Age-Dependent Reduction in Asthmatic Pathology through Reprogramming of Postviral Inflammatory Responses.

Asthma is a chronic disease of childhood, but for unknown reasons, disease activity sometimes subsides as children mature. In this study, we present clinical and animal model evidence suggesting that the age dependency of childhood asthma stems from an evolving host response to respiratory viral infection. Using clinical data, we show that societal suppression of respiratory virus transmission during coronavirus disease 2019 lockdown disrupted the traditional age gradient in pediatric asthma exacerbations, connecting the phenomenon of asthma remission to virus exposure. In mice, we show that asthmatic lung pathology triggered by Sendai virus (SeV) or influenza A virus is highly age-sensitive: robust in juvenile mice (4-6 wk old) but attenuated in mature mice (>3 mo old). Interestingly, allergen induction of the same asthmatic traits was less dependent on chronological age than viruses. Age-specific responses to SeV included a juvenile bias toward type 2 airway inflammation that emerged early in infection, whereas mature mice exhibited a more restricted bronchiolar distribution of infection that produced a distinct type 2 low inflammatory cytokine profile. In the basal state, aging produced changes to lung leukocyte burden, including the number and transcriptional landscape of alveolar macrophages (AMs). Importantly, depleting AMs in mature mice restored post-SeV pathology to juvenile levels. Thus, aging influences chronic outcomes of respiratory viral infection through regulation of the AM compartment and type 2 inflammatory responses to viruses. Our data provide insight into how asthma remission might develop in children.

Basal epithelial stem cells cross an alarmin checkpoint for postviral lung disease.

Epithelial cells are charged with protection at barrier sites, but whether this normally beneficial response might sometimes become dysfunctional still needs definition. Here, we recognized a pattern of imbalance marked by basal epithelial cell growth and differentiation that replaced normal airspaces in a mouse model of progressive postviral lung disease due to the Sendai virus. Single-cell and lineage-tracing technologies identified a distinct subset of basal epithelial stem cells (basal ESCs) that extended into gas-exchange tissue to form long-term bronchiolar-alveolar remodeling regions. Moreover, this cell subset was selectively expanded by crossing a cell-growth and survival checkpoint linked to the nuclear-localized alarmin IL-33 that was independent of IL-33 receptor signaling and instead connected to autocrine chromatin accessibility. This mechanism creates an activated stem-progenitor cell lineage with potential for physiological or pathological function. Thus, conditional loss of Il33 gene function in basal epithelial cells disrupted the homeostasis of the epithelial barrier at skin and gut sites but also markedly attenuated postviral disease in the lung based on the downregulation of remodeling and inflammation. Thus, we define a basal ESC strategy to deploy innate immune machinery that appears to overshoot the primordial goal of self-defense. Our findings reveal new targets to stratify and correct chronic and often deadly postviral disease.

Selective Imaging of Lung Macrophages Using [11C]PBR28-Based Positron Emission Tomography.

PURPOSE: We tested whether the translocator protein (TSPO)-targeted positron emission tomography (PET) tracer, N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine ([11C]PBR28), could distinguish macrophage dominant from neutrophilic inflammation better than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in mouse models of lung inflammation and assessed TSPO association with macrophages in lung tissue from the mouse models and in patients with chronic obstructive pulmonary disease (COPD). PROCEDURES: MicroPET imaging quantified [11C]PBR28 and [18F]FDG lung uptake in wild-type (Wt) C57BL/6J or heterozygous transgenic monocyte-deficient Wt/opT mice at 49 days after Sendai virus (SeV) infection, during macrophage-dominant inflammation, and in Wt mice at 3 days after SeV infection or 24 h after endotoxin instillation during neutrophilic inflammation. Immunohistochemical staining for TSPO in macrophages and neutrophils was performed using Mac3 and Ly6G for cell identification in mouse lung sections and CD68 and neutrophil elastase (NE) in human lung sections taken from explanted lungs from patients with COPD undergoing lung transplantation and donor lungs rejected for transplantation. Differences in tracer uptake among SeV-infected, endotoxin-treated, and uninfected/untreated control mice and in TSPO staining between neutrophils and macrophage populations in human lung sections were tested using analysis of variance. RESULTS: In Wt mice, [11C]PBR28 uptake (% injected dose/ml lung tissue) increased significantly with macrophage-dominant inflammation at 49 days (D49) after SeV infection compared to controls (p = <0.001) but not at 3 days (D49) after SeV infection (p = 0.167). [11C]PBR28 uptake was unchanged at 24 h after endotoxin instillation (p = 0.958). [18F]FDG uptake increased to a similar degree in D3 and D49 SeV-infected and endotoxin-treated Wt mice compared to controls with no significant difference in the degree of increase among the tested conditions. [11C]PBR28 but not [18F]FDG lung uptake at D49 post-SeV infection was attenuated in Wt/opT mice compared to Wt mice. TSPO localized predominantly to macrophages in mouse lung tissue by immunostaining, and TSPO staining intensity was significantly higher in CD68+ cells compared to neutrophils in the human lung sections. CONCLUSIONS: PET imaging with [11C]PBR28 can specifically detect macrophages versus neutrophils during lung inflammation and may be a useful biomarker of macrophage accumulation in lung disease.

TLR3-Activated Monocyte-Derived Dendritic Cells Trigger Progression from Acute Viral Infection to Chronic Disease in the Lung.

Acute infection is implicated as a trigger for chronic inflammatory disease, but the full basis for this switch is uncertain. In this study, we examine this issue using a mouse model of chronic lung disease that develops after respiratory infection with a natural pathogen (Sendai virus). We investigate this model using a combination of TLR3-deficient mice and adoptive transfer of immune cells into these mice versus the comparable responses in wild-type mice. We found that acute and transient expression of TLR3 on monocyte-derived dendritic cells (moDCs) was selectively required to induce long-term expression of IL-33 and consequent type 2 immune-driven lung disease. Unexpectedly, moDC participation was not based on canonical TLR3 signaling and relied instead on a trophic effect to expand the alveolar epithelial type 2 cell population beyond repair of tissue injury and thereby provide an enriched and persistent cell source of IL-33 required for progression to a disease phenotype that includes lung inflammation, hyperreactivity, excess mucus production, and remodeling. The findings thereby provide a framework wherein viral infection activates TLR3 in moDCs as a front-line immune cell niche upstream of lung epithelial cells to drive the type 2 immune response, leading to chronic inflammatory diseases of the lung (such as asthma and chronic obstructive pulmonary disease in humans) and perhaps progressive and long-term postviral disease in general.

Group 2 Innate Lymphoid Cells Must Partner with the Myeloid-Macrophage Lineage for Long-Term Postviral Lung Disease.

Group 2 innate lymphoid cells (ILC2s) are implicated in host defense and inflammatory disease, but these potential functional roles need more precise definition, particularly using advanced technologies to better target ILC2s and engaging experimental models that better manifest both acute infection and chronic, even lifelong, disease. In this study, we use a mouse model that applies an improved genetic definition of ILC2s via IL-7r-conditional Rora gene targeting and takes advantage of a distinct progression from acute illness to chronic disease, based on a persistent type 2 immune response to respiratory infection with a natural pathogen (Sendai virus). We first show that ILC2s are activated but are not required to handle acute illness after respiratory viral infection. In contrast, we find that this type of infection also activates ILC2s chronically for IL-13 production and consequent asthma-like disease traits that peak and last long after active viral infection is cleared. However, to manifest this type of disease, the Csf1-dependent myeloid-macrophage lineage is also active at two levels: first, at a downstream level, this lineage provides lung tissue macrophages (interstitial macrophages and tissue monocytes) that represent a major site of Il13 gene expression in the diseased lung; and second, at an upstream level, this same lineage is required for Il33 gene induction that is necessary to activate ILC2s for participation in disease at all, including IL-13 production. Together, these findings provide a revised scheme for understanding and controlling the innate immune response leading to long-term postviral lung diseases with features of asthma and related progressive conditions.

Respiratory Enterovirus (like Parainfluenza Virus) Can Cause Chronic Lung Disease if Protection by Airway Epithelial STAT1 Is Lost.

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1f/f mice and conventional Stat1-/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1-/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.

Influenza A Virus Infection Causes Chronic Lung Disease Linked to Sites of Active Viral RNA Remnants.

Clinical and experimental observations suggest that chronic lung disease is linked to respiratory viral infection. However, the long-term aspect of this relationship is not yet defined using a virus that replicates at properly high levels in humans and a corresponding animal model. In this study, we show that influenza A virus infection achieves 1 × 106-fold increases in viral load in the lung and dose-dependent severity of acute illness in mice. Moreover, these events are followed by persistence of negative- and positive-strand viral RNA remnants for 15 wk and chronic lung disease for at least 26 wk postinfection. The disease is manifested by focal areas of bronchiolization and mucus production that contain increased levels of viral RNA remnants along with mucin Muc5ac and Il13 mRNA compared with uninvolved areas of the lung. Excess mucus production and associated airway hyperreactivity (but not fibrosis or emphysema) are partially attenuated with loss of IL-13 production or signaling (using mice with IL-13 or STAT6 deficiency). These deficiencies cause reciprocal increases in l17a mRNA and neutrophils in the lung; however, none of these disease endpoints are changed with IL-13/IL-17a compared with IL-13 deficiency or STAT6/IL-17a compared with STAT6 deficiency. The results establish the capacity of a potent human respiratory virus to produce chronic lung disease focally at sites of active viral RNA remnants, likely reflecting locations of viral replication that reprogram the region. Viral dose dependency of disease also implicates high-level viral replication and severity of acute infection as determinants of chronic lung diseases such as asthma and COPD with IL-13-dependent and IL-13/IL-17-independent mechanisms.

Triggering Receptor Expressed on Myeloid Cells-2 Expression Tracks With M2-Like Macrophage Activity and Disease Severity in COPD.

BACKGROUND: Cell and animal models show a key role for Triggering Receptor Expressed on Myeloid Cells (TREM)-2 in chronic airway disease after viral infection, but comparable evidence in humans still needs to be established. METHODS: Lung tissue samples were obtained from lung transplant recipients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD (n = 16), nontransplantable donor lung tissues (n = 7), and resected lung tissues from patients at risk or with GOLD stage I through IV (n = 55) and were assessed for TREM-2 and TREM-1 messenger RNA (mRNA), protein expression, and other markers of a type 2 immune response. RESULTS: TREM2 (but not TREM1) mRNA levels were increased in GOLD stage IV COPD lung tissues compared with non-COPD lung tissues. TREM2 mRNA was coexpressed with its signaling molecule DAP12 and the macrophage marker CD68 and M2-macrophage markers CD206 and CHIT1. TREM-2 protein was also increased in COPD lung tissues and was localized to CD14+ macrophages by flow cytometry and CD68+ and CCR2+ macrophages by tissue immunostaining. In lung samples from patients at risk and with GOLD stage I through IV COPD, TREM2 but not TREM1 mRNA levels were also increased, and the ratio of TREM2/TREM1 mRNA levels was associated with increases in CHIT1 mRNA and decreases in FEV1 and FEV1/FVC. CONCLUSIONS: TREM-2 expression is increased in lung macrophages in COPD, particularly in comparison with TREM-1. Therefore, TREM-2 levels and the ratio of TREM-2/TREM-1 signifies M2 activation in COPD lung tissues and may help to guide therapeutics directed against the type 2 immune response in patients with this disease.

Structural basis for human respiratory syncytial virus NS1-mediated modulation of host responses.

Human respiratory syncytial virus (hRSV) is a major cause of morbidity and mortality in the paediatric, elderly and immune-compromised populations1,2. A gap in our understanding of hRSV disease pathology is the interplay between virally encoded immune antagonists and host components that limit hRSV replication. hRSV encodes for non-structural (NS) proteins that are important immune antagonists3-6; however, the role of these proteins in viral pathogenesis is incompletely understood. Here, we report the crystal structure of hRSV NS1 protein, which suggests that NS1 is a structural paralogue of hRSV matrix (M) protein. Comparative analysis of the shared structural fold with M revealed regions unique to NS1. Studies on NS1 wild type or mutant alone or in recombinant RSVs demonstrate that structural regions unique to NS1 contribute to modulation of host responses, including inhibition of type I interferon responses, suppression of dendritic cell maturation and promotion of inflammatory responses. Transcriptional profiles of A549 cells infected with recombinant RSVs show significant differences in multiple host pathways, suggesting that NS1 may have a greater role in regulating host responses than previously appreciated. These results provide a framework to target NS1 for therapeutic development to limit hRSV-associated morbidity and mortality.

PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

TREM-2 promotes macrophage survival and lung disease after respiratory viral infection.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease.

Novel mode of ISG15-mediated protection against influenza A virus and Sendai virus in mice.

UNLABELLED: ISG15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type I interferon stimulation. Hundreds of host proteins and a number of viral proteins have been shown to be ISGylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. ISG15(-/-) mice exhibit increased susceptibility to viral infection, and in the case of influenza B virus and vaccinia virus, ISG15 conjugation has been shown to restrict virus replication in vivo. A number of studies have also found that ISG15 is capable of antagonizing replication of some viruses in tissue culture. However, recent findings have demonstrated that ISG15 can protect mice from Chikungunya virus infection without affecting the virus burden. In order to better understand the function of ISG15 in vivo, we characterized the pathogenesis of influenza A virus and Sendai virus in ISG15(-/-) mice. We found that ISG15 protects mice from virus induced lethality by a conjugation-dependent mechanism in both of these models. However, surprisingly, we found that ISG15 had minimal effect on virus replication and did not have an obvious role in the modulation of the acute immune response to infection. Instead, we observed an increase in the number of diseased small airways in mice lacking ISG15. This ability of ISG15 to protect mice in a conjugation-dependent, but nonantiviral, manner from respiratory virus infection represents a previously undescribed role for ISG15 and demonstrates the importance of further characterization of ISG15 in vivo. IMPORTANCE: It has previously been demonstrated that ISG15(-/-) mice are more susceptible to a number of viral infections. Since ISG15 is one of the most strongly induced genes after type I interferon stimulation, analysis of ISG15 function has largely focused on its role as an antiviral molecule during acute infection. Although a number of studies have shown that ISG15 does have a small effect on virus replication in tissue culture, few studies have confirmed this mechanism of protection in vivo. In these studies we have found that while ISG15(-/-) mice are more susceptible to influenza A virus and Sendai virus infections, ISGylation does not appear to mediate this protection through the direct inhibition of virus replication or the modulation of the acute immune response. Thus, in addition to showing a novel mode of ISG15 mediated protection from virus infection, this study demonstrates the importance of studying the role of ISG15 in vivo.

Linking acute infection to chronic lung disease. The role of IL-33-expressing epithelial progenitor cells.

Respiratory infection is a common feature of the major human airway diseases, such as asthma and chronic obstructive pulmonary disease, but the precise link between acute infection and chronic lung disease is still undefined. In a mouse model of this process, parainfluenza virus infection is followed by long-term induction of IL-33 expression and release and in turn innate immune cell generation of IL-13 and consequent airway disease signified by excess mucus formation. IL-33 induction was traceable to a subset of secretoglobin-positive airway epithelial cells linked to progenitor/stem cell function. In corresponding studies of humans with chronic obstructive pulmonary disease, an increase in IL-33 production was also detected in concert with up-regulation of IL-13 and airway mucus formation. In this case, increased IL-33 production was localized to a subset of airway basal cells that maintain an endogenous capacity for increased pluripotency and ATP-regulated release of IL-33 even ex vivo. The results provide evidence of a sustainable epithelial cell population that may be activated by environmental danger signals to release IL-33 and thereby lead to IL-13-dependent disease. The progenitor nature of this IL-33-expressing ATP-responsive cell population could explain an acquired susceptibility to chronic airway disease. The findings may therefore provide a new paradigm to explain the role of viral infection and the innate immune system in chronic lung disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production. Further studies are needed to address the basis for this type of postviral reprogramming and the means to correct it and thereby restore airway mucosal immune function to normal.

Interferon response and respiratory virus control are preserved in bronchial epithelial cells in asthma.

BACKGROUND: Some investigators find a deficiency in IFN production from airway epithelial cells infected with human rhinovirus in asthma, but whether this abnormality occurs with other respiratory viruses is uncertain. OBJECTIVE: To assess the effect of influenza A virus (IAV) and respiratory syncytial virus (RSV) infection on IFN production and viral level in human bronchial epithelial cells (hBECs) from subjects with and without asthma. METHODS: Primary-culture hBECs from subjects with mild to severe asthma (n = 11) and controls without asthma (hBECs; n = 7) were infected with live or ultraviolet-inactivated IAV (WS/33 strain), RSV (Long strain), or RSV (A/2001/2-20 strain) with multiplicity of infection 0.01 to 1. Levels of virus along with IFN-ß and IFN-λ and IFN-stimulated gene expression (tracked by 2'-5'-oligoadenylate synthetase 1 and myxovirus (influenza virus) resistance 1 mRNA) were determined up to 72 hours postinoculation. RESULTS: After IAV infection, viral levels were increased 2-fold in hBECs from asthmatic subjects compared with nonasthmatic control subjects (P < .05) and this increase occurred in concert with increased IFN-λ1 levels and no significant difference in IFNB1, 2'-5'-oligoadenylate synthetase 1, or myxovirus (influenza virus) resistance 1mRNA levels. After RSV infections, viral levels were not significantly increased in hBECs from asthmatic versus nonasthmatic subjects and the only significant difference between groups was a decrease in IFN-λ levels (P < .05) that correlated with a decrease in viral titer. All these differences were found only at isolated time points and were not sustained throughout the 72-hour infection period. CONCLUSIONS: The results indicate that IAV and RSV control and IFN response to these viruses in airway epithelial cells is remarkably similar between subjects with and without asthma.

High-throughput screening normalized to biological response: application to antiviral drug discovery.

The process of conducting cell-based phenotypic screens can result in data sets from small libraries or portions of large libraries, making accurate hit picking from multiple data sets important for efficient drug discovery. Here, we describe a screen design and data analysis approach that allow for normalization not only between quadrants and plates but also between screens or batches in a robust, quantitative fashion, enabling hit selection from multiple data sets. We independently screened the MicroSource Spectrum and NCI Diversity Set II libraries using a cell-based phenotypic high-throughput screening (HTS) assay that uses an interferon-stimulated response element (ISRE)-driven luciferase-reporter assay to identify interferon (IFN) signal enhancers. Inclusion of a per-plate, per-quadrant IFN dose-response standard curve enabled conversion of ISRE activity to effective IFN concentrations. We identified 45 hits based on a combined z score ≥2.5 from the two libraries, and 25 of 35 available hits were validated in a compound concentration-response assay when tested using fresh compound. The results provide a basis for further analysis of chemical structure in relation to biological function. Together, the results establish an HTS method that can be extended to screening for any class of compounds that influence a quantifiable biological response for which a standard is available.

Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease.

Chronic obstructive lung disease is characterized by persistent abnormalities in epithelial and immune cell function that are driven, at least in part, by infection. Analysis of parainfluenza virus infection in mice revealed an unexpected role for innate immune cells in IL-13-dependent chronic lung disease, but the upstream driver for the immune axis in this model and in humans with similar disease was undefined. We demonstrate here that lung levels of IL-33 are selectively increased in postviral mice with chronic obstructive lung disease and in humans with very severe chronic obstructive pulmonary disease (COPD). In the mouse model, IL-33/IL-33 receptor signaling was required for Il13 and mucin gene expression, and Il33 gene expression was localized to a virus-induced subset of airway serous cells and a constitutive subset of alveolar type 2 cells that are both linked conventionally to progenitor function. In humans with COPD, IL33 gene expression was also associated with IL13 and mucin gene expression, and IL33 induction was traceable to a subset of airway basal cells with increased capacities for pluripotency and ATP-regulated release of IL-33. Together, these findings provide a paradigm for the role of the innate immune system in chronic disease based on the influence of long-term epithelial progenitor cells programmed for excess IL-33 production.

Monitoring in vivo changes in lung microstructure with ³He MRI in Sendai virus-infected mice.

Recently, a Sendai virus (SeV) model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human chronic obstructive pulmonary disease (COPD) and asthma, but the effect on distal lung parenchyma has not been investigated. The aim of our study is to image the time course and regional distribution of mouse lung microstructural changes in vivo after SeV infection. (1)H and (3)He diffusion magnetic resonance imaging (MRI) were successfully performed on five groups of C57BL/6J mice. (1)H MR images provided precise anatomical localization and lung volume measurements. (3)He lung morphometry was implemented to image and quantify mouse lung geometric microstructural parameters at different time points after SeV infection. (1)H MR images detected the SeV-induced pulmonary inflammation in vivo; spatially resolved maps of acinar airway radius R, alveolar depth h, and mean linear intercept Lm were generated from (3)He diffusion images. The morphometric parameters R and Lm in the infected group were indistinguishable from PBS-treated mice at day 21, increased slightly at day 49, and were increased with statistical significance at day 77 (p = 0.02). Increases in R and Lm of infected mice imply that there is a modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Our results indicate that (3)He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and that the Sendai mouse model has the potential to be a valid murine model of COPD.

Detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness.

BACKGROUND: A specific diagnosis of a lower respiratory viral infection is often difficult despite frequent clinical suspicion. This low diagnostic yield may be improved by use of sensitive detection methods and biomarkers. METHODS: The prevalence, clinical predictors and inflammatory mediator profile of respiratory viral infection in serious acute respiratory illness were investigated. Sequential bronchoalveolar lavage (BAL) fluids from all patients hospitalised with acute respiratory illness over 12 months (n=283) were tested for the presence of 17 respiratory viruses by multiplex PCR assay and for newly discovered respiratory viruses (bocavirus, WU and KI polyomaviruses) by single-target PCR. BAL samples also underwent conventional testing (direct immunoflorescence and viral culture) for respiratory virus at the clinician's discretion. 27 inflammatory mediators were measured in a subset of the patients (n=64) using a multiplex immunoassay. RESULTS: 39 respiratory viruses were detected in 37 (13.1% of total) patients by molecular testing, including rhinovirus (n=13), influenza virus (n=8), respiratory syncytial virus (n=6), human metapneumovirus (n=3), coronavirus NL63 (n=2), parainfluenza virus (n=2), adenovirus (n=1) and newly discovered viruses (n=4). Molecular methods were 3.8-fold more sensitive than conventional methods. Clinical characteristics alone were insufficient to separate patients with and without respiratory virus. The presence of respiratory virus was associated with increased levels of interferon gamma-inducible protein 10 (IP-10) (p<0.001) and eotaxin-1 (p=0.017) in BAL. CONCLUSIONS: Respiratory viruses can be found in patients with serious acute respiratory illness by use of PCR assays more frequently than previously appreciated. IP-10 may be a useful biomarker for respiratory viral infection.

Comparison of the Eragen Multi-Code Respiratory Virus Panel with conventional viral testing and real-time multiplex PCR assays for detection of respiratory viruses.

High-throughput multiplex assays for respiratory viruses are an important step forward in diagnostic virology. We compared one such assay, the PLx Multi-Code Respiratory Virus Panel (PLx-RVP), manufactured by Eragen Biosciences, Inc. (Madison, WI), with conventional virologic testing, consisting of fluorescent-antibody staining plus testing with the R-mix system and fibroblast tube cultures. The test set consisted of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been positive by conventional testing for a balanced selection of common respiratory viruses. Specimens yielding discrepant results were evaluated using a panel of respiratory virus PCR assays developed, characterized, and validated with clinical specimens. PLx-RVP increased the total rate of detection of viruses by 35.8%, and there was a 25.7% increase in the rate of detection of positive specimens. Reference PCR assay results corroborated the PLx-RVP result for 54 (82%) of 66 discrepancies with conventional testing. Of the 12 specimens with discrepancies between PLx-RVp and the reference PCRs, 6 were positive for rhinovirus by PLx-RVP and the presence of rhinovirus was confirmed by nucleotide sequencing. The remaining six specimens included five in which the PLx-RVP failed to detect parainfluenza virus and one in which the detection of influenza A virus by PLx-RVP could not be confirmed by the reference PCR. Taking the results of the reference PCR assay results into account, the sensitivities of the PLx-RVP for individual viruses ranged from 94 to 100% and the specificities ranged from 99 to 100%. We conclude that PLx-RVP is a highly accurate system for the detection of respiratory viruses and significantly improves the rate of detection of these viruses compared to that by conventional virologic testing.

KI polyomavirus detected in respiratory tract specimens from patients in St. Louis, Missouri.

BACKGROUND: Studies have reported the presence of KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in respiratory secretions of young patients. So far, evidence has not supported a link between infections with either virus and respiratory tract disease; however, there has not been a large comparison of KIPyV-infected patients to age-matched patient groups. METHODS: A retrospective study comparing clinical aspects of KIPyV-positive patients with respiratory syncytial virus (RSV)-positive, WUPyV-positive, and respiratory-virus negative patients. Using real-time polymerase chain reaction, 2599 respiratory samples from patients ranging from 1 day to 88 years of age were tested for KIPyV. Electronic medical records were reviewed for 65 cases, for a comparison group consisting of 195 patients negative for common respiratory viruses, and for 56 WUPyV-positive patients drawn from the same population. Twelve patients testing positive for KIPyV as the sole pathogen were matched to 36 RSV-positive patients and clinical features of both groups were compared. RESULTS: Seventy-two (2.8%) respiratory samples were positive for KIPyV. Another virus was detected in 71% of the KIPyV-positive samples. Analysis showed no statistically significant differences in clinical manifestations between KIPyV-positive patients and patients negative for common respiratory viruses, however, clinical characteristics of KIPyV-positive patients were less severe than those of patients positive for RSV. KIPyVpositive patients >or=3 years of age were usually immunocompromised in contrast to the younger children with KIPyV. CONCLUSIONS: This study did not demonstrate a link between KIPyV infection and symptomatic respiratory disease. Patients positive for KIPyV exhibited less severe clinical symptoms than patients positive for RSV.