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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.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).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 × 10These 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.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.

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.

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. In this work, 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.

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.

Summary 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.

Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/β). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.

Interferon responses in the nasopharyngeal epithelium are critical to prevent severe COVID-19. Epithelial cells alone are unable to stop SARS-CoV-2 growth, but sufficient early interferon can be provided by non-epithelial cells or a co-infecting virus. Autoantibodies block early interferon, enhancing the risk of severe COVID-19., Interferons establish innate antiviral immunity. Two recent papers in JEM by Lopez et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20211211) and Cheemarla et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20210583) show that an appropriate supply of antiviral interferon enables epithelial cells of the nasopharyngeal mucosa to inhibit SARS-CoV-2 growth and that interferon-induced mucosal genes serve as biomarkers of infection.

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., Single-cell analysis of human airway epithelial cells infected with SARS-CoV-2 provides novel insights into viral replication, cell tropism, and host-viral interactions. This study reveals novel polyadenylated viral transcripts, preferential tropism for ciliated cells that later extends to other epithelial cell types, and cell-intrinsic expression of type I and type III IFNs and IL6 induced by infection, resulting in expression of interferon-stimulated genes in both infected and bystander cells.

SARS-CoV-2 replication in the upper respiratory tract initiates infection and leads to viral transmission. Cheemarla et al. show that a dynamic innate immune response can curtail viral replication early in infection, and that recent exposure to rhinovirus accelerates innate defenses and blocks viral replication., 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., Graphical Abstract

Improved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) for detection of SARS-CoV-2 using rapid DNA electrophoresis and alternatively using multiplex viral sequencing (mVseq). For RNA specimens extracted from nasopharyngeal (NP) swabs in viral transport media (VTM), our assays achieved a sensitivity for SARS-CoV-2 detection corresponding to cycle threshold (Ct) of 37.2 based on testing of these specimens using quantitative reverse transcription PCR (RT-qPCR). For NP swab-VTM specimens without prior RNA extraction, sensitivity was reduced to Ct of 31.6, which was due to lower concentration of SARS-CoV-2 genome copies in VTM compared to RNA-extracted samples. Assay turnaround time was 60 minutes using rapid gel electrophoresis, 90 minutes using Agilent Bioanalyzer, and 24-48 hours using Illumina sequencing, the latter of which required a second PCR to produce a sequence-ready library using m-RT-PCR products as the template. Our assays can be employed for high-throughput sequencing-based detection of SARS-CoV-2 directly from a clinical specimen without RNA isolation, while ease-of-use and low cost of the electrophoresis-based readout enables screening, particularly in resource-constrained settings.

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 (Charite) 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. This is a comparative analysis of the performance of the primer–probe sets from four open-source molecular diagnostic assays for SARS-CoV-2 recommended by the World Health Organization.

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 reopen1. 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 viruses2. 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.One Sentence SummaryWe describe a host-response based screening strategy to identify undiagnosed cases of COVID-19 and to expand capacity for PCR-based testing.

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, angiogenesis, 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.

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 polymerase chain reaction (qRT-PCR) assays are being used by clinical, research, and public health laboratories. However, it is currently unclear if results from different tests are comparable. Our goal was to evaluate the primer-probe sets used in four common diagnostic assays available on the World Health Organization (WHO) website. To facilitate this effort, we generated RNA transcripts to be used as assay standards and distributed them to other laboratories for internal validation. We then used these (1) RNA transcript standards, (2) full-length SARS-CoV-2 RNA, and (3) pre-COVID-19 nasopharyngeal swabs, and (4) clinical samples from COVID-19 patients to determine analytical efficiency and sensitivity of the qRT-PCR primer-probe sets. We show that all primer-probe sets can be used to detect SARS-CoV-2, but there are clear differences in the ability to differentiate between true negatives and positives with low amounts of virus. We found that several primer-probe sets cross-react with SARS-CoV-2-negative nasopharyngeal swabs. However, background cross-reactivity by the 2019-nCoV_N2 set issued by the US Centers for Disease Control and Prevention did not interfere with outcomes of the combined 9N19 and 9N29 assay when testing COVID-19 clinical samples. Our findings characterize the limitations of currently used primer-probe sets and can assist other laboratories in selecting appropriate assays for the detection of SARS-CoV-2.

Summary The novel coronavirus SARS-CoV-2 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 United States, 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 effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance., Graphical Abstract, Highlights • Connecticut’s COVID-19 outbreak resulted from multiple domestic virus introductions • SARS-CoV-2 genomic data indicate that coast-to-coast spread occurred in the United States • Risk of introduction by domestic air travel exceeded international travel in March • Restrictions on international travel did not significantly alter risk estimates, Using genomics and air travel information, the spread of SARS-CoV-2 in the United States from coast to coast is shown to be more a consequence of domestic introductions than of international travel.

Experimental evolution studies offer the possibility to examine whether closely related populations converge versus diverge in phenotype and genotype, when challenged to adapt under identical selection pressures. Human rhinoviruses (RV) are largely responsible for common cold illnesses, but little is known on whether different strains of these RNA viruses tend to evolve similarly when cultured on cells in laboratory tissue culture. Here we compared and contrasted evolution of two RV-A serotype strains (RV-1A, RV-1B) grown for ∼25 passages on mouse-derived LA-4 cells, expanding on a previous study concerning temperaturedependent innate immunity in mouse-adapted rhinovirus strains. Results showed faster adaptation of the population founded by RV-1B, consistent with classic theory predicting more rapid improvement in populations poorly adapted to their environment. Moreover, we observed increased molecular divergence between the two lineages as they adapted to mouse cells, demonstrated by selection targeting different viral capsid genes and different substitutions in affected viral replication genes, across the two populations. Our findings identify the genetic changes associated with human rhinovirus adaptation to a novel mouse host, which furthers the understanding of loci contributing to RV host jumps in mammals and efforts to develop mice as useful animal models for studying RV infection and pathogenicity.

BACKGROUND: Despite the high burden of respiratory infection and the importance of early and accurate diagnosis, there is no simple diagnostic test to rule in viral infection as a cause of respiratory symptoms. METHODS: We performed RNA sequencing on human nasal epithelial cells following stimulation of the intracellular viral recognition receptor RIG-I. Next, we evaluated whether measuring identified host mRNAs and proteins from patient nasopharyngeal swabs could predict the presence of a respiratory virus in the sample. RESULTS: Our first study showed that a signature of 3 mRNAs, CXCL10, IFIT2, and OASL, predicted respiratory virus detection with an accuracy of 97% (95% confidence interval [CI], 0.9–1.0), and identified proteins correlating with virus detection. In a second study, elevated CXCL11 or CXCL10 protein levels identified samples containing respiratory viruses, including viruses not on the initial test panel. Overall area under the curve (AUC) values were: CXCL11 AUC = 0.901 (95% CI, 0.86–0.94); CXCL10 AUC = 0.85 (95% CI, 0.80–0.91). CONCLUSIONS: Host antiviral mRNAs and single host proteins detectable using nasopharyngeal swabs accurately predict the presence of viral infection. This approach holds promise for developing rapid, cost-effective tests to improve management of patients with respiratory illnesses.

The novel coronavirus SARS-CoV-2 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 United States, 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 effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

The respiratory immune response consists of multiple tiers of cellular responses that are engaged in a sequential manner in order to control infections. The stepwise engagement of effector functions with progressively increasing host fitness costs limits tissue damage. In addition, specific mechanisms are in place to promote disease tolerance in response to respiratory infections. Environmental factors, obesity and the ageing process can alter the efficiency and regulation of this tiered response, increasing pathology and mortality as a result. In this Review, we describe the cell types that coordinate pathogen clearance and tissue repair through the serial secretion of cytokines, and discuss how the environment and comorbidity influence this response.

Most strains of rhinovirus (RV), the common cold virus, replicate better at cool temperatures found in the nasal cavity (33-35 °C) than at lung temperature (37 °C). Recent studies found that although 37 °C temperature suppressed RV growth largely by engaging the type 1 IFN response in infected epithelial cells, a significant temperature dependence to viral replication remained in cells devoid of IFN induction or signaling. To gain insight into IFN-independent mechanisms limiting RV replication at 37 °C, we studied RV infection in human bronchial epithelial cells and H1-HeLa cells. During the single replication cycle, RV exhibited temperature-dependent replication in both cell types in the absence of IFN induction. At 37 °C, earlier signs of apoptosis in RV-infected cells were accompanied by reduced virus production. Furthermore, apoptosis of epithelial cells was enhanced at 37 °C in response to diverse stimuli. Dynamic mathematical modeling and B cell lymphoma 2 (BCL2) overexpression revealed that temperature-dependent host cell death could partially account for the temperature-dependent growth observed during RV amplification, but also suggested additional mechanisms of virus control. In search of a redundant antiviral pathway, we identified a role for the RNA-degrading enzyme RNAseL. Simultaneous antagonism of apoptosis and RNAseL increased viral replication and dramatically reduced temperature dependence. These findings reveal two IFN-independent mechanisms active in innate defense against RV, and demonstrate that even in the absence of IFNs, temperature-dependent RV amplification is largely a result of host cell antiviral restriction mechanisms operating more effectively at 37 °C than at 33 °C.

Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33–35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I–like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, trancripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.

Leukocytes navigate through complex chemoattractant arrays, and in so doing, they must migrate from one chemoattractant source to another. By evaluating directional persistence and chemotaxis during neutrophil migration under agarose, we show that cells migrating away from a local chemoattractant, against a gradient, display true chemotaxis to distant agonists, often behaving as if the local gradient were without effect. We describe two interrelated properties of migrating cells that allow this to occur. First, migrating leukocytes can integrate competing chemoattractant signals, responding as if to the vector sum of the orienting signals present. Second, migrating cells display memory of their recent environment: cells' perception of the relative strength of orienting signals is influenced by their history, so that cells prioritize newly arising or newly encountered attractants. We propose that this cellular memory, by promoting sequential chemotaxis to one attractant after another, is in fact responsible for the integration of competitive orienting signals over time, and allows combinations of chemoattractants to guide leukocytes in a step-by-step fashion to their destinations within tissues.

Cells migrating within tissues may encounter multiple chemoattractant signals in complex spatial and temporal patterns. To understand leukocyte navigation in such settings, we have explored the migratory behavior of neutrophils in model scenarios where they are presented with two chemoattractant sources in various configurations. We show that, over a wide range of conditions, neutrophils can migrate “down” a local chemoattractant gradient in response to a distant gradient of a different chemoattractant. Furthermore, cells can chemotax effectively to a secondary distant agonist after migrating up a primary gradient into a saturating, nonorienting concentration of an initial attractant. Together, these observations suggest the potential for cells' step-by-step navigation from one gradient to another in complex chemoattractant fields. The importance of such sequential navigation is confirmed here in a model system in which neutrophil homing to a defined domain (a) requires serial responses to agonists presented in a defined spatial array, and (b) is a function of both the agonist combination and the sequence in which gradients are encountered. We propose a multistep model of chemoattractant-directed migration, which requires that leukocytes display multiple chemoattractant receptors for successful homing and provides for combinatorial determination of microenvironmental localization.

Many diseases may be caused by the combination of a specific host genetic background and a viral infection. Here, Foxman and Iwasaki describe how viral infections may be involved in type 1 diabetes, asthma and inflammatory bowel disease. New technologies have widened our view of 'complex diseases': those with both genetic and environmental risk factors. In this Review, we explore recent genetic and virological evidence implicating host–virus interactions in three diseases: type 1 diabetes, inflammatory bowel disease and asthma. The viruses implicated in these diseases cause mucosal infections that affect most of the population but are asymptomatic or mild in many hosts. These findings place a new emphasis on common viral infections as important environmental factors in the pathogenesis of complex diseases, and they compel the field to pursue a better understanding of host interactions with the human virome.

Early delivery attributable to preterm labor remains a significant cause of neonatal morbidity and mortality. Obstetricians often face the challenge of deciding whether to admit a woman to the hospital when she presents with possible preterm labor. In recent years, there has been much emphasis on developing laboratory tests that can help clinicians predict the likelihood of “true” preterm labor. The fetal fibronectin (fFN) assay has attracted interest because of its reported high negative predictive value for preterm labor. We implemented the Rapid Fetal Fibronectin lateral immunodiffusion assay on the TLiIQTM System (Adeza Biomedical) in the Brigham and Women’s Hospital clinical laboratory in October 2001. To determine how the test results would influence clinical decision-making in our hospital, we created a survey to be completed by clinicians at the time of test requisition. In this report, we compare the survey results, which reflect the pretest intention of the ordering clinicians, to the clinical course of tested patients. fFN, a uniquely glycosylated form of the abundant plasma protein fibronectin, is found in the placenta and amniotic fluid, primarily at the uteroplacental junction, and is released into the upper vagina near the onset of labor (1). Although the presence of fFN in vaginal fluids does not necessarily indicate the onset of labor (positive predictive value, 15–25%), its absence rules out labor within 7 days with a very high negative predictive value (97–99.5%) (1)(2)(3). Therefore, a negative result indicates a low likelihood of delivery, but a positive test should not be interpreted as an indication of labor or a reason for admission. From January to June 2002, all physicians requesting a fFN test at Brigham and Women’s Hospital were asked to complete a brief survey with the test requisition. On this survey, the physician was asked to …

Chemotaxis is a complex response of a cell to an external stimulus. It involves detecting and measuring the concentration of the chemoattractant, biochemical transmission of the information, and the motility and adhesive changes associated with the response. This unit describes a number of chemotaxis assays that can be used to identify chemoattractants individually and in large-scale screenings, to distinguish chemotaxis from chemokinesis, and to analyze cellular behavioral and biochemical responses. Some of these assays such as the filter, under agarose, and small population assays, can be used to monitor the behavior of large groups of cells; the bridge, pipet, and upshift assays can be used to analyze the responses of single cells.

Leukocytes express multiple chemoattractant receptors that can trigger adhesion and direct their migration. Regulation of such proadhesive and migratory responses must often occur in a complex cytokine milieu in vivo, in which multiple receptors may be engaged simultaneously or sequentially, Here we have examined the interplay between interleukin-8 (IL-8) receptor and formyl peptide receptor (fPR)-stimulation and its consequences for leukocyte adhesion and chemotactic responses. IL-8 has no significant effect on fMLP-stimulated adhesion and migration of human neutrophils, indicating that leukocytes have the potential to respond to sequential proadhesive and chemoattractant stimuli during homing and targeted migration. In contrast, fMLP at > or = 10 nM totally abrogated proadhesive and chemoattractant responses to IL-8, a trnas effect to which the fPR itself is relatively resistant. N-formyl peptides are released by invasive bacteria and lysed cells, and the dominance of the fPR may ensure that signals from these terminal phagocyte targets can override host-derived recruitment signaling through IL-8 and other chemokine receptors. Asymmetric inhibition of adhesion-triggering responses is also observed in lymphoid cells transfected with IL-8 receptor A and fPR, but in this cellular context chemotactic responses are bidirectionally abrogated, suggesting the potential for downstream desensitization of motility programs as well. Cross talk between chemoattractant receptors and their signaling pathways may help target leukocyte migration in the context of complex chemoattractant arrays in vivo.