A 58-year-old woman was admitted to the hospital 3 weeks after lumbar surgery because of diplopia and fever. Left leg pain and a rash had preceded the fever and diplopia. A diagnosis was made.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a zoonosis caused by several species of Borrelia burgdorferi sensu lato and transmitted by the bite of infected ticks of the Ixodes ricinus complex. Lyme borreliosis in North America and Europe differs in certain respects, likely reflecting the different Borrelia species that cause human disease in these locations. The earliest manifestation of Lyme borreliosis is the skin lesion erythema migrans, which develops at the tick bite site, typically 7-14 days after the bite. Some untreated patients will then (within the first few weeks or months after onset of the infection) develop additional erythema migrans skin lesions or other clinical manifestations such as borrelial lymphocytoma, nervous system involvement or carditis. Several months or even years after infection onset, Lyme arthritis or acrodermatitis chronica atrophicans may develop. The diagnosis of typical erythema migrans is clinical, whereas for all other manifestations the diagnosis is supported via serological testing. Treatment with an appropriate antibiotic will result in resolution of clinical symptoms in most patients; however, some patients experience prolonged subjective symptoms, which usually improve over time. Repeated courses of antimicrobials are not beneficial except in rare cases when there is objective evidence of treatment failure.

OBJECTIVE:To investigate SARS-CoV-2 viral shedding duration in autoimmune patients using B cell depleting (BCD) therapy or tumor necrosis factor inhibitors (TNFi) and immunocompetent comparators. METHODS:We conducted a matched cohort analysis among participants in POSITIVES, a prospective study enrolling outpatients with acute COVID-19 within five days of diagnosis. Anterior nasal swabs were self-collected thrice weekly over two weeks and then weekly until two negative polymerase chain reaction (PCR) results. We compared autoimmune cases on BCD or TNFi at baseline with immunocompetent comparators, matched using a propensity score based on age, sex, race and ethnicity, number of COVID-19 vaccinations, calendar time, and antiviral use. Survival methods with Cox proportional hazards modeling was used to compare the time to undetectable PCR. RESULTS:We enrolled 16 BCD users and 30 TNFi users, each matched to 33 and 53 immunocompetent comparators, respectively. The most common autoimmune disease was rheumatoid arthritis (31% among BCD and 53% among TNFi). About 60% used an antiviral, and the mean number of COVID-19 vaccinations was 4.3. At the time of infection, BCD users had significantly longer viral shedding duration than comparators (median 15 vs 7 days; hazard ratio [HR] 0.43, 95% confidence interval [CI] 0.22-0.86; P = 0.017). The median time to undetectable viral load was similar between TNFi users and comparators (10 vs 8 days; HR 0.77, 95% CI 0.48-1.23; P = 0.27). CONCLUSION:BCD users had eight days longer viral shedding duration than immunocompetent comparators; TNFi users and comparators had similar viral shedding duration. These findings inform clinical and public health strategies for autoimmune patients with COVID-19.

Wastewater monitoring enables non-invasive, population-scale tracking of community infections independent of healthcare-seeking behavior and clinical diagnosis. Metagenomic sequencing extends this capability by enabling broad, pathogen-agnostic detection, genomic characterization, and identification of novel or unexpected threats. Here, we present data from CASPER (the Coalition for Agnostic Sequencing of Pathogens from Environmental Reservoirs), a U.S.-based wastewater metagenomic sequencing network designed for deep, untargeted pathogen monitoring at national scale. This release includes 1,206 samples collected between December 2023 and December 2025 from 27 sites across nine states, covering 13 million people. Deep sequencing (~1 billion read pairs per sample) generated 1.2 trillion read pairs (347 terabases), enabling detection of even rare taxa, with CASPER representing 66% of all untargeted wastewater sequencing data currently available on the NCBI Sequence Read Archive. Virus abundance trends correlate with nationwide wastewater PCR and clinical data for SARS-CoV-2, influenza A, and respiratory syncytial virus, while the pathogen-agnostic approach captures emerging threats, including avian influenza H5N1 during initial dairy cattle outbreaks, West Nile virus, and measles, among hundreds of viral taxa. As the largest publicly available untargeted wastewater sequencing dataset to date, CASPER provides a shared and growing resource for pathogen surveillance and microbial ecology. ### Competing Interest Statement D.H.O. received support for this project from Inkfish and Heart of Racing. D.H.O. is a managing partner of Pathogenuity LLC, a consultancy that advises on topics including environmental monitoring for pathogens. P.C.S. hold several patents related to diagnostic and surveillance technologies and is a co-founder and equity holder in Delve Biosciences and Lyra Labs, a board member and equity holder in Polaris Genomics, and an equity holder of NextGenJane. P.C.S was formerly a co-founder of Sherlock Biosciences and board member of Danaher Corporation, until December 2024. All potential conflicts are managed in accordance with institutional policy. ### Funding Statement L.J.J. was supported by the Draper Scholar program at The Charles Stark Draper Laboratory. J.K., O.S.H., R.F-O., S.L.G., W.J.B., H.B., D.P.R., K.S., J.D.F., and M.R.M. received support for this work from Coefficient Giving via a gift to SecureBio. C.R., A.T-M., E.E.C., M.C.J., and D.H.O. were supported by Inkfish and Heart of Racing. L.J.J., J.P., and P.C.S. were supported by the CDC Pathogen Genomics Centers of Excellence (contract INTF5104H78W22195346) and a CDC Broad Agency Announcement (contract 75D30123C17983). J.E.L. and G.A. were supported by a subcontract under CDC Broad Agency Announcement contract 75D30123C17983. H.M.S-G. and A.A. were supported in part by the National Institute on Drug Abuse of the National Institutes of Health under Award Number U01DA053941, and by the University of Miami Initiative on Virology and Infectious Disease and SecureBio. R.P. was supported by the Illinois Department of Public Health and the Chicago Department of Public Health. This work used Expanse at the San Diego Supercomputer Center through allocation BIO240238 to J.A.R. from the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, which is supported by U.S. National Science Foundation grants #2138259, #2138286, #2138307, #2137603, and #2138296. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Clinical data was collected under a waiver of consent as non-identifiable aggregate variables with oversight from Massachusetts General Brigham Institutional Review Board (IRB: 2023P001716). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Sequencing data generated by the CASPER consortium included in this release are available on the NCBI SRA under BioProject PRJNA1247874. Prior to public release, human-derived reads were masked using NCBI's Human Read Removal Tool (Katz et al. 2021). Additional deep sequencing data from Los Angeles, collected under CASPER, are available under BioProject PRJNA1198001 (Grimm, Rothman, et al. 2025). Near real-time visualization of human-infecting virus trends from CASPER sampling sites is available through a public dashboard ("Wastewater Surveillance Dashboards", n.d.), which uses a different bioinformatic pipeline (github.com/dhoconno/nvd; O'Connor et al., 2026) than described here. Wastewater PCR data used for validation are publicly available through the CDC NWSS data portal: SARS-CoV-2 (dataset j9g8-acpt), Influenza A (dataset ymmh-divb), and RSV (dataset 45cq-cw4i). A subset of these data were generated by WastewaterSCAN (Boehm et al. 2024, 2026) and are shared under a CC BY-NC 4.0 license; see https://data.wastewaterscan.org/about for terms of use. Tabular data underlying manuscript figures, PMMoV RT-qPCR measurements, and clinical respiratory virus testing data from Massachusetts General Hospital are available in the paper's GitHub repository (github.com/securebio/casper-paper).

Respiratory syncytial virus (RSV) continues to circulate at high levels despite the introduction of new monoclonal antibodies (mAbs) and vaccines targeting the prefusion F (pre-F) protein. We analyzed the viral genome sequences of 133 RSV clinical samples collected during the 2022-2023 and 2023-2024 seasons, and selected representative isolates for phenotypic testing. We selected four RSV A and four RSV B replication-competent, sequence-verified stocks that were assessed for replication kinetics in vitro and neutralization sensitivity by a panel of F-targeting mAbs and polyclonal sera using a rabbit vaccination model. Isolates retained sensitivity to all mAbs tested. We detected no mutations in mAb binding sites in the isolates tested. RSV-A isolates were slightly less susceptible to multiple mAbs than RSV-B isolates. Antigenic cartography revealed a separation of antibody responses by subtype: RSV-A isolates clustered together and aligned with lower neutralization by antibodies such as MPE8 and 101F, whereas RSV-B isolates formed a distinct cluster associated with higher mAb susceptibility. In a rabbit vaccination model, RSV-A-only sera efficiently neutralized all RSV-A isolates and the RSV-B1 reference strain but showed diminished activity against contemporary RSV-B isolates. RSV-B-only sera displayed balanced neutralization across both subtypes. Combined RSV-A+B immunization produced uniformly strong responses to all isolates, suggesting that multivalent exposure may overcome subtype-specific antibody polarization. Collectively, our results demonstrate consistent antigenic divergence of RSV subtypes and underscore the importance of considering genetic and phenotypic divergence for F-directed immunoprophylaxis. Importance:RSV remains a major cause of lower respiratory tract infections. Although new mAbs and vaccines have been approved in recent years, the impact of circulating genetic diversity on these therapeutics is incompletely understood. In this study, we demonstrate how recent RSV isolates respond to a panel of mAbs and whether sera from rabbits vaccinated with mRNA vaccines effectively neutralize these isolates. We reveal that RSV-A isolates are more resistant to neutralization by mAbs than RSV-B isolates, and the bivalent vaccine elicits broader neutralizing responses than the monovalent vaccine. This study provides insights into how viral diversity may influence antibody-mediated protection and suggests that recent RSV-B isolates may respond differently from the laboratory-adapted strains commonly used in research.

New SARS-CoV-2 variants have undergone repeated selective sweeps since the beginning of the COVID-19 pandemic, but the fitness advantages and mechanisms driving these sweeps are not fully understood. We developed a probabilistic modeling framework to analyze pandemic growth, infectivity, and immune escape, explicitly accounting for seven immune exposure histories in 5,732 experiments and estimating the effects of 835 mutations. We found infectivity was important for early variants, but as gains became zero-sum, growth became driven by consistent increase in immune escape conferred by a primarily additive effect of mutational accumulation. While phenotypic tradeoffs exist for individual mutations, successful viral strains boast assemblages of mutations that do not sacrifice infectivity for escape. Thus, during an apparent transition to endemicity, SARS-CoV-2 evolution ascended along an evolutionary ridge in the mutational space defined by infectivity and escape, with infectivity reaching an early peak and antigenicity continuing to evolve.

Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a zoonosis caused by several species of Borrelia burgdorferi sensu lato and transmitted by the bite of infected ticks of the Ixodes ricinus complex. Lyme borreliosis in North America and Europe differs in certain respects, likely reflecting the different Borrelia species that cause human disease in these locations. The earliest manifestation of Lyme borreliosis is the skin lesion erythema migrans, which develops at the tick bite site, typically 7–14 days after the bite. Some untreated patients will then (within the first few weeks or months after onset of the infection) develop additional erythema migrans skin lesions or other clinical manifestations such as borrelial lymphocytoma, nervous system involvement or carditis. Several months or even years after infection onset, Lyme arthritis or acrodermatitis chronica atrophicans may develop. The diagnosis of typical erythema migrans is clinical, whereas for all other manifestations the diagnosis is supported via serological testing. Treatment with an appropriate antibiotic will result in resolution of clinical symptoms in most patients; however, some patients experience prolonged subjective symptoms, which usually improve over time. Repeated courses of antimicrobials are not beneficial except in rare cases when there is objective evidence of treatment failure. This Primer by Strle and colleagues summarizes the epidemiology, pathophysiology, diagnosis and treatment of Lyme borreliosis.

BACKGROUND:Regional blood donor screening for Babesia has been conducted in the United States since 2019, using highly sensitive and specific nucleic acid testing (NAT). Currently, there are no recommendations regarding the management of asymptomatic blood donors who test positive for Babesia. METHODS:A multidisciplinary expert panel was convened to develop guidance for the management of asymptomatic Babesia-infected blood donors. Additionally, a survey was distributed through the Infectious Diseases Society of America (IDSA) Emerging Infections Network (EIN) to evaluate how a geographically diverse group of infectious diseases specialists would approach this problem. RESULTS:The expert panel recommends that all Babesia NAT-positive blood donors should be referred for clinical evaluation and retesting using peripheral blood smear (PBS) and Babesia PCR. The panel also recommends observation rather than treatment for a reactive molecular test alone. Antimicrobial therapy should be considered for PBS-positive cases. Donors should be instructed to seek medical care if symptoms develop. The EIN survey results are consistent with these recommendations. CONCLUSIONS:Since blood donors comprise healthy, immunocompetent adults, findings of incidental Babesia infections in asymptomatic donors are frequently self-limited. Longitudinal studies show that molecular evidence of infection in blood donors clears in almost all without intervention.

Antimicrobial resistance in Neisseria gonorrhoeae is an urgent public health threat. Resistance-guided therapy can assure appropriate treatment and reintroduce alternative therapeutic options by identifying genetic predictors of resistance. Mosaicism at codons 375-377 of the penA gene is associated with cefixime resistance. Rapid, field-deployable assays for predicting cefixime susceptibility are lacking. We used a machine-learning algorithm to develop a CRISPR Cas13a-based assay to detect the absence of mosaicism at codons 375-377 of the penA gene, combined with isothermal amplification in a single reaction. We integrated the assay onto a portable fluorescence-based platform. We evaluated performance using cultured isolates and compared results with PCR genotyping and phenotypic antimicrobial susceptibility testing. We also assessed the feasibility of reagent lyophilization of an existing porA N. gonorrhoeae detection assay, which can support cold-chain-independent deployment. Among 40 N. gonorrhoeae isolates, the Cas13a penA assay demonstrated 100% concordance with PCR genotyping and 92.5% (95% confidence interval 79.6%-98.4%) concordance with phenotypic cefixime susceptibility. Median time to detection of the penA mosaic assay was 12 min (interquartile range [IQR] 5 min). The lyophilized porA N. gonorrhoeae detection system detected all 12 isolates with a median time to detection of 45 min (IQR 40-45) compared to 45 min (IQR 35-50) for the positive aqueous control, although peak fluorescence was higher for the aqueous control (P < 0.01). The Cas13a assay was rapid and demonstrated strong correlation with genotypic and phenotypic cefixime susceptibility in N. gonorrhoeae, while a lyophilized assay retained functionality. IMPORTANCE:Antimicrobial resistance in Neisseria gonorrhoeae is an urgent threat to public health. Recent reports have highlighted the continued rise in ceftriaxone resistance, our last-line empiric treatment option. Molecular assays that detect the genetic determinants of resistance can improve antibiotic stewardship and increase the therapeutic options for cases of gonorrhea. As a result, those tests can reduce the pressure toward the emergence of ceftriaxone resistance. However, most molecular assays cannot be deployed in low-resource settings due to a lack of infrastructure. We report on the development of a point-of-care assay for predicting resistance to an oral first-line treatment option, cefixime. The assay provided results in under 30 min and demonstrated strong correlation with phenotypic and genotypic resistance. Furthermore, we report on the proof-of-concept freeze-drying of assay reagents, which could permit cold-chain-independent use. Collectively, our study provides the groundwork for developing and deploying such molecular resistance assays in low-resource settings.

Background:Nirmatrelvir-ritonavir (N-R) reduces morbidity and mortality from COVID-19 in high-risk individuals; however, N-R use has been associated with risk of SARS-CoV-2 virologic rebound. The mechanisms contributing to virologic rebound after N-R treatment are currently unknown. One plausible mechanism is that antiviral treatment may alter the development of immune responses to SARS-CoV-2 infection, thereby contributing to rebound after cessation of therapy. Methods:We profiled immune responses in a case-ascertained, longitudinal, prospective cohort of ambulatory individuals with COVID-19. Participants were grouped according to whether virological rebound occurred and whether they had received N-R treatment. We assessed antibody, T-cell, and innate responses. Results:We observed no differences in the binding or neutralizing antibody, T-cell, and innate immune responses between participants with virologic rebound compared to participants without virologic rebound. N-R use was associated with slightly weaker antibody responses overall, even after adjustment for immunosuppression. Conclusion:Virologic rebound after N-R treatment is likely driven by non-immune mechanisms. Funding:The project was supported by the National Institutes of Health (R01 AI 138801) and the Massachusetts Consortium on Pathogen Readiness. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Highly pathogenic avian influenza A (H5) virus continues to spread in animals with ongoing concern for potential human transmission. We describe an adaptable H5 screening approach that integrates clinical laboratory workflows with research-developed assays. This strategy efficiently screened thousands of samples, providing a practical and scalable model for H5 surveillance.

Background: Highly pathogenic H5 avian influenza A has caused sporadic human infections, increasing the risk for potential human–to–human spread. In 2024, the U.S. experienced outbreaks among poultry and cattle, prompting enhanced surveillance. Objective: To evaluate an H5 testing algorithm in subjects with respiratory symptoms presenting for routine care during low influenza A virus circulation. Design: Observational study using clinical– and research–developed nucleic acid amplification tests (NAATs) and pooled screening methods. Setting: Academic medical center in Boston, MA. Participants: 5,400 symptomatic individuals contributing 6,935 respiratory specimens from June 23 to August 28, 2024. Measurements: Specimens underwent initial respiratory pathogen testing per clinical protocols, which did not routinely include influenza due to low summer–month prevalence. Influenza A–positive specimens were subtyped using a clinical assay for H5 assessment. SARS–CoV–2–negative specimens not tested for influenza were screened in pooled batches. Positive pools were deconvoluted to individual specimens and screened for H5 using quantitative polymerase chain reaction. Results: Influenza A was detected in 40 of 6,935 specimens (0.6%), comprising 35 of 5,400 unique subjects (0.7%). No H5 infections were identified. Of the 35 influenza–positive individuals, 10 cases (29%) were found through research–specific screening of SARS–CoV–2–negative specimens. No deaths attributed to influenza were recorded. Limitations: Single center design, convenience sampling, absence of ocular specimens, and minimal sampling in high–risk areas may limit generalizability. Conclusion: Expanded influenza testing using pooled NAATs successfully identified low–prevalence influenza A and ruled out H5 in this cohort. These data support targeted influenza screening to enhance surveillance for emerging subtypes rather than a broad–based clinical testing strategy for influenza A testing. ### Competing Interest Statement J.A.B. has received funding to his institution for other research projects from Pfizer Inc., Analog Devices Inc., and the Steven & Alexandra Cohen Foundation. He has been a consultant for Diasorin, Roche Diagnostics and Flightpath Biosciences. P.C.S. is a co-founder and shareholder of Delve Bio. She was a co-founder and shareholder of Sherlock Biosciences (sold to Orasure in December 2024) and was a non-executive board member and shareholder of Danaher Corporation (stepping down in December 2024). S.E.T. has received research funding from SeLux Diagnostics and has served as a consultant for CarbX. All others report no disclosures. ### Funding Statement This study was funded by Massachusetts Department of Public Health and Center for Disease Control and Prevention through the US Public Health Pathogens Genomics Centers of Excellence award (MADPH Contract Number INTF5104H78W22195363). Additional support was provided from CDC through two Broad Agency Announcement contract subawards from the Broad Institute of MIT and Harvard (Federal Contract Numbers 75D30123C17983 and 75D30122C15113) and the Office of Advanced Molecular Detection, through Cooperative Agreement Number CK22-2204. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Board of Mass General Brigham gave ethical approval for this work (Mass General Brigham Human Research Protection Program). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

Avian influenza viruses (AIVs) are zoonotic pathogens that pose an increasing global threat due to their potential for significant economic losses in agriculture, spillover into humans, and the risk of a pandemic should human-to-human transmission occur. These concerns underscore the need for rapid, sensitive and specific tools to detect and differentiate circulating AIV subtypes and clades. Current AIV diagnostic methods rely on specialized equipment and trained personnel, limiting their use in the field and in low-resource settings. Here, we extended SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a CRISPR-based platform, to detect and subtype AIVs. We designed, optimized, and validated SHINE assay for the H5 AIV detection using both fluorescence and lateral flow readout, achieving 100% specificity with PCR-based assays when tested on seasonal influenza-positive clinical samples, and a limit of detection of 121.7 copies/μL on vaccine-derived H5 viral seedstocks. To expand the scope of avian influenza detection, we also designed and validated a SHINE assay targeting the 2.3.4.4b A(H5N1) lineage, in response to the ongoing H5N1 outbreak in cattle in the United States, and a SHINE assay specific to Eurasian H7 lineage to discriminate against North American H7 lineage. Together, these SHINE assays offer a promising platform for AIV diagnosis and surveillance, particularly in settings with limited laboratory infrastructure.

Lyme disease is a tick-borne spirochetosis with diverse clinical manifestations. Genotypic and phenotypic variation among Borrelia burgdorferi strains correlates with variable manifestations of Lyme disease in humans; this diversity is attributed in part to variation in surface-exposed lipoproteins, which are targets of the human antibody response and contribute to tissue adhesion, immune evasion, and other host interactions. Many B. burgdorferi lipoproteins are encoded as multi-copy gene families, such as the OspE/F-like leader peptide (Elp) protein family, which inhibits classical complement activation by binding complement C1s. To characterize Elp allelic variants, we adapted the Pseudomonas syringae ice nucleation protein (INP) system to present B. burgdorferi lipoproteins on the surface of Escherichia coli. Using this system, we identified interactions with classical complement proteins and mapped binding regions, then validated interactions using recombinant proteins and B. burgdorferi surface display. We also discovered a novel potential interaction between Elp proteins and the mammalian basement membrane protein perlecan, thus revealing a bifunctional nature of Elps. Our findings indicate that Elps have undergone functional diversification while maintaining classical complement inhibition mediated by potent and conserved C1s binding and demonstrate that E. coli surface display offers an efficient, cost-effective, and relatively high-throughput approach to characterize B. burgdorferi lipoproteins.

Recurrent waves of viral infection necessitate vaccines and therapeutics that remain effective against emerging viruses. Our ability to evaluate interventions is currently limited to assessments against past or circulating variants, which likely differ in their immune escape potential compared with future variants. To address this, we developed EVE-Vax, a computational method for designing antigens that foreshadow immune escape observed in future viral variants. We designed 83 SARS-CoV-2 spike proteins that transduced ACE2-positive cells and displayed neutralization resistance comparable to variants that emerged up to 12 months later in the COVID-19 pandemic. Designed spikes foretold antibody escape from B.1-BA.4/5 bivalent booster sera seen in later variants. The designed constructs also highlighted the increased neutralization breadth elicited by nanoparticle-based, compared with mRNA-based, boosters in non-human primates. Our approach offers targeted panels of synthetic proteins that map the immune landscape for early vaccine and therapeutic evaluation against future viral strains.

Pathogen genomic analysis is central to tracking, understanding, and containing outbreaks, but complexity and high costs of state-of-the-art (SOTA) phylogenetic tools limit global access and impact. We introduce Delphy, an exact reformulation of Bayesian phylogenetics designed to transform its speed, scalability and accessibility while retaining SOTA accuracy. Delphy's central data structure, an Explicit Mutation Annotated Tree, exploits the high sequence similarity in large-scale epidemic datasets for efficient tree exploration and convergence. By reproducing key analyses from recent major epidemics (Ebola, Zika, SARS-CoV-2, mpox, and H5N1), we demonstrate SOTA accuracy with up to 1,000x speedups. Assessing Delphy's scalability, we show that a simulated dataset of 100,000 sequences can be analyzed in under a day—the largest such computation to date. We distribute Delphy as a client-side web application, enabling users worldwide to turn raw data into interactive results within minutes, without the data ever leaving the user's machine. Delphy automatically identifies key viral lineages and mutations, as well as their emergence and prevalence through time, all with quantified uncertainties derived from a solid theoretical foundation. Delphy shows the power of Bayesian phylogenetics as a fast, accessible frontline tool for tackling future outbreaks. ### Competing Interest Statement P.V., B.F., M.S., K.Y., I.S., M.D.M. and P.C.S. are inventors on Patent Application No. PCT/US2024/050993 filed for this work; Delphy is free for all academic use. P.C.S. is a co-founder of, shareholder in Delve Bio; she was formerly a co-founder of and shareholder in Sherlock Biosciences, Inc and a Board member of and shareholder in Danaher Corporation. B.F. is the Founder of Fathom Information Design, a design and software development firm in Boston.

Memory lymphocytes are durable cells that persist in the absence of antigen, but few human B cell subsets have been characterized in terms of durability. The relative durability of eight non-overlapping human B cell sub-populations covering 100% of all human class-switched B cells was interrogated. Only two long-lived B cell populations persisted in the relative absence of antigen. In addition to canonical germinal center-derived switched-memory B cells with an IgD-CD27+CXCR5+ phenotype, a second, non-canonical, but distinct memory population of IgD-CD27-CXCR5+ DN1 B cells was also durable, exhibited a unique TP63-linked transcriptional and anti-apoptotic signature, had low levels of somatic hypermutation, but was more clonally expanded than canonical switched-memory B cells. DN1 B cells likely evolved to preserve immunological breadth and may represent the human counterparts of rodent extrafollicular memory B cells that, unlike canonical memory B cells, can enter germinal centers and facilitate B cell and antibody evolution.

BACKGROUNDSARS-CoV-2 has evolved subvariants since the emergence of the Omicron variant in 2021. Whether these changes impact viral shedding and transmissibility is not known.METHODSPOSITIVES is a prospective longitudinal cohort of individuals with mild SARS-CoV-2 infection. Ambulatory, immunocompetent participants who did not receive antivirals self-administered 6 anterior nasal swabs over 15 days. Samples were analyzed by qPCR to quantify viral RNA, semiquantitative viral culture to detect shedding of replication-competent virus, and whole-genome sequencing to classify subvariants. Our predictor of interest was Omicron subvariants: BA.1x, BA.2x, BA.4/5x, XBB.x, and JN.x. Outcomes included RNA levels and duration of shedding replication-competent virus. We additionally explored whether symptoms are a valid marker for ending isolation.RESULTSThe median peak nasal SARS-CoV-2 RNA (6.0-6.3 log10 RNA copies/mL), median days to peak RNA (4-5 days), median days to undetectable viral RNA (12-14 days), and median days to negative viral culture (4-8 days) were similar across Omicron subvariants. Number and duration of symptoms were also similar. For all subvariants, a sizeable percentage (range 27.5%-56.0%) shed replication-competent virus after fever resolution and improvement of symptoms.CONCLUSIONDespite ongoing viral evolution, key aspects of viral dynamics of SARS-CoV-2 infection, including the duration of shedding replication-competent virus, have not substantially changed across Omicron subvariants. Replication-competent shedding of these subvariants is detected for a large proportion of people who meet criteria for ending isolation.FUNDINGNIH (U19 AI110818, R01 AI176287, K24 HL166024), the Massachusetts Consortium on Pathogen Readiness, and the Massachusetts General Hospital Department of Medicine.