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CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance., Competing Interests: Disclosure Statement J.A.-S., P.C.S., and C.M. are inventors on a pending patent application held by the Broad Institute (international patent application number PCT/US2021/049145), which covers the SHINE technology. P.C.S. is a cofounder of, shareholder in, and consultant to Sherlock Biosciences, Inc., and Delve Bio, as well as a board member of and shareholder in Danaher Corp. C.M. is a cofounder of Carver Biosciences, a startup company developing Cas13-based antivirals, and holds equity in Carver Biosciences., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Respiratory syncytial virus (RSV) circulation dropped markedly early in the COVID-19 pandemic, followed by a resurgence with heightened case counts. The "immunity debt" hypothesis proposes that the RSV-naїve pediatric population increased during the period of low transmission. However, the evidence supporting this hypothesis is limited, and the role of changing testing practices in the perceived surge has not been comprehensively evaluated., Methods: We conducted a multicenter, retrospective analysis of 342 530 RSV encounters and 980 546 RSV diagnostic tests occurring at 32 US pediatric hospitals in 2013-2023. We used interrupted time series analysis to estimate pandemic-associated changes in RSV patient and test volume and to quantify changes in the proportions of patients requiring hospitalization, intensive care, or mechanical ventilation. We quantified the fraction of the shifts in case counts and in the age of diagnosed patients attributable to changes in testing., Results: RSV patient volume increased 2.4-fold (95% confidence interval [CI]: 1.7, 3.5) in 2021-2023 relative to the pre-pandemic phase and was accompanied by an 18.9-fold increase (95% CI: 15.0, 23.9) in RSV test volume. Shifts in patient volume and in patient age were largely attributable to increased testing. The proportions of patients with RSV that required hospitalization, intensive care, or mechanical ventilation declined significantly across all patient age groups., Conclusions: A surge in RSV testing, rather than in viral circulation, likely underlies the increased case counts observed in 2021-2023. These findings warrant a critical assessment of the immunity debt hypothesis and highlight the importance of considering the testing denominator when surveillance strategies are dynamic., Competing Interests: Potential conflicts of interest. P. C. S. is a cofounder of, shareholder in, and advisor to Sherlock Biosciences, Inc; a board member of and shareholder in the Danaher Corporation; a cofounder of and shareholder in Delve Bio; and a shareholder in NextGenJane and TruGenomix. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Sequencing of SARS-CoV-2 from rapid diagnostic tests (RDTs) can bolster viral genomic surveillance efforts; however, approaches to maximise and standardise pathogen genome recovery from RDTs remain underdeveloped. We aimed to systematically optimise the elution of genetic material from RDT components and to evaluate the efficacy of RDT sequencing for outbreak investigation., Methods: In this laboratory and cohort-based study we seeded RDTs with inactivated SARS-CoV-2 to optimise the elution of genomic material from RDT lateral flow strips. We measured the effect of changes in buffer type, time in buffer, and rotation on PCR cycle threshold (Ct) value. We recruited individuals older than 18 years residing in the greater Boston area, MA, USA, from July 18 to Nov 5, 2022, via email advertising to students and staff at Harvard University, MA, USA, and via broad social media advertising. All individuals recruited were within 5 days of a positive diagnostic test for SARS-CoV-2; no other relevant exclusion criteria were applied. Each individual completed two RDTs and one PCR swab. On Dec 29, 2022, we also collected RDTs from a convenience sample of individuals who were positive for SARS-CoV-2 and associated with an outbreak at a senior housing facility in MA, USA. We extracted all returned PCR swabs and RDT components (ie, swab, strip, or buffer); samples with a Ct of less than 40 were subject to amplicon sequencing. We compared the efficacy of elution and sequencing across RDT brands and components and used RDT-derived sequences to infer transmission links within the outbreak at the senior housing facility. We conducted metagenomic sequencing of negative RDTs from symptomatic individuals living in the senior housing facility., Findings: Neither elution duration of greater than 10 min nor rotation during elution impacted viral titres. Elution in Buffer AVL (Ct=31·4) and Tris-EDTA Buffer (Ct=30·8) were equivalent (p=0·34); AVL outperformed elution in lysis buffer and 50% lysis buffer (Ct=40·0, p=0·0029 for both) as well as Universal Viral Transport Medium (Ct=36·7, p=0·079). Performance of RDT strips was poorer than that of matched PCR swabs (mean Ct difference 10·2 [SD 4·3], p<0·0001); however, RDT swabs performed similarly to PCR swabs (mean Ct difference 4·1 [5·2], p=0·055). No RDT brand significantly outperformed another. Across sample types, viral load predicted the viral genome assembly length. We assembled greater than 80% complete genomes from 12 of 17 RDT-derived swabs, three of 18 strips, and four of 11 residual buffers. We generated outbreak-associated SARS-CoV-2 genomes using both amplicon and metagenomic sequencing and identified multiple introductions of the virus that resulted in downstream transmission., Interpretation: RDT-derived swabs are a reasonable alternative to PCR swabs for viral genomic surveillance and outbreak investigation. RDT-derived lateral flow strips yield accurate, but significantly fewer, viral reads than matched PCR swabs. Metagenomic sequencing of negative RDTs can identify viruses that might underlie patient symptoms., Funding: The National Science Foundation, the Hertz Foundation, the National Institute of General Medical Sciences, Harvard Medical School, the Howard Hughes Medical Institute, the US Centers for Disease Control and Prevention, the Broad Institute and the National Institute of Allergy and Infectious Diseases., Competing Interests: Declaration of interests PCS is a cofounder of, shareholder in, and advisor to Sherlock Biosciences; a board member of and shareholder in the Danaher Corporation; a cofounder of and shareholder in Delve Bio; and a shareholder in NextGenJane and TruGenomix. MS is a cofounder of, shareholder in, and advisor to Rhinostics; a shareholder of and advisor to Nexus Laboratories; and a consultant for Vectis Consulting. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85 megabases of the genome. Using 332 functionally confirmed CRE-gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome., (© 2024. The Author(s).)

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat ( Rousettus aegyptiacus ) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis., (© 2024. The Author(s).)

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata in a cross-sectional study of febrile patients and healthy controls in a low malaria burden area. Using 16S and untargeted sequencing, we detected viral, bacterial, or eukaryotic pathogens in 23% (38/163) of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15.5% and 3.8% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model that can distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs (F1 score: 0.823). These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance., (© 2024. The Author(s).)

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4 + T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4 + and CD8 + T cell epitopes to maximize vaccine effectiveness., Competing Interests: Declaration of interests S.W.-G., D.-Y.C., S.S., K.R.C., N.H., S.A.C., J.G.A., M.S., and P.C.S. are named co-inventors on a patent application related to this work, filed by The Broad Institute, that is being made available in accordance with the COVID-19 technology licensing framework to maximize access to university innovations. N.H. is a founder of Neon Therapeutics (now BioNTech US), was a member of its scientific advisory board, and holds shares. N.H. is also an advisor for IFM Therapeutics. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer, and PrognomIQ. J.G.A. is a past employee of Neon Therapeutics (now BioNTech US). P.C.S. is a cofounder of and consultant to Sherlock Biosciences and Delve Biosciences and a board member of Danaher Corporation and holds equity in the companies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

While genome-wide association studies (GWAS) and positive selection scans identify genomic loci driving human phenotypic diversity, functional validation is required to discover the variant(s) responsible. We dissected the IVD gene locus-which encodes the isovaleryl-CoA dehydrogenase enzyme-implicated by selection statistics, multiple GWAS, and clinical genetics as important to function and fitness. We combined luciferase assays, CRISPR/Cas9 genome-editing, massively parallel reporter assays (MPRA), and a deletion tiling MPRA strategy across regulatory loci. We identified three regulatory variants, including an indel, that may underpin GWAS signals for pulmonary fibrosis and testosterone, and that are linked on a positively selected haplotype in the Japanese population. These regulatory variants exhibit synergistic and opposing effects on IVD expression experimentally. Alleles at these variants lie on a haplotype tagged by the variant most strongly associated with IVD expression and metabolites, but with no functional evidence itself. This work demonstrates how comprehensive functional investigation and multiple technologies are needed to discover the true genetic drivers of phenotypic diversity., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ebola virus (EBOV) causes Ebola virus disease (EVD), marked by severe hemorrhagic fever; however, the mechanisms underlying the disease remain unclear. To assess the molecular basis of EVD across time, we performed RNA sequencing on 17 tissues from a natural history study of 21 rhesus monkeys, developing new methods to characterize host-pathogen dynamics. We identified alterations in host gene expression with previously unknown tissue-specific changes, including downregulation of genes related to tissue connectivity. EBOV was widely disseminated throughout the body; using a new, broadly applicable deconvolution method, we found that viral load correlated with increased monocyte presence. Patterns of viral variation between tissues differentiated primary infections from compartmentalized infections, and several variants impacted viral fitness in a EBOV/Kikwit minigenome system, suggesting that functionally significant variants can emerge during early infection. This comprehensive portrait of host-pathogen dynamics in EVD illuminates new features of pathogenesis and establishes resources to study other emerging pathogens., Competing Interests: P.C.S. is a co-founder and shareholder of Sherlock Biosciences and Delve Bio, a board member and shareholder of Danaher Corporation, and has filed IP related to genomic sequencing and diagnostic technologies. A.K.S. reports compensation for consulting and/or scientific advisory board (SAB) membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, Third Rock Ventures, Hovione, Relation Therapeutics, FL82, FL86, Empress Therapeutics, IntrECate Biotherapeutics, Senda Biosciences, and Dahlia Biosciences unrelated to this work. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Aera Therapeutics. F.Z. is a scientific advisor for Octant., (© 2023 The Authors.)

Neisseria gonorrhoeae is one of the most common bacterial sexually transmitted infections. The emergence of antimicrobial-resistant N. gonorrhoeae is an urgent public health threat. Currently, the diagnosis of N. gonorrhoeae infection requires expensive laboratory infrastructure, while antimicrobial susceptibility determination requires bacterial culture, both of which are infeasible in low-resource areas where the prevalence of infection is highest. Recent advances in molecular diagnostics, such as s pecific h igh-sensitivity e nzymatic r eporter un lock ing (SHERLOCK) using CRISPR-Cas13a and isothermal amplification, have the potential to provide low-cost detection of pathogen and antimicrobial resistance. We designed and optimized RNA guides and primer sets for SHERLOCK assays capable of detecting N. gonorrhoeae via the por A gene and of predicting ciprofloxacin susceptibility via a single mutation in the gyrase A ( gyr A) gene. We evaluated their performance using both synthetic DNA and purified N. gonorrhoeae isolates. For por A , we created both a fluorescence-based assay and lateral flow assay using a biotinylated fluorescein reporter. Both methods demonstrated sensitive detection of 14 N . gonorrhoeae isolates and no cross-reactivity with 3 non-gonococcal Neisseria isolates. For gyr A, we created a fluorescence-based assay that correctly distinguished between 20 purified N. gonorrhoeae isolates with phenotypic ciprofloxacin resistance and 3 with phenotypic susceptibility. We confirmed the gyr A genotype predictions from the fluorescence-based assay with DNA sequencing, which showed 100% concordance for the isolates studied. We report the development of Cas13a-based SHERLOCK assays that detect N. gonorrhoeae and differentiate ciprofloxacin-resistant isolates from ciprofloxacin-susceptible isolates. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, disproportionately affects resource-limited settings. Such areas, however, lack the technical capabilities for diagnosing the infection. The consequences of poor or absent diagnostics include increased disease morbidity, which, for gonorrhea, includes an increased risk for HIV infection, infertility, and neonatal blindness, as well as an overuse of antibiotics that contributes to the emergence of antibiotic resistance. We used a novel CRISPR-based technology to develop a rapid test that does not require laboratory infrastructure for both diagnosing gonorrhea and predicting whether ciprofloxacin can be used in its treatment, a one-time oral pill. With further development, that diagnostic test may be of use in low-resource settings., Competing Interests: P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, as well as a board member of and shareholder in Danaher Corporation. J.E.L. previously served as a consultant to SHERLOCK Biosciences. J.B. has received research funding from Analog Devices, Inc., Zeus Scientific, Immunetics, Pfizer, DiaSorin, and bioMerieux and has been a paid consultant to T2 Biosystems, DiaSorin, and Roche Diagnostics. The remaining authors have nothing to disclose.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens.  • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens.  • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines.  • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing.  • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions.  • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV., Competing Interests: SJL has received research funding paid to her institution from Gilead Sciences. VC has received travel grants, advisor honorarium and research grant from Merck Sharp & Dohme, ViiV Healthcare and Gilead Sciences. FCS has been a consultant to ViiV Healthcare, Gilead Sciences and Merck Sharp & Dohme, and received research grants paid to her institution from Gilead Sciences. CC has received honoraria and conference travels grants from Merck Sharp & Dohme, Gilead Sciences, and ViiV Healthcare. DD has received honoraria for participation in advisory boards and conference travel grants ViiV Healthcare, Gilead Sciences, Janssen Pharmaceuticals, and Merck Sharp & Dohme. LF has received NIH research grants paid to her institution. RKG has received honoraria for participation on advisory boards from Gilead-Sciences and GlaxoSmithKline. RDK has received research grants from Gilead Sciences paid to his institution. DRK is a consultant to and has received honoraria from AbbVie, Gilead Sciences, GlaxoSmithKline, Janssen Pharmaceuticals, Merck, Roche, and ViiV Healthcare. DRK has also received honoraria from Gilead for expert testimony and speaking fees from Gilead Sciences and Janssen Pharmaceuticals and has received research support paid to his institution from Gilead Sciences, Merck, and ViiV Healthcare. AGM received travel grants, honoraria and study grants from Gilead Sciences, Merck Sharp & Dohme, ViiV Healthcare, GlaxoSmithKline, Roche, and Astra Zeneca. RP has received research grants paid to his institution from Merck Sharp & Dohme and ViiV Healthcare and consulting fees from Gilead Sciences, Merck Sharp & Dohme, GlaxoSmithKline, Atea Pharmaceuticals, Roche, and Shinogi Pharmaceuticals. PCS is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, and a board member of and shareholder in Danaher Corporation. JMS has received research support, honorarium, or consulting fees from the following: Abbvie, Merck, Gilead Sciences, GlaxoSmithKline, Tibotec-Janssen, Teva, Virology Education and ViiV Healthcare. He has received travel support and stipends for advisory work for the World Health Organization. WS has received speaking honoraria from GlaxoSmithKline, ViiV Healthcare, Merck Sharp & Dohme, Pfizer, and Abbott Pharmaceuticals. AMJW has received research support paid to her institution by Gilead Sciences and has consulted for Gilead Sciences and ViiV Healthcare. JOW receives funding from grants and contracts to his institution from NIH and CDC pertaining to work on HIV molecular epidemiology. HFG has received grants paid to his institution from Gilead Sciences, and Roche, and has received consulting fees from Merck, Gilead Sciences, ViiV Healthcare, Janssen Pharmaceuticals, GlaxoSmithKline, Johnson and Johnson, and Novartis. RWS has received honoraria for participation in advisory boards from Gilead Sciences and GlaxoSmithKline and speaking honoraria from Gilead Sciences and ViiV Healthcare., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection., Competing Interests: P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, as well as a board member of and shareholder in Danaher Corporation. K.S. served as a consultant for T2 Biosystems, Roche, BioMerieux, and NYS Biodefense Fund, for the development of a diagnostic assay in Lyme borreliosis, and is currently employed at Takeda. F.S. served on the scientific advisory board for Roche on Lyme disease serological diagnostics and on the scientific advisory board for Pfizer on Lyme disease vaccine, and is an unpaid member of the steering committee of the ESCMID Study Group on Lyme Borreliosis/ESGBOR. J.A.B. has received research funding from Analog Devices Inc., Zeus Scientific, Immunetics, Pfizer, DiaSorin and bioMerieux, and has been a paid consultant to T2 Biosystems, DiaSorin, and Roche Diagnostics. G.P.W. reports receiving research grants from Institute for Systems Biology, Biopeptides, Corp., and Pfizer, Inc. He has been an expert witness in malpractice cases involving Lyme disease and babesiosis; and is an unpaid board member of the non-profit American Lyme Disease Foundation. J.E.L previously served as a consultant to Sherlock Biosciences., (Copyright: © 2023 Lemieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Effective infectious disease surveillance in high-risk regions is critical for clinical care and pandemic preemption; however, few clinical diagnostics are available for the wide range of potential human pathogens. Here, we conduct unbiased metagenomic sequencing of 593 samples from febrile Nigerian patients collected in three settings: i) population-level surveillance of individuals presenting with symptoms consistent with Lassa Fever (LF); ii) real-time investigations of outbreaks with suspected infectious etiologies; and iii) undiagnosed clinically challenging cases. We identify 13 distinct viruses, including the second and third documented cases of human blood-associated dicistrovirus, and a highly divergent, unclassified dicistrovirus that we name human blood-associated dicistrovirus 2. We show that pegivirus C is a common co-infection in individuals with LF and is associated with lower Lassa viral loads and favorable outcomes. We help uncover the causes of three outbreaks as yellow fever virus, monkeypox virus, and a noninfectious cause, the latter ultimately determined to be pesticide poisoning. We demonstrate that a local, Nigerian-driven metagenomics response to complex public health scenarios generates accurate, real-time differential diagnoses, yielding insights that inform policy., (© 2023. Springer Nature Limited.)

Viral genomic surveillance has been integral in the global response to the SARS-CoV-2 pandemic. Surveillance efforts rely on the availability of representative clinical specimens from ongoing testing activities. However, testing practices have recently shifted due to the widespread availability and use of rapid antigen tests, which could lead to gaps in future monitoring efforts. As such, genomic surveillance strategies must adapt to include laboratory workflows that are robust to sample type. To that end, we compare the results of RT-qPCR and viral genome sequencing using samples from positive BinaxNOW COVID-19 Antigen Card swabs (N = 555) to those obtained from nasopharyngeal (NP) swabs used for nucleic acid amplification testing (N = 135). We show that swabs obtained from antigen cards are comparable in performance to samples from NP swabs, providing a viable alternative and allowing for the potential expansion of viral genomic surveillance to outpatient clinic as well as other settings where rapid antigen tests are often used., (© 2023. The Author(s).)

Synthetic biomarkers, bioengineered sensors that generate molecular reporters in diseased microenvironments, represent an emerging paradigm in precision diagnostics. Despite the utility of DNA barcodes as a multiplexing tool, their susceptibility to nucleases in vivo has limited their utility. Here we exploit chemically stabilized nucleic acids to multiplex synthetic biomarkers and produce diagnostic signals in biofluids that can be 'read out' via CRISPR nucleases. The strategy relies on microenvironmental endopeptidase to trigger the release of nucleic acid barcodes and polymerase-amplification-free, CRISPR-Cas-mediated barcode detection in unprocessed urine. Our data suggest that DNA-encoded nanosensors can non-invasively detect and differentiate disease states in transplanted and autochthonous murine cancer models. We also demonstrate that CRISPR-Cas amplification can be harnessed to convert the readout to a point-of-care paper diagnostic tool. Finally, we employ a microfluidic platform for densely multiplexed, CRISPR-mediated DNA barcode readout that can potentially evaluate complex human diseases rapidly and guide therapeutic decisions., (© 2023. The Author(s).)

Long non-coding RNAs (lncRNAs) are involved in numerous biological processes and are pivotal mediators of the immune response, yet little is known about their properties at the single-cell level. Here, we generate a multi-tissue bulk RNAseq dataset from Ebola virus (EBOV) infected and not-infected rhesus macaques and identified 3979 novel lncRNAs. To profile lncRNA expression dynamics in immune circulating single-cells during EBOV infection, we design a metric, Upsilon, to estimate cell-type specificity. Our analysis reveals that lncRNAs are expressed in fewer cells than protein-coding genes, but they are not expressed at lower levels nor are they more cell-type specific when expressed in the same number of cells. In addition, we observe that lncRNAs exhibit similar changes in expression patterns to those of protein-coding genes during EBOV infection, and are often co-expressed with known immune regulators. A few lncRNAs change expression specifically upon EBOV entry in the cell. This study sheds light on the differential features of lncRNAs and protein-coding genes and paves the way for future single-cell lncRNA studies., (© 2023. The Author(s).)

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Conserved genomic sequences disrupted in humans may underlie uniquely human phenotypic traits. We identified and characterized 10,032 human-specific conserved deletions (hCONDELs). These short (average 2.56 base pairs) deletions are enriched for human brain functions across genetic, epigenomic, and transcriptomic datasets. Using massively parallel reporter assays in six cell types, we discovered 800 hCONDELs conferring significant differences in regulatory activity, half of which enhance rather than disrupt regulatory function. We highlight several hCONDELs with putative human-specific effects on brain development, including HDAC5 , CPEB4 , and PPP2CA . Reverting an hCONDEL to the ancestral sequence alters the expression of LOXL2 and developmental genes involved in myelination and synaptic function. Our data provide a rich resource to investigate the evolutionary mechanisms driving new traits in humans and other species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continually evolving resulting in variants with increased transmissibility, more severe disease, reduced effectiveness of treatments or vaccines, or diagnostic detection failure. The SARS-CoV-2 Delta variant (B.1.617.2 and AY lineages) was the dominant circulating strain in the United States from July to mid-December 2021, followed by the Omicron variant (B.1.1.529 and BA lineages). Coronavirus disease 2019 (COVID-19) has been associated with neurological sequelae including loss of taste/smell, headache, encephalopathy, and stroke, yet little is known about the impact of viral strain on neuropathogenesis. Detailed postmortem brain evaluations were performed for 22 patients from Massachusetts, including 12 who died following infection with Delta variant and 5 with Omicron variant, compared to 5 patients who died earlier in the pandemic. Diffuse hypoxic injury, occasional microinfarcts and hemorrhage, perivascular fibrinogen, and rare lymphocytes were observed across the 3 groups. SARS-CoV-2 protein and RNA were not detected in any brain samples by immunohistochemistry, in situ hybridization, or real-time quantitative PCR. These results, although preliminary, demonstrate that, among a subset of severely ill patients, similar neuropathological features are present in Delta, Omicron, and non-Delta/non-Omicron variant patients, suggesting that SARS-CoV-2 variants are likely to affect the brain by common neuropathogenic mechanisms., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Association of Neuropathologists, Inc. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection is poorly understood, partly because few studies have systematically applied genomic analysis to distinguish reinfection from persistent RNA detection related to initial infection. We aimed to evaluate the characteristics of SARS-CoV-2 reinfection and persistent RNA detection using independent genomic, clinical, and laboratory assessments., Methods: All individuals at a large academic medical center who underwent a SARS-CoV-2 nucleic acid amplification test (NAAT) ≥45 days after an initial positive test, with both tests between 14 March and 30 December 2020, were analyzed for potential reinfection. Inclusion criteria required having ≥2 positive NAATs collected ≥45 days apart with a cycle threshold (Ct) value <35 at repeat testing. For each included subject, likelihood of reinfection was assessed by viral genomic analysis of all available specimens with a Ct value <35, structured Ct trajectory criteria, and case-by-case review by infectious diseases physicians., Results: Among 1569 individuals with repeat SARS-CoV-2 testing ≥45 days after an initial positive NAAT, 65 (4%) met cohort inclusion criteria. Viral genomic analysis characterized mutations present and was successful for 14/65 (22%) subjects. Six subjects had genomically supported reinfection, and 8 subjects had genomically supported persistent RNA detection. Compared to viral genomic analysis, clinical and laboratory assessments correctly distinguished reinfection from persistent RNA detection in 12/14 (86%) subjects but missed 2/6 (33%) genomically supported reinfections., Conclusions: Despite good overall concordance with viral genomic analysis, clinical and Ct value-based assessments failed to identify 33% of genomically supported reinfections. Scaling-up genomic analysis for clinical use would improve detection of SARS-CoV-2 reinfections., Competing Interests: Potential conflicts of interest. M. N. A. is an equity holder and consultant to Day Zero Diagnostics (company does not work on SARS-CoV-2). P. C. S. is a co-founder of, shareholder in, and scientific advisor to Sherlock Biosciences, Inc (2019–present); she is also a Board member of and shareholder in Danaher Corporation (2019–present), and co-founder and consultant to Delve Bio (2022–present). P. C. S. has filed IP related to genome sequencing and analysis. P. C. S. also reports that Sherlock Biosciences has licensed technology from the author and her lab on CRISPR-based diagnostics and has received consulting fees from the same; stock or stock options with Polaris Genomics (investor and future scientific advisory board [SAB] member) and NextGen Jane (former SAB member and investor). I. V. B. reports an unrelated NIH grant number R01AI042006-24S1. E. S. S. reports unrelated grants or contracts from CDC, Assistant Secretary for Preparedness and Response, MIT/Quanta; payment or honoraria as a writer for Up To Date (2022 to current) and for a single lecture on COVID Infection Prevention to Vertex Pharmaceuticals, 2020; roles as President of Massachusetts Infectious Diseases Society, Vice-Chair of Public Policy and Government Affairs Committee, Society for Healthcare Epidemiology of America, and Member of Boston Biosafety Committee, Boston Public Health Commission. K. A. B, reports NIH training grant 5T32 AI007387-32, unrelated to this work. C. M. D. reports grants paid to institution (NIH/National Institute of Child Health and Human Development [NICHD] grant number K08 HD101342; Harvard University Center for AIDS Research [CFAR]; and MGH Executive Committee on Research), payments to institution from CDC; and contracts to institution from International AIDS Vaccine Initiative and NIH/IMPAACT Network. R. C. L. reports grant from CDC: U01-CK000633; UpToDate: Royalties for Authorship; payment or honoraria from CDC Foundation: Editorial Services; a leadership or fiduciary role on the board of Greater Boston Physicians for Social Responsibility. J. E. L. reports consulting fees 2019–2020 from Sherlock Biosciences; and honorarium from Virology Education. E. R. reports participation as PPD member of the Moderna Clinical Endpoint Adjudication Committee. S. E. T. reports royalties from UpToDate; travel support for attending Duke Clinical Research Institute meeting on preparing for the next pandemic; and reports that her husband is a partner and has equity in an economic consulting firm (Analysis Group Inc.) that provides consulting services for life science companies among many others. E. P. H. reports grants or contracts unrelated to this work and paid to institution from NIH and MGH; royalties paid to author from UpToDate. All other authors report no conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Identifying the dissemination patterns and impacts of a virus of economic or health importance during a pandemic is crucial, as it informs the public on policies for containment in order to reduce the spread of the virus. In this study, we integrated genomic and travel data to investigate the emergence and spread of the SARS-CoV-2 B.1.1.318 and B.1.525 (Eta) variants of interest in Nigeria and the wider Africa region. By integrating travel data and phylogeographic reconstructions, we find that these two variants that arose during the second wave in Nigeria emerged from within Africa, with the B.1.525 from Nigeria, and then spread to other parts of the world. Data from this study show how regional connectivity of Nigeria drove the spread of these variants of interest to surrounding countries and those connected by air-traffic. Our findings demonstrate the power of genomic analysis when combined with mobility and epidemiological data to identify the drivers of transmission, as bidirectional transmission within and between African nations are grossly underestimated as seen in our import risk index estimates., (© 2023. The Author(s).)

Background: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area., Methods: We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs., Results: We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections., Conclusions: We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread., Competing Interests: Potential conflicts of interest. M. S. is cofounder of, shareholder in, and advisor to Rhinostics, Inc and reports a patent for an anterior nares swab (US provisional patent application 63/085,571). M. G., D. T., S. R., and E. W. are employed by Integrated DNA Technologies. D. T. reports stock or stock options from Danaher Corporation (payments to author). S. R. reports stock or stock options from Danaher Corporation. C. M. K. is cofounder of Biosens8, Inc and reports grants or contracts unrelated to this work from the NIH National Institute of General Medical Sciences, Defense Advanced Research Projects Agency, Uniformed Services University, and BU; reports consulting fees paid to author from Adventus Research + Consulting, Inc; is a member of the Biomedical Engineering Society Board; and has a leadership or fiduciary role with the American Institute for Medical and Biological Engineering. J. H. C. is a paid consultant for Cell Signaling Technologies and reports funding unrelated to this work from Mass CPR/Evergrande. W. P. H. serves on scientific advisory boards for Biobot Analytics, Inc and Merck; received consulting fees from Biobot Analytics; contributed expert witness testimony on the expected course of the pandemic for Analysis Group; and reports stock or stock options from Biobot Analytics. P. C. S. is a founder of and shareholder in Sherlock Biosciences, reports consulting fees from Sherlock Biosciences, reports patents for CRISPR technology (PCT/US2018/022764 and PCT/US2019/061577), and is on the board and serves as shareholder for the Danaher Corporation. B. A. P. reports lecture honoraria from the Harvard Secondary School Program and a patent for CRISPR technology (WO 2022/051667). D. H. H. reports funding from an institution unrelated to this work (CDC 1 U01CK000632-01-00 GeoSentinel); consultancy payments to the author from Major League Soccer, the Professional Golf Association of America, Equinox, and Xenophon Strategies, Inc; and participation as chair of a data and safety monitoring board for COVEDZ. M. T. G. reports stock or stock options from Danaher Corporation. T. B. reports grants or contracts unrelated to this work from Gilead Sciences, Inc and Fujifilm Pharmaceuticals U.S.A., Inc. M. H. reports consulting fees as an independent laboratory consultant for Nichols Management Group, LLC. All remaining authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)