict of interest statement There are no conflicts of interest to report., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate infection with the SARS-CoV-2 USA-WA1/2020 strain increased the risk of pneumococcal (type 2 strain D39) coinfection in a time-dependent, but sex-independent, manner in the transgenic K18-hACE2 mouse model of COVID-19. Bacterial coinfection increased lethality when the bacteria was initiated at 5 or 7 d post-virus infection (pvi) but not at 3 d pvi. Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Smith, Williams, Plunkett, Selvaraj, Lane, Zalduondo, Xue, Vogel, Channappanavar, Jonsson and Smith.)

Objective: Pediatric inpatient bed availability is increasingly constrained by the prolonged hospitalizations of children with medical complexity. The sickest of these patients are chronic critically ill and often have protracted intensive care unit (ICU) stays. Numbers and characteristics of infants with chronic critical illness are unclear, which undermines resource planning in ICU's and general pediatric wards. The goal of this study was to describe infants with chronic critical illness at six academic institutions in the United States., Study Design: Infants admitted to six academic medical centers were screened for chronic, critical illness based on a combination of prolonged and repeated hospitalizations, use of medical technology, and chronic multiorgan involvement. Data regarding patient and hospitalization characteristics were collected., Results: Just over one-third (34.8%) of pediatric inpatients across the six centers who met eligibility criteria for chronic critical illness were <12 months of age. Almost all these infants received medical technology (97.8%) and had multiorgan involvement (94.8%). Eighty-six percent (115/134) had spent time in an ICU during the current hospitalization; 31% were currently in a neonatal ICU, 34% in a pediatric ICU, and 17% in a cardiac ICU. Among infants who had been previously discharged home ( n  = 55), most had been discharged with medical technology (78.2%) and nearly all were still using that technology during the current readmission. Additional technologies were commonly added during the current hospitalization., Conclusion: Advanced strategies are needed to plan for hospital resource allocation for infants with chronic critical illness. These infants' prolonged hospitalizations begin in the neonatal ICU but often transition to other ICUs and general inpatient wards. They are commonly discharged with medical technology which is rarely weaned but often escalated during subsequent hospitalizations. Identification and tracking of these infants, beginning in the neonatal ICU, will help hospitals anticipate and strategize for inpatient bed management., Key Points: · 35% of inpatients with chronic critical illness are infants.. · Nearly 90% of these infants spend some time in an intensive care unit.. · 78% are discharged with medical technology.., Competing Interests: None declared., (Thieme. All rights reserved.)

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus , the bank vole (45%); Apodemus flavicollis , the yellow-necked mouse (29%); and Apodemus agrarius , the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus , A. glareolus , and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis , one Mus musculus , and one Sorex minutus . We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus , 1 M. musculus , and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans?

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known., 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) 2021.)

The innate immune response is critical for recognizing and controlling infections through the release of cytokines and chemokines. However, severe pathology during some infections, including SARS-CoV-2, is driven by hyperactive cytokine release, or a cytokine storm. The innate sensors that activate production of proinflammatory cytokines and chemokines during COVID-19 remain poorly characterized. In the present study, we show that both TLR2 and MYD88 expression were associated with COVID-19 disease severity. Mechanistically, TLR2 and Myd88 were required for β-coronavirus-induced inflammatory responses, and TLR2-dependent signaling induced the production of proinflammatory cytokines during coronavirus infection independent of viral entry. TLR2 sensed the SARS-CoV-2 envelope protein as its ligand. In addition, blocking TLR2 signaling in vivo provided protection against the pathogenesis of SARS-CoV-2 infection. Overall, our study provides a critical understanding of the molecular mechanism of β-coronavirus sensing and inflammatory cytokine production, which opens new avenues for therapeutic strategies to counteract the ongoing COVID-19 pandemic.

Beamline I22 at Diamond Light Source is dedicated to the study of soft-matter systems from both biological and materials science. The beamline can operate in the range 3.7 keV to 22 keV for transmission SAXS and 14 keV to 20 keV for microfocus SAXS with beam sizes of 240 µm × 60 µm [full width half-maximum (FWHM) horizontal (H) × vertical (V)] at the sample for the main beamline, and approximately 10 µm × 10 µm for the dedicated microfocusing platform. There is a versatile sample platform for accommodating a range of facilities and user-developed sample environments. The high brilliance of the insertion device source on I22 allows structural investigation of materials under extreme environments (for example, fluid flow at high pressures and temperatures). I22 provides reliable access to millisecond data acquisition timescales, essential to understanding kinetic processes such as protein folding or structural evolution in polymers and colloids., (open access.)

Understanding the ecology of rodent-borne hantaviruses is critical to assessing the risk of spillover to humans. Longitudinal surveys have suggested that hantaviral prevalence in a given host population is tightly linked to rodent ecology and correlates with changes in the species composition of a rodent community over time and/or habitat composition. We tested two hypotheses to identify whether resource addition and/or habitat composition may affect hantavirus prevalence among two sympatric reservoir hosts in a neotropical forest: (i) increased food resources will alter the rodent community and thus hantaviral prevalence; and (ii) host abundance and viral seroprevalence will be associated with habitat composition. We established a baseline of rodent-virus prevalence in three grid pairs of distinct habitat compositions and subjected one grid of each pair to resource augmentation. Increased rodent species diversity was observed on grids where food was added versus untreated control grids during the first post-treatment sampling session. Resource augmentation changed species community composition, yet it did not affect the prevalence of hantavirus in the host population over time, nor was there evidence of a dilution effect. Secondly, we show that the prevalence of the virus in the respective reservoir hosts was associated with habitat composition at two spatial levels, independent of resource addition, supporting previous findings that habitat composition is a primary driver of the prevalence of hantaviruses in the neotropics.

COVID-19 is characterized by excessive production of pro-inflammatory cytokines and acute lung damage associated with patient mortality. While multiple inflammatory cytokines are produced by innate immune cells during SARS-CoV-2 infection, we found that only the combination of TNF-α and IFN-γ induced inflammatory cell death characterized by inflammatory cell death, PANoptosis. Mechanistically, TNF-α and IFN-γ co-treatment activated the JAK/STAT1/IRF1 axis, inducing nitric oxide production and driving caspase-8/FADD-mediated PANoptosis. TNF-α and IFN-γ caused a lethal cytokine shock in mice that mirrors the tissue damage and inflammation of COVID-19, and inhibiting PANoptosis protected mice from this pathology and death. Furthermore, treating with neutralizing antibodies against TNF-α and IFN-γ protected mice from mortality during SARS-CoV-2 infection, sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other infectious and autoinflammatory diseases by limiting tissue damage/inflammation., Competing Interests: Declaration of Interests St. Jude Children’s Research hospital filed a provisional patent application on TNF-α and IFN-γ signaling described in this study, listing R.K. and T.-D.K. as inventors (serial no. 63/106,012)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF-α and IFN-γ specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the JAK/STAT1/IRF1 axis activated by TNF-α and IFN-γ co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF-α and IFN-γ-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF-α and IFN-γ in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF-α and IFN-γ synergism play key pathological roles., Competing Interests: DECLARATION OF INTERESTS St. Jude Children’s Research hospital filed a provisional patent application on TNF-α and IFN-γ signaling described in this study, listing R.K. and T.-D.K. as inventors (Serial No. 63/106,012).

Hantaviruses rewire the host cell and induce extensive membrane rearrangements for their replication and the morphogenesis of the virion. Transmission electron microscopy (TEM) is a powerful technique for imaging these pathological membrane changes especially when combined with large volume electron tomography. Excellent preservation of membrane structure can be obtained when chemical fixation is combined with cryofixation via high pressure freezing making the samples amenable to serial-section tomographic reconstruction. Taking advantage of this, we have optimized a hybrid method that employs aldehyde fixation, a step that is essential for virus inactivation, followed by high-pressure freezing for ultrastructural study of Hantaan (HTN) and Andes (AND) virus infected Vero E6 cells. HTNV and ANDV are two species of the Orthohantavirus , from the Old and New World, respectively, and the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in humans. We applied the method for the qualitative assessment of the perturbation of the endomembrane system induced by HTNV and ANDV in infected vs. mock-infected cells. Screening of serial-sections revealed consistency of membrane preservation across large volumes indicating potential of these samples for tomographic studies. Images revealed large-scale perturbations of the endomembrane system following HTNV-infection that included the dilation of the rough endoplasmic reticulum and fragmentation of the Golgi apparatus. Infected cells exhibited a tendency to accumulate large numbers of vacuoles that were especially apparent in ANDV. In summary, our hybrid method provides a path for the study of BSL-3 pathogens using cutting edge 3D-imaging technologies., (Copyright © 2020 Parvate, Sengupta, Williams, Xue, Chu, Stahelin and Jonsson.)

CHD remains one of the leading causes of mortality of children in the United States. There is limited research about the experience of parents from the diagnosis of their child with CHD through the death of their child. A prior study has shown that adults with heart failure go through a series of four transitions: 1) learning the diagnosis, 2) reframing the new normal, 3) taking control of the illness, and 4) understanding death is inevitable. In our qualitative study, we performed semi-structured interviews with parents who have a child die of CHD to determine whether the four transitions in adults apply to parents of children with CHD. We found that these four transitions were present in the parents we interviewed and that there were two novel transitions, one that proceeded the first Jones et al transition ("Prenatal diagnosis") and one that occurred after the final Jones et al transition ("Adjustment after death"). It is our hope that identification of these six transitions will help better support families of children with CHD.

In Ukraine, a retrospective review of clinical case reports by public health officials suggest that human cases of febrile illnesses associated with hemorrhage may be due to infections of Crimean-Congo hemorrhagic fever virus (CCHFV) and Old World hantaviruses. In a serosurvey of 966 healthy individuals in the Lviv Oblast, Ukraine, bordering Poland, we found that 1.6% showed cross-reactivity to hantaviral antigens by an immunofluorescence assay (IFA) and 1.7% of the study participants had antibodies cross-reactive to CCHFV by enzyme-linked immunosorbent assay (ELISA). Demographic variables and history of exposures obtained through questionnaires were assessed by logistic regression models for association with seroprevalence for both viruses with no significant risk factors found. Analysis of spatial distribution identified two clusters of samples positive for antibodies to both hantaviruses and CCHFV, which, however, were not statistically significant (p > 0.05). In general, the study results suggest that the population of the study area is exposed to hantaviruses and CCHFV. Further surveillance for respective pathogens in Ukraine is warranted and prospective surveillance of febrile patients with unidentified febrile illness., (Copyright © 2020 Lozynskyi, Shulgan, Zarichna, Ben, Kessler, Cao, Nesterova, Glass, Spruill-Harrell, Taylor, Williams and Jonsson.)

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99-100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10 -5 -10 -7 per genome and 10 -4 -10 -5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein., (Copyright © 2020 Taylor, Williams, Wongsurawat, Jenjaroenpun, Nookaew and Jonsson.)

Coronaviruses have caused several zoonotic infections in the past two decades, leading to significant morbidity and mortality globally. Balanced regulation of cell death and inflammatory immune responses is essential to promote protection against coronavirus infection; however, the underlying mechanisms that control these processes remain to be resolved. Here we demonstrate that infection with the murine coronavirus mouse hepatitis virus (MHV) activated the NLRP3 inflammasome and inflammatory cell death in the form of PANoptosis. Deleting NLRP3 inflammasome components or the downstream cell death executioner gasdermin D (GSDMD) led to an initial reduction in cell death followed by a robust increase in the incidence of caspase-8- and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated inflammatory cell deathafter coronavirus infection. Additionally, loss of GSDMD promoted robust NLRP3 inflammasome activation. Moreover, the amounts of some cytokines released during coronavirus infection were significantly altered in the absence of GSDMD. Altogether, our findings show that inflammatory cell death, PANoptosis, is induced by coronavirus infection and that impaired NLRP3 inflammasome function or pyroptosis can lead to negative consequences for the host. These findings may have important implications for studies of coronavirus-induced disease., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Zheng et al.)

This was the first study to examine the experience of parents who discover their child was living with anorexia nervosa (AN), thus fulfilling a critical gap in the eating disorder literature. Gadamerian hermeneutic inquiry was the guiding philosophy and method used to investigate this topic. Dialogues with parents revealed the ambiguity inherent within discovery; the isolation, betrayal, and loss felt by parents; and the complicated family dynamics occurring during the process of discovering one's child has AN. As such, when discoveries are made, parents play a vital role in the development and functioning of the family's response to the situation. This research offers health care providers a better understanding of the difficult times parents and caregivers experience when discovering their child has AN.

There are currently no effective chemotherapeutic drugs approved for the treatment of diffuse intrinsic pontine glioma (DIPG), an aggressive pediatric cancer resident in the pons region of the brainstem. Radiation therapy is beneficial but not curative, with the condition being uniformly fatal. Analysis of the genomic landscape surrounding DIPG has revealed that activin receptor-like kinase-2 (ALK2) constitutes a potential target for therapeutic intervention given its dysregulation in the disease. We adopted an open science approach to develop a series of potent, selective, orally bioavailable, and brain-penetrant ALK2 inhibitors based on the lead compound LDN-214117 . Modest structural changes to the C-3, C-4, and C-5 position substituents of the core pyridine ring afforded compounds M4K2009 , M4K2117 , and M4K2163 , each with a superior potency, selectivity, and/or blood-brain barrier (BBB) penetration profile. Robust in vivo pharmacokinetic (PK) properties and tolerability mark these inhibitors as advanced preclinical compounds suitable for further development and evaluation in orthotopic models of DIPG.

The COVID-19 pandemic swept through New York City swiftly and with devastating effect. The crisis put enormous pressure on all hospital services, including the clinical ethics consultation team. This report describes the recent experience of the ethics consultants and Columbia University Irving Medical Center during the COVID-19 surge and compares the case load and characteristics to the corresponding period in 2019. By reporting this experience, we hope to supplement the growing body of COVID-19 scientific literature and provide details of the human toll the virus took on our hospitals and communities. We also aim to highlight the role of the clinical ethics consultant as well as areas of policy and law that may need to be addressed in order to be better prepared for a future public health crisis., (Copyright 2020 The Journal of Clinical Ethics. All rights reserved.)

Individuals living or working in moldy buildings complain of a variety of health problems including pain, fatigue, increased anxiety, depression, and cognitive deficits. The ability of mold to cause such symptoms is controversial since no published research has examined the effects of controlled mold exposure on brain function or proposed a plausible mechanism of action. Patient symptoms following mold exposure are indistinguishable from those caused by innate immune activation following bacterial or viral exposure. We tested the hypothesis that repeated, quantified doses of both toxic and nontoxic mold stimuli would cause innate immune activation with concomitant neural effects and cognitive, emotional, and behavioral symptoms. We intranasally administered either 1) intact, toxic Stachybotrys spores; 2) extracted, nontoxic Stachybotrys spores; or 3) saline vehicle to mice. As predicted, intact spores increased interleukin-1β immunoreactivity in the hippocampus. Both spore types decreased neurogenesis and caused striking contextual memory deficits in young mice, while decreasing pain thresholds and enhancing auditory-cued memory in older mice. Nontoxic spores also increased anxiety-like behavior. Levels of hippocampal immune activation correlated with decreased neurogenesis, contextual memory deficits, and/or enhanced auditory-cued fear memory. Innate-immune activation may explain how both toxic mold and nontoxic mold skeletal elements caused cognitive and emotional dysfunction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rodent-borne hantaviruses have been reported in many of the countries surrounding Ukraine; however, to date we have no knowledge of the viral strains circulating in Ukraine within reservoirs such as the striped field mouse ( Apodemus agrarius ), the yellow-necked field mouse ( Apodemus flavicollis ), and the bank vole ( Myodes glareolus ). To determine the prevalence of hantaviruses in Ukraine, we captured 1,261 mammals, of which 1,109 were rodents, in 58 field sites within the province of Volyn in western Ukraine. Foci of the striped field mouse tended to occur in the eastern and southern parts of the province, whereas the bank vole were clustered in western and northern regions. The striped field mouse and bank vole had detectable serum antibodies to Puumala virus (PUUV) or Dobrava virus (DOBV) antigens at 7% or 2%, respectively, using an indirect immunofluorescence assay. Antibody prevalence among the bank vole males and females was equivalent, whereas for the striped field mouse, the prevalence among males was 5% versus 1% for females. In two bank vole specimens, we were able to detect partial nucleotide sequences that showed identity to PUUV. In summary, this study suggests that two human pathogens, PUUV and DOBV, cocirculate in the bank vole and the striped field mouse, respectively, in Ukraine. Future studies will focus on new rodent collections that will enable obtaining the complete genome sequences of the PUUV and DOBV strains circulating in Ukraine to provide guidance on the design of optimal molecular diagnostics that can enable insight into the potential contribution of hantaviruses to human disease in Ukraine.

Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009 , an analogue of the previously reported ALK2 inhibitor LDN-214117 . Although highly selective for ALK2 over the TGF-βR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives ( 26a-c ), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

Objective: To explore how medical decision-making for children with medical complexity (CMC) occurs in the context of foster care (FC)., Methods: Together with a medical FC agency, we identified 15 CMC in medical FC and recruited eligible care team members (biological and foster parents, medical FC nurses, caseworkers in medical FC/child welfare, and pediatricians) for each child. Semistructured interviews were conducted, and conventional content analysis was applied to transcripts., Results: Fifty-eight interviews were completed with 2-5 care team members/child. Serious decision-making related to surgeries and medical technology was common. Themes regarding medical decision-making for CMC in FC emerged: 1) Protocol: decision-making authority is dictated by court order and seriousness of decision, 2) Process: decision-making is dispersed among many team members, 3) Representing the child's interests: the majority of respondents stated that the foster parent represents the child's best interests, while the child welfare agency should have legal decision-making authority, and 4) Perceived barriers: serious medical decision-making authority is often given to individuals who spend little time with the child., Conclusions: Medical decisions for CMC can have uncertain risk/benefit ratios. For CMC in FC, many individuals have roles in these nuanced decisions; those with ultimate decision-making authority may have minimal interaction with the child. Pediatricians can assist by clarifying who has legal decision-making authority, facilitating team communication to promote truly informed consent, and serving as a resource to decision-makers. Further research should explore how to adapt the traditional model of shared decision-making to meet the needs of this population., (Copyright © 2019 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

To further understanding of the structure and morphology of the Orthohantavirus , family Hantaviridae , we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus .

Background: Medical foster care (MFC) offers a family-home setting for children with medical complexity (CMC) who cannot be cared for by their parents. We explored MFC as a placement option for CMC via in-depth interviews with the individuals providing and monitoring care., Methods: In collaboration with an MFC agency, we recruited care team members for 15 CMC. Semistructured interviews were audiotaped and transcribed. Descriptive analyses were performed on care team composition and roles, the placement process, and child medical, placement, and quality of life (QoL) characteristics. Foster parents completed child QoL questionnaires. Conventional content analysis was applied to participant suggestions for MFC improvements., Results: Fifty-eight interviews were conducted; a median of 4 care team members interviewed per child. An extensive network of individuals and systems are involved in providing care. Each child received care from multiple subspecialists (median = 5). Most children were technology dependent (87%), developmentally delayed (87%), and entered MFC from the hospital (73%) because of medical neglect (86%). Nearly half were in care for >2 years. Changes in placement and/or care team were common. QoL scoring showed impairments in multiple domains, whereas respondent interviews described positive aspects of QoL. Participants provided suggestions to improve care within MFC., Conclusions: MFC is a promising placement option for CMC. Because many CMC are entering MFC directly from the hospital and require ongoing care from pediatric subspecialists, pediatricians should be familiar with MFC, the placement process, and the various systems and individuals involved. Pediatricians can play important roles in ensuring that children in MFC receive coordinated and high-quality care., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2019 by the American Academy of Pediatrics.)

The treatment of atypical anorexia nervosa (AN) poses new research and practice challenges for the field of eating disorders. The objective of this study was to describe frontline practitioners' perceptions of differences between adolescents living with atypical versus typical AN, as well as the intervention challenges they experience when working with these adolescents and their families. We followed the principles of fundamental qualitative description and recruited a purposeful sample of practitioners treating adolescent eating disorders to complete a one-on-one semi-structured interview. Conventional content analysis and the constant comparison technique were used for data analysis. A total of 23 practitioners from four countries participated in this study. Practitioners described that adolescents with atypical AN present with higher pre-morbid weights and rates of weight-based teasing compared to their AN peers. Clinical challenges perceived by practitioners to be specific to working with adolescents with atypical AN included: addressing conflicting messages about eating disorders and weight loss, empathizing with a justified fear of weight gain, and increased risk for parental and therapist collusion with the eating disorder. Findings have implications for delivering interventions to adolescents seeking care for atypical AN.

Background: Atypical anorexia nervosa (AN) has received minimal empirical attention regarding effective diagnosis and treatment. Family-based treatment (FBT) might be a promising treatment for atypical AN, yet it is unclear as to what adaptations are needed to the current manualized FBT for AN model. The objective of the current study was to identify how FBT practitioners applied FBT for atypical AN for adolescents in their clinical practice, and if there were any implementation challenges and adaptations to the model for this population., Methods: The current study employed fundamental qualitative description, with the aim of capturing practitioners' reflections on working with adolescents with atypical AN in clinical practice. A purposeful sample of practitioners with training in FBT were recruited and each participant completed an individual, semi-structured interview. Data was analyzed using conventional content analysis., Results: A total of 23 practitioners participated in this study. The results indicate that practitioners maintained some fidelity to manualized FBT in treating atypical AN, but they differed in their discussions around target weights, what constitutes weight restoration, and the dosage for FBT phases. Salient practice challenges included operationalizing the Diagnostic and Statistical Manual of Mental Disorders - 5th Edition (DSM-5) definition of atypical AN, identifying a 'goal weight' for adolescents and activating parents to take charge of the re-nourishment process., Conclusions: The results of this qualitative study demonstrate practitioner reflections on the delivery and adaptations of FBT for adolescents with atypical AN. These reflections highlight the need to establish the delivery of coherent and consistent treatment and messaging with patients and families. Further, practitioners' reflections highlight common strategies to increase the sense of urgency in parents to support their child with atypical AN., Competing Interests: The current study was approved by the University of Calgary Institutional Ethics Board.Dr. Le Grange is the Co-Director of the Training Institute for Child and Adolescent Eating Disorders, LLC, and receives royalties from Routledge and Guilford Press. Dr. Loeb receives royalties from Routledge, and is a faculty member of and consultant for the Training Institute for Child and Adolescent Eating Disorders.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.