High burden of viruses and bacterial pathobionts drives heightened nasal innate immunity in children.

Studies during the COVID-19 pandemic showed that children had heightened nasal innate immune responses compared with adults. To evaluate the role of nasal viruses and bacteria in driving these responses, we performed cytokine profiling and comprehensive, symptom-agnostic testing for respiratory viruses and bacterial pathobionts in nasopharyngeal samples from children tested for SARS-CoV-2 in 2021-22 (n = 467). Respiratory viruses and/or pathobionts were highly prevalent (82% of symptomatic and 30% asymptomatic children; 90 and 49% for children <5 years). Virus detection and load correlated with the nasal interferon response biomarker CXCL10, and the previously reported discrepancy between SARS-CoV-2 viral load and nasal interferon response was explained by viral coinfections. Bacterial pathobionts correlated with a distinct proinflammatory response with elevated IL-1ß and TNF but not CXCL10. Furthermore, paired samples from healthy 1-year-olds collected 1-2 wk apart revealed frequent respiratory virus acquisition or clearance, with mucosal immunophenotype changing in parallel. These findings reveal that frequent, dynamic host-pathogen interactions drive nasal innate immune activation in children.

Considerations for viral co-infection studies in human populations.

When respiratory viruses co-circulate in a population, individuals may be infected with multiple pathogens and experience possible virus-virus interactions, where concurrent or recent prior infection with one virus affects the infection process of another virus. While experimental studies have provided convincing evidence for within-host mechanisms of virus-virus interactions, evaluating evidence for viral interference or potentiation using population-level data has proven more difficult. Recent studies have quantified the prevalence of co-detections using populations drawn from clinical settings. Here, we focus on selection bias issues associated with this study design. We provide a quantitative account of the conditions under which selection bias arises in these studies, review previous attempts to address this bias, and propose unbiased study designs with sample size estimates needed to ascertain viral interference. We show that selection bias is expected in cross-sectional co-detection prevalence studies conducted in clinical settings, except under a strict set of assumptions regarding the relative probabilities of being included in a study limited to individuals with clinical disease under different viral states. Population-wide studies that collect samples from participants irrespective of their clinical status would meanwhile require large sample sizes to be sufficiently powered to detect viral interference, suggesting that a study's timing, inclusion criteria, and the expected magnitude of interference are instrumental in determining feasibility.

Double-take: SARS-CoV-2 has evolved to evade human innate immunity, twice.

Sequential replacement of the dominant SARS-CoV-2 virus by new variants has been a striking feature of the COVID-19 pandemic. In two recent articles, Bouhaddou et al. and Kehrer et al. demonstrate that, like the original virus, the SARS-CoV-2 omicron strain has progressively evolved to evade host innate immune defenses.

Causal identification of single-cell experimental perturbation effects with CINEMA-OT.

Recent advancements in single-cell technologies allow characterization of experimental perturbations at single-cell resolution. While methods have been developed to analyze such experiments, the application of a strict causal framework has not yet been explored for the inference of treatment effects at the single-cell level. Here we present a causal-inference-based approach to single-cell perturbation analysis, termed CINEMA-OT (causal independent effect module attribution + optimal transport). CINEMA-OT separates confounding sources of variation from perturbation effects to obtain an optimal transport matching that reflects counterfactual cell pairs. These cell pairs represent causal perturbation responses permitting a number of novel analyses, such as individual treatment-effect analysis, response clustering, attribution analysis, and synergy analysis. We benchmark CINEMA-OT on an array of treatment-effect estimation tasks for several simulated and real datasets and show that it outperforms other single-cell perturbation analysis methods. Finally, we perform CINEMA-OT analysis of two newly generated datasets: (1) rhinovirus and cigarette-smoke-exposed airway organoids, and (2) combinatorial cytokine stimulation of immune cells. In these experiments, CINEMA-OT reveals potential mechanisms by which cigarette-smoke exposure dulls the airway antiviral response, as well as the logic that governs chemokine secretion and peripheral immune cell recruitment.

Viral interference during influenza A-SARS-CoV-2 coinfection of the human airway epithelium and reversal by oseltamivir.

To gain insight into interactions among respiratory viruses, we modeled influenza A virus (IAV) - SARS-CoV-2 coinfections using differentiated human airway epithelial cultures. Replicating IAV induced a more robust interferon response than SARS-CoV-2 and suppressed SARS-CoV-2 replication in both sequential and simultaneous infections, whereas SARS-CoV-2 did not enhance host cell defense during influenza infection or suppress IAV replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the host antiviral response and restored SARS-CoV-2 replication. These results demonstrate how perturbations in one viral infection can impact its effect on a coinfecting virus.

PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection.

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial1-4. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR-Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. Its robust activity extended to other highly pathogenic coronaviruses, was functionally conserved in bats and mice, and interfered with the uptake of SARS-CoV-2 in both the endocytic and the TMPRSS2-dependent fusion routes. Whole-cell 4Pi single-molecule switching nanoscopy together with bipartite nano-reporter assays found that PLSCR1 directly targeted SARS-CoV-2-containing vesicles to prevent spike-mediated fusion and viral escape. A PLSCR1 C-terminal ß-barrel domain-but not lipid scramblase activity-was essential for this fusogenic blockade. Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people3,4, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Counterintuitive effect of antiviral therapy on influenza A-SARS-CoV-2 coinfection due to viral interference.

The resurgence of influenza and continued circulation of SARS-CoV-2 raise the question of how these viruses interact in a co-exposed host. Here we studied virus-virus and host-virus interactions during influenza A virus (IAV) -SARS-CoV-2 coinfection using differentiated cultures of the human airway epithelium. Coexposure to IAV enhanced the tissue antiviral response during SARS-CoV-2 infection and suppressed SARS-CoV-2 replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the antiviral response and paradoxically restored SARS-CoV-2 replication. These results highlight the importance of diagnosing coinfections and compel further study of how coinfections impact the outcome of antiviral therapy.

Respiratory viruses: New frontiers-a Keystone Symposia report.

Respiratory viruses are a common cause of morbidity and mortality around the world. Viruses like influenza, RSV, and most recently SARS-CoV-2 can rapidly spread through a population, causing acute infection and, in vulnerable populations, severe or chronic disease. Developing effective treatment and prevention strategies often becomes a race against ever-evolving viruses that develop resistance, leaving therapy efficacy either short-lived or relevant for specific viral strains. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Respiratory Viruses: New Frontiers." Researchers presented new insights into viral biology and virus-host interactions to understand the mechanisms of disease and identify novel treatment and prevention approaches that are effective, durable, and broad.

Nasal host response-based screening for undiagnosed respiratory viruses: a pathogen surveillance and detection study.

BACKGROUND: Symptomatic patients who test negative for common viruses are an important possible source of unrecognised or emerging pathogens, but metagenomic sequencing of all samples is inefficient because of the low likelihood of finding a pathogen in any given sample. We aimed to determine whether nasopharyngeal CXCL10 screening could be used as a strategy to enrich for samples containing undiagnosed viruses. METHODS: In this pathogen surveillance and detection study, we measured CXCL10 concentrations from nasopharyngeal swabs from patients in the Yale New Haven health-care system, which had been tested at the Yale New Haven Hospital Clinical Virology Laboratory (New Haven, CT, USA). Patients who tested negative for a panel of respiratory viruses using multiplex PCR during Jan 23-29, 2017, or March 3-14, 2020, were included. We performed host and pathogen RNA sequencing (RNA-Seq) and analysis for viral reads on samples with CXCL10 higher than 1 ng/mL or CXCL10 testing and quantitative RT-PCR (RT-qPCR) for SARS-CoV-2. We used RNA-Seq and cytokine profiling to compare the host response to infection in samples that were virus positive (rhinovirus, seasonal coronavirus CoV-NL63, or SARS-CoV-2) and virus negative (controls). FINDINGS: During Jan 23-29, 2017, 359 samples were tested for ten viruses on the multiplex PCR respiratory virus panel (RVP). 251 (70%) were RVP negative. 60 (24%) of 251 samples had CXCL10 higher than 150 pg/mL and were identified for further analysis. 28 (47%) of 60 CXCL10-high samples were positive for seasonal coronaviruses. 223 (89%) of 251 samples were PCR negative for 15 viruses and, of these, CXCL10-based screening identified 32 (13%) samples for further analysis. Of these 32 samples, eight (25%) with CXCL10 concentrations higher than 1 ng/mL and sufficient RNA were selected for RNA-Seq. Microbial RNA analysis showed the presence of influenza C virus in one sample and revealed RNA reads from bacterial pathobionts in four (50%) of eight samples. Between March 3 and March 14, 2020, 375 (59%) of 641 samples tested negative for 15 viruses on the RVP. 32 (9%) of 375 samples had CXCL10 concentrations ranging from 100 pg/mL to 1000 pg/mL and four of those were positive for SARS-CoV-2. CXCL10 elevation was statistically significant, and a distinguishing feature was found in 28 (8%) of 375 SARS-CoV-2-negative samples versus all four SARS-CoV-2-positive samples (p=4·4 × 10-5). Transcriptomic signatures showed an interferon response in virus-positive samples and an additional neutrophil-high hyperinflammatory signature in samples with high amounts of bacterial pathobionts. The CXCL10 cutoff for detecting a virus was 166·5 pg/mL for optimal sensitivity and 1091·0 pg/mL for specificity using a clinic-ready automated microfluidics-based immunoassay. INTERPRETATION: These results confirm CXCL10 as a robust nasopharyngeal biomarker of viral respiratory infection and support host response-based screening followed by metagenomic sequencing of CXCL10-high samples as a practical approach to incorporate clinical samples into pathogen discovery and surveillance efforts. FUNDING: National Institutes of Health, the Hartwell Foundation, the Gruber Foundation, Fast Grants for COVID-19 research from the Mercatus Center, and the Huffman Family Donor Advised Fund.

Complement Plays a Critical Role in Inflammation-Induced Immunoprophylaxis Failure in Mice.

Complement impacts innate and adaptive immunity. Using a model in which the human KEL glycoprotein is expressed on murine red blood cells (RBCs), we have shown that polyclonal immunoprophylaxis (KELIg) prevents alloimmunization to transfused RBCs when a recipient is in their baseline state of heath but with immunoprophylaxis failure occurring in the presence of a viral-like stimulus. As complement can be detected on antibody coated KEL RBCs following transfusion, we hypothesized that recipient complement synergizes with viral-like inflammation to reduce immunoprophylaxis efficacy. Indeed, we found recipient C3 and C1q were critical to immunoprophylaxis failure in the setting of a viral-like stimulus, with no anti-KEL IgG alloantibodies generated in C3-/- or C1q-/- mice following KELIg treatment and KEL RBC transfusion. Differences in RBC uptake were noted in mice lacking C3, with lower consumption by splenic and peripheral blood inflammatory monocytes. Finally, no alloantibodies were detected in the setting of a viral-like stimulus following KELIg treatment and KEL RBC transfusion in mice lacking complement receptors (CR1/2-/-), narrowing key cells for immunoprophylaxis failure to those expressing these complement receptors. In-vitro studies showed complement fixed opsonized RBCs were significantly less likely to bind to B-cells from CR1/2-/- than wild type mice, potentially implicating lowered B-cell activation threshold in the presence of complement as being responsible for these findings. We thus propose a two-hit model for inflammation-induced immunoprophylaxis failure, where the first "hit" is recipient inflammation and the second "hit" is complement production/sensing. These results may have translational relevance to antigen-antibody interactions in humans.

Dynamic innate immune response determines susceptibility to SARS-CoV-2 infection and early replication kinetics.

Initial replication of SARS-CoV-2 in the upper respiratory tract is required to establish infection, and the replication level correlates with the likelihood of viral transmission. Here, we examined the role of host innate immune defenses in restricting early SARS-CoV-2 infection using transcriptomics and biomarker-based tracking in serial patient nasopharyngeal samples and experiments with airway epithelial organoids. SARS-CoV-2 initially replicated exponentially, with a doubling time of ∼6 h, and induced interferon-stimulated genes (ISGs) in the upper respiratory tract, which rose with viral replication and peaked just as viral load began to decline. Rhinovirus infection before SARS-CoV-2 exposure accelerated ISG responses and prevented SARS-CoV-2 replication. Conversely, blocking ISG induction during SARS-CoV-2 infection enhanced viral replication from a low infectious dose. These results show that the activity of ISG-mediated defenses at the time of SARS-CoV-2 exposure impacts infection progression and that the heterologous antiviral response induced by a different virus can protect against SARS-CoV-2.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.

Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection.

The interferon response is a potent antiviral defense mechanism, but its effectiveness depends on its timing relative to viral replication. Here, we report viral replication and host response kinetics in patients at the start of SARS-CoV-2 infection and explore the impact of these kinetics experimentally. In both longitudinal patient nasopharyngeal samples and airway epithelial organoids, we found that SARS-CoV-2 initially replicated exponentially with a doubling time of ~6hr, and induced interferon stimulated genes (ISGs) with delayed timing relative to viral replication. Prior exposure to rhinovirus increased ISG levels and blocked SARS-CoV-2 replication. Conversely, inhibiting ISG induction abrogated interference by rhinovirus and enhanced SARS-CoV-2 replication rate. These results demonstrate the importance of initial interferon-mediated defenses in determining the extent to which SARS-CoV-2 can replicate at the start of infection and indicate that biological variables that alter the airway interferon response, including heterologous induction of innate immunity by other viruses, could profoundly impact SARS-CoV-2 susceptibility and transmission.

An in vivo atlas of host-pathogen transcriptomes during Streptococcus pneumoniae colonization and disease.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

Interference between rhinovirus and influenza A virus: a clinical data analysis and experimental infection study.

BACKGROUND: During the 2009 pandemic of an emerging influenza A virus (IAV; H1N1pdm09), data from several European countries indicated that the spread of the virus might have been interrupted by the annual autumn rhinovirus epidemic. We aimed to investigate viral interference between rhinovirus and IAV with use of clinical data and an experimental model. METHODS: We did a clinical data analysis and experimental infection study to investigate the co-occurrence of rhinovirus and IAV in respiratory specimens from adults (≥21 years) tested with a multiplex PCR panel at Yale-New Haven Hospital (CT, USA) over three consecutive winter seasons (Nov 1 to March 1, 2016-17, 2017-18, and 2018-19). We compared observed versus expected co-detections using data extracted from the Epic Systems electronic medical record system. To assess how rhinovirus infection affects subsequent IAV infection, we inoculated differentiated primary human airway epithelial cultures with rhinovirus (HRV-01A; multiplicity of infection [MOI] 0·1) or did mock infection. On day 3 post-infection, we inoculated the same cultures with IAV (H1N1 green fluorescent protein [GFP] reporter virus or H1N1pdm09; MOI 0·1). We used reverse transcription quantitative PCR or microscopy to quantify host cell mRNAs for interferon-stimulated genes (ISGs) on day 3 after rhinovirus or mock infection and IAV RNA on days 4, 5, or 6 after rhinovirus or mock infection. We also did sequential infection studies in the presence of BX795 (6 µM), to inhibit the interferon response. We compared ISG expression and IAV RNA and expression of GFP by IAV reporter virus. FINDINGS: Between July 1, 2016, and June 30, 2019, examination of 8284 respiratory samples positive for either rhinovirus (n=3821) or IAV (n=4463) by any test method was used to establish Nov 1 to March 1 as the period of peak virus co-circulation. After filtering for samples within this time frame meeting the inclusion criteria (n=13 707), there were 989 (7·2%) rhinovirus and 922 (6·7%) IAV detections, with a significantly lower than expected odds of co-detection (odds ratio 0·16, 95% CI 0·09-0·28). Rhinovirus infection of cell cultures induced ISG expression and protected against IAV infection 3 days later, resulting in an approximate 50 000-fold decrease in IAV H1N1pdm09 viral RNA on day 5 post-rhinovirus inoculation. Blocking the interferon response restored IAV replication following rhinovirus infection. INTERPRETATION: These findings show that one respiratory virus can block infection with another through stimulation of antiviral defences in the airway mucosa, supporting the idea that interference from rhinovirus disrupted the 2009 IAV pandemic in Europe. These results indicate that viral interference can potentially affect the course of an epidemic, and this possibility should be considered when designing interventions for seasonal influenza epidemics and the ongoing COVID-19 pandemic. FUNDING: National Institutes of Health, National Institute of General Medical Sciences, and the Yale Department of Laboratory Medicine.

Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets.

The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the critical need for accurate and rapid diagnostic assays to prompt clinical and public health interventions. Currently, several quantitative reverse transcription-PCR (RT-qPCR) assays are being used by clinical, research and public health laboratories. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR analytical efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charité) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium.

SARS-CoV-2, the causative agent of COVID-19, has tragically burdened individuals and institutions around the world. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon-stimulated genes in both infected and bystander cells. We observe similar gene expression changes from a COVID-19 patient ex vivo. In addition, we developed a new computational method termed CONditional DENSity Embedding (CONDENSE) to characterize and compare temporal gene dynamics in response to infection, which revealed genes relating to endothelin, angio-genesis, interferon, and inflammation-causing signaling pathways. In this study, we conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and a COVID-19 patient and revealed genes, cell types, and cell state changes associated with infection.

Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology.

Since its emergence and detection in Wuhan, China in late 2019, the novel coronavirus SARS-CoV-2 has spread to nearly every country around the world, resulting in hundreds of thousands of infections to date. The virus was first detected in the Pacific Northwest region of the United States in January, 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the U.S., we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated impacts of federal travel restrictions. This study provides evidence for widespread, sustained transmission of SARS-CoV-2 within the U.S. and highlights the critical need for local surveillance.

Host response-based screening to identify undiagnosed cases of COVID-19 and expand testing capacity.

The COVID-19 pandemic has created unprecedented challenges in diagnostic testing. At the beginning of the epidemic, a confluence of factors resulted in delayed deployment of PCR-based diagnostic tests, resulting in lack of testing of individuals with symptoms of the disease. Although these tests are now more widely available, it is estimated that a three- to ten-fold increase in testing capacity will be required to ensure adequate surveillance as communities reopen(1). In response to these challenges, we evaluated potential roles of host-response based screening in the diagnosis of COVID-19. Previous work from our group showed that the nasopharyngeal (NP) level of CXCL10, a protein produced as part of the host response to viral infection, is a sensitive predictor of respiratory virus infection across a wide spectrum of viruses(2). Here, we show that NP CXCL10 is elevated during SARS-CoV-2 infection and use a CXCL10-based screening strategy to identify four undiagnosed cases of COVID-19 in Connecticut in early March. In a second set of samples tested at the Yale New Haven Hospital, we show that NP CXCL10 had excellent performance as a rule-out test (NPV 0.99, 95% C.I. 0.985-0.997). Our results demonstrate how biomarker-based screening could be used to leverage existing PCR testing capacity to rapidly enable widespread testing for COVID-19.