ABSTRACT The recent development of new respiratory syncytial virus (RSV) prophylactics for the prevention of severe lower respiratory tract infections in infants and older adults promises in lowering disease burden in these vulnerable populations. However, it remains unclear if periodic breakthrough infections in these populations may drive the emergence of resistant isolates or clades and what factors might contribute to these breakthroughs. In this retrospective cohort study, we performed whole-genome sequencing of RSV isolates from infants and adults during the past two RSV seasons (2023-2025) to assess viral and clinical correlates of nirsevimab breakthrough. RSV infections from nirsevimab breakthrough cases were associated with less severe clinical outcomes in the first, but not second, season after administration. While breakthrough isolates did not share any Fusion glycoprotein mutations in predicted antigenic sites, they largely belonged to only a few circulating clades that were responsible for driving temporally distinct pediatric transmission clusters. To determine if these transmission clusters and breakthrough infections were in part driven by differences in the Fusion proteins of these clades, we compared the relative fusogenicity and neutralization susceptibility of Fusion proteins from contemporary circulating clades. Notably, RSV-A clade A.D.3 exhibited modestly reduced susceptibility to nirsevimab neutralization, though it wasn’t associated with any transmission clusters or breakthrough infections. Collectively, these data suggest that clade associations with prophylactic breakthrough are driven by pediatric transmission clusters rather than clade-associated resistance, though continued surveillance will be vital as prophylactic coverage continues to rise.

Flow cytometry analysis showing that anti–Siglec-10 antibodies do not alter ITGA3, ITGB1, or Siglec-10 ligand levels on PDAC cells

Treatment of severe invasive Streptococcus pyogenes infections has historically relied on β-lactam antibiotics with adjunctive clindamycin. Rising rates of lincosamide resistance have led to adoption of linezolid as an alternative adjunctive therapy. This report describes a clinical S. pyogenes isolate carrying a multi-drug resistance gene, cfr(C), known to confer the phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) resistance phenotype in other bacterial species. Strain GAS-390756 is a pharyngeal isolate collected from a child with pharyngitis in the United States. Whole-genome sequence analysis demonstrated that the isolate belonged to emm type 89 and carried an integrative and conjugative element with erm(B) and cfr(C) cargo genes (ICESpy390756). Antimicrobial susceptibility testing demonstrated the isolate was resistant to erythromycin and clindamycin but susceptible to linezolid, consistent with the activity of erm(B) but not cfr(C). Comparative analysis of cfr(C)'s genomic context suggested that the promoter sequence may have been lost during ICESpy390756's acquisition of the gene. While GAS-390756 remains susceptible to linezolid, the expansion of cfr(C)'s host range into S. pyogenes and its presence on a novel mobile element are concerning. In light of known expression mechanisms for other silent antimicrobial resistance genes, this observation carries epidemiologic and public health importance. IMPORTANCE:Treatment of severe invasive Streptococcus pyogenes infection has traditionally relied on the adjunctive use of clindamycin with β-lactam antibiotic therapy. However, rising rates of clindamycin non-susceptibility have led clinicians to adopt linezolid as an alternative therapy. We identified S. pyogenes isolated from a child with pharyngitis that carried the cfr(C) gene. This gene causes resistance to multiple antibiotics, including clindamycin and linezolid, in other bacterial species. At the time of this study, we found no other instances of the cfr(C) gene carried by S. pyogenes in either published reports or the public genome sequence database. While this isolate remained sensitive to linezolid, the expanded host range of cfr(C) found in a novel potentially mobile genomic element may herald the eventual emergence of broader antibiotic resistance in S. pyogenes.

Flow cytometry analysis showing high expression of ITGA3 and ITGB1 on PDAC cells

Tumor-associated macrophages (TAM) in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) exhibit immunosuppressive phenotypes and impaired phagocytic activity, facilitating tumor progression and immune evasion. In this study, we identified integrin α3β1, composed of ITGA3 and ITGB1 subunits, as a sialylated glycoprotein ligand for Siglec-10, an inhibitory glyco-immune checkpoint receptor highly expressed on TAMs in PDAC. The interaction between Siglec-10 on TAMs and α3β1 on PDAC cells suppressed macrophage-mediated phagocytosis, thereby promoting immune evasion. Consistently, disrupting Siglec-10 interactions using mAbs significantly enhanced macrophage phagocytosis of PDAC cells and alleviated myeloid cell-mediated inhibition of T-cell proliferation and activation in vitro. In both a xenograft mouse model engrafted with human macrophages and a human Siglec-10 transgenic mouse model, targeting Siglec-10 with mAbs reduced PDAC growth. These findings suggest that Siglec-10 interactions are key mediators of TAM-driven immune evasion in PDAC and highlight the therapeutic potential of targeting these interactions to restore antitumor immunity. SIGNIFICANCE:Pancreatic tumor cells exploit integrin α3β1 to engage the immunosuppressive checkpoint receptor Siglec-10 on myeloid cells, driving immune evasion, which can be targeted with antibody-mediated blockade of Siglec-10 to restore antitumor immunity.

Correlation analysis in TCGA showing associations between ITGA3 or ITGB1 and Siglec-10 and its ligand-forming enzymes (ST3GAL1, B3GNT3)

Analysis showing expression of human Siglec-10 on myeloid cells in B-hSIGLEC10 mice compared with C57BL/6 controls

We investigated sequences of the penicillin-binding protein 2x (PBP2x) among 902 Streptococcus pyogenes isolates from children with pharyngitis. Twenty-five pharyngeal isolates carried variants associated with reduced β-lactam susceptibility. Penicillin and ampicillin susceptibility testing confirmed reduced susceptibility among PBP2x variant isolates relative to those with the wild-type sequence.

Sialic acid-containing glycans (sialoglycans) on pathological cells interact with Siglecs, glyco-immune checkpoint receptors expressed on myeloid cells, suppressing the cytotoxic functions of these immune cells. Using targeted glycomic analyses and gene editing, we show that HIV infection reprograms the glycosylation machinery of infected cells to increase the expression of the sialoglycan ligands for Siglec-3, −7, and −9. These ligands engage Siglecs on myeloid cells, impairing their ability to target HIV-infected cells. Selective disruption of these interactions using 10-1074-SiaD, an HIV-specific antibody conjugated to sialidase, an enzyme that removes sialic acids, significantly enhances monocyte- and neutrophil-mediated killing of HIV-infected cells in autologous assays. Treatment with 10-1074-SiaD in female humanized mice infected with HIV reduces viral load and decreases inflammation. These findings reveal an immune evasion mechanism exploited by HIV to evade myeloid cell immune surveillance and highlight the potential of targeting sialoglycan-Siglec interactions to improve immune clearance of HIV-infected cells. Immune evasion mechanisms of initial HIV infection are incompletely understood. Here, the authors show that HIV rewires the glycosylation machinery of infected myeloid cells, forming a glycan shield that engages glyco-immune checkpoints and inhibits cell function, and thus targeted killing of infected cells.

Human immunodeficiency virus (HIV) relies upon a broad array of host factors in order to replicate and evade the host antiviral response. Cleavage and polyadenylation specificity factor 6 (CPSF6) is one such host factor that is recruited by incoming HIV-1 cores to regulate trafficking, nuclear import, uncoating, and integration site selection. Despite these well-described roles, the impact of CPSF6 perturbation on HIV-1 infectivity varies considerably by cell type. Here, we report that CPSF6 knock-out in primary CD4+ T cells leads to increased permissivity to HIV-1 infection due to broad transcriptional reprogramming. Knock-out of CPSF6 results in widespread differential gene expression, including downregulation of genes involved in the innate immune response and enhanced expression of the HIV-1 co-receptors. Accordingly, these cells are less responsive to interferon and express lower levels of antiretroviral restriction factors, including TRIM5α. These transcriptional changes are linked to global shortening of mRNA 3' untranslated regions (UTRs) through changes in alternative polyadenylation (APA), which are triggered by disruption of the CPSF6-containing Cleavage Factor Im (CFIm) complex. Furthermore, we find that recruitment of CPSF6 by HIV-1 cores is sufficient to perturb CPSF6 function, leading to 3' UTR shortening and subsequent transcriptional rewiring. These results suggest a model in which HIV-1 transcriptionally reprograms target cells through recruitment of CPSF6 to incoming cores to circumvent the antiviral response and enhance permissivity to infection.

The impact of remdesivir on SARS-CoV-2 diversity and evolution in vivo has remained unclear. In this single-center, retrospective cohort study, we assessed SARS-CoV-2 diversification and diversity over time in a cohort of hospitalized patients who did or did not receive remdesivir. Whole-genome sequencing was performed on 98 paired specimens collected from 49 patients before and after remdesivir administration. The genetic divergence between paired specimens was not significantly different in this cohort compared with that in a control group of patients who did not receive the drug. However, when we focused on minority variants, several positions showed preferential diversification after remdesivir treatment, some of which were associated with specific variants of concern. Most notably, remdesivir administration resulted in strong selection for a nonsynonymous mutation in nsp12, G671S, previously associated with enhanced viral fitness. This same mutation was found to be enriched in a second cohort of 143 inpatients with specimens collected after remdesivir administration compared with controls. Only one other mutation previously implicated in remdesivir resistance (nsp12:V792I) was found to be preferentially selected for after remdesivir administration. These data suggest that SARS-CoV-2 variants with enhanced replicative fitness may be selected for in the presence of antiviral therapy as an indirect means to overcome this selective pressure.

In humans, mutations in sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) lead to the development of a type I interferonopathy known as Aicardi-Goutières syndrome (AGS). AGS can present with a variety of severe phenotypes in patients, and a hallmark of this disease is chronic activation of type I interferon (IFN) signaling. However, the mechanism through which type I IFN signaling is activated in the absence of functional SAMHD1 is not known. Here, we investigated the molecular pathways that lead to type I IFN signaling activation in the absence of SAMHD1. Our investigations revealed that chronic activation of type I IFN signaling in SAMHD1-knockout (KO) monocytes is cyclic GMP-AMP synthase (cGAS)-dependent. Analysis of other nucleic acid sensors showed that type I IFN signaling in SAMHD1-KO cells is not dependent on melanoma differentiation-associated protein 5 (MDA5) or retinoic acid-inducible gene I (RIG-I). In agreement with our observation that type I IFN signaling is dependent on cGAS, two inhibitors of the cGAS-stimulator of IFN genes pathway, G140 and H151, effectively prevented type I IFN activation in SAMHD1-KO monocytes. We also found that type I IFN signaling in SAMHD1-KO monocytes is dependent on type I IFN receptor expression. Further exploration revealed mitochondrial malfunction in SAMHD1-KO monocytes that is likely to leak mitochondrial components into the cytoplasm. Overall, our work suggests that genetic knock out of SAMHD1 leads to mitochondrial disfunction, resulting in the presence of mitochondrial DNA in the cytoplasm, which triggers cGAS and the type I IFN response.

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, most of these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased toward the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published functional screens and proteomics data to reveal (i) common pathways that were identified in all OMICs datasets-including regulation of Wnt signaling and gap junctions, (ii) pathways uniquely identified in this screen-including NADH oxidation, or (iii) pathways supported by this screen and proteomics data but not published functional screens-including arachionate production and MAPK signaling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 32 proteins that were determined to impact viral replication and 27 impacting late stages of infection, respectively. Additionally, a subset of proteins was tested across other coronaviruses revealing a subset of proviral factors that were conserved across pandemic SARS-CoV-2, epidemic SARS-CoV-1 and MERS-CoV, and the seasonal coronavirus OC43-CoV. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m 6 A during HIV-1 replication have produced conflicting results. Since m 6 A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m 6 A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m 6 A or 5-methylcytosine (m 5 C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m 6 A writer complex auxiliary proteins VIRMA and WTAP, and the m 6 A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m 6 A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells. m 6 A is the most common chemical modification on cellular and viral RNA and regulates its stability, localization, and translation. m 6 A modification and its regulation varies dramatically between cell types and cell states. In this study, we investigated the role of m 6 A factors during HIV-1 infection of physiologically relevant primary CD4+ T cells. Using CRISPR-Cas9 to knockout 46 cellular genes implicated in RNA modification, we found only the m 6 A writer complex auxiliary members WTAP and VIRMA, and the reader YTHDF2, significantly affected HIV-1 replication in these cells. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in marginal changes in HIV-1 infection, despite larger reductions in total cellular m 6 A levels. Our findings suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.

Prevalence of emerging fungal infections is increasing, particularly among immunocompromised persons, children, and older persons. We report 108 cases of Scheffersomyces spartinae infection in pediatric patients from Karachi and other cities in Pakistan, of which 107 were identified from blood cultures. Cultures were initially misidentified as Clavispora lusitaniae by a biochemical assay before speciation as S. spartinae by whole-genome sequencing. All isolates were from children <12 years of age, and >69% were from children <1 month of age. Isolates were genetically distinct across regions of Pakistan; however, genetic diversity was low in isolates from patients in Karachi and nearby Nawabshah and had median differences of just 9 pairwise nucleotide variants. This study demonstrates S. spartinae is a potentially emerging pathogen in neonates and young infants in Pakistan. The findings highlight the limitations of phenotypic identification for detecting emerging fungal infections and underscore the value of molecular identification approaches.

Early detection of HIV is crucial for reducing transmission and ensuring timely initiation of antiretroviral therapy (ART), significantly improving patient outcomes. Although diagnostic tests have advanced from first-generation antibody detection assays to fourth-generation immunoassays that detect both HIV antibodies and the p24 antigen, these are limited to clinical labs. Their longer processing times, high costs, and the requirement for multiple patient visits highlight the need for rapid, affordable point-of-care (POC) diagnostics. This study introduces a nanomechanical cantilever-based biosensor for the rapid detection of HIV-1 p24 antigen, a key marker for early diagnosis. The platform demonstrated remarkable sensitivity, detecting p24 at concentrations as low as 100 fg/mL in solution and 1 pg/mL in human serum, and was quantitative within several orders of magnitude. After functionalizing the microcantilevers with two broadly cross-reactive monoclonal antibodies (ANT-152 and C65690M), the system was able to detect p24 from a wide range of HIV-1 subtypes. Furthermore, this biosensor was found to be compatible with various blood processing methods and with a direct electronic output. This platform's high sensitivity, specificity, and applicability across multiple HIV subtypes underscores its potential for future development into a next-generation POC diagnostic tool.

N6-methyladenosine (m6A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m6A during HIV-1 replication have produced conflicting results. Since m6A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m6A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m6A or 5-methylcytosine (m5C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m6A writer complex auxiliary proteins VIRMA and WTAP, and the m6A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m6A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m6A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.

The Coronavirus disease 2019 (COVID-19) pandemic had a profound global impact, yet children exhibited distinct clinical and epidemiological patterns compared to adults. Pediatric cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were generally characterized by milder disease, lower hospitalization rates, and few long-term sequelae. However, a subset of children developed severe complications such as multisystem inflammatory syndrome in children (MIS-C), highlighting the heterogeneity in disease presentation. Differences in immune system maturity and comorbidities likely contribute to the age-dependent manifestation of SARS-CoV-2 and other respiratory viruses. Persistent SARS-CoV-2 infection, particularly in immunocompromised individuals, has been implicated in the emergence of new viral variants with immune escape characteristics due to ongoing viral replication in the presence of selective pressure. While SARS-CoV-2 evolution in persistently infected adults has been well-documented, it is less clear how the virus evolves during persistent infection in the pediatric population. To address this question, we performed viral whole genome sequencing of longitudinal specimens collected from immunocompetent and immunocompromised pediatric COVID-19 patients. Similarly to what has been observed in adult cohorts, mutations associated with enhanced viral fitness and immune escape arose intra-host over time. Intra-host diversity accumulated at similar rates in immunocompetent and immunocompromised children, though more mutations overall were observed in the immunocompromised cohort due to the longer infection time courses. Overall, we identified similar viral evolutionary trends over the course of infection despite clinical differences in pediatric COVID-19 manifestation and severity. This similarity suggests that persistent infection in children may be an additional, but not unique, source of ongoing viral diversification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.

BACKGROUND. Survivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung -derived pro -inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans. METHODS. We measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single -cell RNA -sequencing. RESULTS. Microglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro -inflammatory cytokines. Peak levels of pro -inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines. CONCLUSION. Prolonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia -related cognitive dysfunction.