Multicenter integrated analysis of noncoding CRISPRi screens.

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.

Increased Pediatric Respiratory Syncytial Virus Case Counts Following the Emergence of SARS-CoV-2 Can Be Attributed to Changes in Testing.

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.

Increased pediatric RSV case counts following the emergence of SARS-CoV-2 are attributable to increased testing.

Background: The incidence of respiratory syncytial virus (RSV) dropped markedly early in the COVID-19 pandemic, followed by a resurgence with heightened case counts. The "immunity debt" hypothesis proposes that the RSV-naive pediatric population increased during the period of low transmission, resulting in a subsequent increased risk of infection. However, the evidence supporting this hypothesis is limited, and no studies have comprehensively evaluated the role of changing respiratory viral testing practices in the perceived surge. Methods: We conducted a multicenter, retrospective analysis of 342,530 RSV encounters and 980,546 RSV diagnostic tests occurring at 32 United States pediatric hospitals between 2013 and 2023. We used interrupted time series analysis to estimate pandemic-associated changes in RSV patient and testing volume, and to quantify changes in the proportions of patients admitted from the emergency department (ED), admitted to the intensive care unit (ICU), and receiving mechanical ventilation. We quantified the fraction of the observed shifts in case counts and in the age of diagnosed patients attributable to changes in RSV testing practices. Finally, we analyzed 524,404 influenza virus encounters and 1,768,526 influenza diagnostic tests to address the specificity of the findings to RSV. Findings: RSV patient volume increased 2.4-fold (95% 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. Over two-thirds of the apparent shifts in patient volume and in patient age were attributable to increased testing, which was concentrated among older pediatric patients. The proportions of patients with RSV requiring hospitalization, intensive care, or mechanical ventilation declined significantly across all patient age groups. These declines were not observed for patients with influenza virus. Interpretation: A surge in RSV testing, rather than in viral circulation, likely underlies the increased case counts observed in 2021-2023. We identify expected consequences of increased testing, including the diagnosis of less severe cases and a shift in the patient age distribution. These findings warrant a critical assessment of the immunity debt hypothesis, while highlighting the importance of considering the testing denominator when surveillance strategies are dynamic. Funding: National Institutes of Health & Howard Hughes Medical Institute.

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.

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.

Reproducible single cell annotation of programs underlying T-cell subsets, activation states, and functions.

T-cells recognize antigens and induce specialized gene expression programs (GEPs) enabling functions including proliferation, cytotoxicity, and cytokine production. Traditionally, different classes of helper T-cells express mutually exclusive responses - for example, Th1, Th2, and Th17 programs. However, new single-cell RNA sequencing (scRNA-Seq) experiments have revealed a continuum of T-cell states without discrete clusters corresponding to these subsets, implying the need for new analytical frameworks. Here, we advance the characterization of T-cells with T-CellAnnoTator (TCAT), a pipeline that simultaneously quantifies pre-defined GEPs capturing activation states and cellular subsets. From 1,700,000 T-cells from 700 individuals across 38 tissues and five diverse disease contexts, we discover 46 reproducible GEPs reflecting the known core functions of T-cells including proliferation, cytotoxicity, exhaustion, and T helper effector states. We experimentally characterize several novel activation programs and apply TCAT to describe T-cell activation and exhaustion in Covid-19 and cancer, providing insight into T-cell function in these diseases.

Clinical Performance of Cas13a-based Point-of-Care Lateral Flow Assay for Detecting Neisseria gonorrhoeae.

Background: Diagnosis of Neisseria (N.) gonorrhoeae is dependent on nucleic acid amplification testing (NAAT), which is not available in resource-limited settings where the prevalence of infection is highest. Recent advances in molecular diagnostics leveraging the high specificity of CRISPR enzymes can permit field-deployable, point-of-care lateral flow assays. We previously reported on the development and in vitro performance of a lateral flow assay for detecting N. gonorrhoeae. Here we aimed to pair that assay with point-of-care DNA extraction techniques and assess the performance on clinical urine specimens. Methods: We collected an additional urine specimen among individuals enrolling in an ongoing clinical trial at the Massachusetts General Hospital Sexual Health Clinic who presented with symptoms of urethritis or cervicitis (urethral or vaginal discharge, dysuria, or dyspareunia). We then assessed thermal, detergent, and combination DNA extraction conditions, varying the duration of heat at 95°C and concentration of Triton X. We assessed the efficacy of the various DNA extraction methods by quantitative polymerase chain reaction (qPCR). Once an extraction method was selected, we incubated samples for 90 minutes to permit isothermal recombinase polymerase amplification. We then assessed the performance of lateral flow Cas13a-based detection using our previously designed porA probe and primer system for N. gonorrhoeae detection, comparing lateral flow results with NAAT results from clinical care. Results: We assessed DNA extraction conditions on 3 clinical urine specimens. There was no consistent significant difference in copies per microliter of DNA obtained using more or less heat. On average, we noted that 0.02% triton combined with 5 minutes of heating to 95°C resulted in the highest DNA yield, however, 0.02% triton alone resulted in a quantity of DNA that was above the previously determined analytic sensitivity of the assay. Given that detergent-based extraction is more easily deployable, we selected that as our method for extraction. We treated 23 clinical specimens with 0.02% triton, which we added to the Cas13a detection system. We ran all lateral flow detections in duplicate. The Cas13a-based assay detected 8 of 8 (100%) positive specimens, and 0 of 15 negative specimens. Conclusion: Using point-of-care DNA extraction, isothermal amplification, and Cas13a-based detection, our point-of-care lateral flow N. gonorrhoeae assay correctly identified 23 clinical urine specimens as either positive or negative. Further evaluation of this assay among larger samples and more diverse sample types is warranted.

Optimisation and evaluation of viral genomic sequencing of SARS-CoV-2 rapid diagnostic tests: a laboratory and cohort-based study.

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.

The HLA-II immunopeptidome of SARS-CoV-2.

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.

Functional dissection of complex and molecular trait variants at single nucleotide resolution.

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics. The majority of these variants have individually weak effects and lie in non-coding gene-regulatory elements where we lack a complete understanding of how single nucleotide alterations modulate transcriptional processes to affect human phenotypes. To address this, we measured the activity of 221,412 trait-associated variants that had been statistically fine-mapped using a Massively Parallel Reporter Assay (MPRA) in 5 diverse cell-types. We show that MPRA is able to discriminate between likely causal variants and controls, identifying 12,025 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% can plausibly be explained by the disruption of a known transcription factor (TF) binding motif. We dissect the mechanisms of 136 variants using saturation mutagenesis and assign impacted TFs for 91% of variants without a clear canonical mechanism. Finally, we provide evidence that epistasis is prevalent for variants in close proximity and identify multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants underlying complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Household Transmission during the Omicron Era in Massachusetts: A Prospective, Case-Ascertained Study using Genomic Epidemiology.

Background: Households are a major setting for SARS-CoV-2 infections, but there remains a lack of knowledge regarding the dynamics of viral transmission, particularly in the setting of widespread pre-existing SARS-CoV-2 immunity and evolving variants. Methods: We conducted a prospective, case-ascertained household transmission study in the greater Boston area in March-July 2022. Anterior nasal swabs, along with clinical and demographic data, were collected for 14 days. Nasal swabs were tested for SARS-CoV-2 by PCR. Whole genome sequencing was performed on high-titer samples. Results: We enrolled 33 households in a primary analysis set, with a median age of participants of 25 years old (range 2-66); 98% of whom had received at least 2 doses of a COVID-19 vaccine. 58% of households had a secondary case during follow up and the secondary attack rate (SAR) for contacts infected was 39%. We further examined a strict analysis set of 21 households that had only 1 PCR+ case at baseline, finding an SAR of 22.5%. Genomic epidemiology further determined that there were multiple sources of infection for household contacts, including the index case and outside introductions. When limiting estimates to only highly probable transmissions given epidemiologic and genomic data, the SAR was 18.4%. Conclusions: Household contacts of a person newly diagnosed with COVID-19 are at high risk for SARS-CoV-2 infection in the following 2 weeks. This is, however, not only due to infection from the household index case, but also because the presence of an infected household member implies increased SARS-CoV-2 community transmission. Further studies to understand and mitigate household transmission are needed.

Investigating the etiologies of non-malarial febrile illness in Senegal using metagenomic sequencing.

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.

Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses.

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.