Human Metapneumovirus Infection Research Articles

G378 Demographics and selected clinical features of paediatric human metapneumovirus infection

Aims This study aimed to characterise seasonal variation, co-infection rates, susceptible groups (by gender, age and co-morbidities) and selected clinical features of childhood human metapneumovirus (HMPV) infection. Methods The study was a retrospective analysis of 656 HMPV-positive respiratory samples collected from paediatric patients aged 0–15 years processed from January 2010 to December 2013 using real time PCR assays. In addition, 200 HMPV-positive samples from January 2012 to January 2013 were cross-referenced with electronic discharge summaries and descriptive statistical analyses of selected clinical features were performed. Results 377 of 656 (57%) HMPV-positive samples were from male patients. HMPV was most frequently detected in children aged 6–9 months and the median age of patients studied was 15 months. Seasonal patterns of HMPV infection varied from year to year. The peak in HMPV-positive isolates occurred between February and May. 117 of 656 (17.5%) samples were positive for other respiratory viruses in addition to HMPV. The most common co-infections were due to rhinovirus (58/656, 8.84%) and adenovirus (36/656, 5.49%). 81 (40%) of the 200 fully-characterised patients were hospitalised, 7 (3.5%) of whom required intensive care (ICU) or high dependency (HDU) admission. Rashes were reported in 14 (7%) and febrile seizures in 9 (4.5%) of 200 patients. These clinical features were more common in children with co-infections [6/40 (15%) and 4/40 (10%) respectively]. Conclusions Male gender is suggested as a predisposing factor for HMPV infection, along with younger age. Seasonal variation of HMPV infection in Scottish children appears different to the reported winter peaks of other studies, with peak incidence occurring between February and May in our dataset. Co-infection is common, and most frequently associated with rhinovirus or adenovirus. Rashes and febrile seizures are relatively common in HMPV-positive patients, especially those co-infected with other respiratory pathogens. Human metapneumovirus is a significant cause of morbidity in children. Further, larger-scale epidemiological research appears warranted, along with work to develop new therapies aimed at targeting HMPV.

Role of Type I Interferon Signaling in Human Metapneumovirus Pathogenesis and Control of Viral Replication

Type I IFN signaling, which is initiated through activation of the alpha interferon receptor (IFNAR), regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses, including human metapneumovirus (HMPV), have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to the control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR(-/-)) mice to assess the effect of IFNAR signaling on HMPV replication and the CD8(+) T cell response. HMPV-infected IFNAR(-/-) mice had a higher peak of early viral replication but cleared the virus with kinetics similar to those of wild-type (WT) mice. However, IFNAR(-/-) mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8(+) T cells of IFNAR(-/-) mice after HMPV infection expressed levels of the inhibitory receptor programmed death 1 (PD-1) similar to those of WT mice. However, despite lower expression of inhibitory programmed death ligand 1 (PD-L1), HMPV-specific CD8(+) T cells of IFNAR(-/-) mice were more functionally impaired than those of WT mice and upregulated the inhibitory receptor Tim-3. Analysis of the antigen-presenting cell subsets in the lungs revealed that the expansion of PD-L1(low) dendritic cells (DCs), but not PD-L1(high) alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8(+) T cell impairment. Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8(+) T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8(+) T cell impairment via PD-1-PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune responses to HMPV by using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8(+) T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor Tim-3 may contribute to CD8(+) T cell impairment.

Human Metapneumovirus Infections in Hematopoietic Cell Transplant Recipients: Seasonality and Factors Associated with Progression to Lower Respiratory Tract Disease

Human Metapneumovirus Infections in Hematopoietic Cell Transplant Recipients: Seasonality and Factors Associated with Progression to Lower Respiratory Tract Disease

Adoptive T-Cell Therapy to Prevent and Treat Human Metapneumovirus (hMPV) Infections Post Hematopoietic Stem Cell Transplant (HSCT)

Adoptive T-Cell Therapy to Prevent and Treat Human Metapneumovirus (hMPV) Infections Post Hematopoietic Stem Cell Transplant (HSCT)

Role of dietary antioxidants in human metapneumovirus infection

Human metapneumovirus (hMPV) is a major cause of respiratory tract infections in children, elderly and immunocompromised hosts, for which no vaccine or treatment are currently available. Oxidative stress and inflammatory responses represent important pathogenic mechanism(s) of hMPV infection. Here, we explored the potential protective role of dietary antioxidants in hMPV infection. Treatment of airway epithelial cells with resveratrol and quercetin during hMPV infection significantly reduced cellular oxidative damage, inflammatory mediator secretion and viral replication, without affecting viral gene transcription and protein synthesis, indicating that inhibition of viral replication occurred at the level of viral assembly and/or release. Modulation of proinflammatory mediator expression occurred through the inhibition of transcription factor nuclear factor (NF)-κB and interferon regulatory factor (IRF)-3 binding to their cognate site of endogenous gene promoters. Our results indicate the use of dietary antioxidants as an effective treatment approach for modulating hMPV induced lung oxidative damage and inflammation.

Diagnosis of community-acquired acute respiratory illness: From conventional microbiological methods to molecular detection (multiplex)

Investigations of the etiologic agents of community-acquired acute respiratory illness may lead to better treatment decisions and patient outcomes. In a routine care setting, we assessed the diagnostic performance of a multiplex PCR assay with respect to conventional microbiological methods, in a continuous series of adult cases of community-acquired acute respiratory illness. We enrolled 279 adult patients hospitalised for community-acquired acute respiratory illness at Tours University Hospital during the winter of 2011–2012. Respiratory samples (mostly nasopharyngeal aspirates) were studied prospectively by indirect immunofluorescence assay and multiplex PCR, that enable detection of 8 viruses and 21 respiratory pathogens respectively. In total, 255 of the 279 (91.4%) samples had interpretable results by both methods. At least one respiratory pathogen was detected by multiplex PCR in 171 specimens (65%). Overall, 130 (76%) of the 171 positive samples were positive for only one respiratory pathogen, 37 (22%) samples were positive for two pathogens and four (2%) were positive for three pathogens. With indirect immunofluorescence assay, a respiratory virus was detected in 27 of the 255 (11%) specimens. Indirect immunofluorescence assay detected some of the influenza virus A (15/51, 29%) infections identified by multiplex PCR and some (7/15, 47%) human metapneumovirus and (5/12, 42%) respiratory syncytial virus infections, but it did not detect all the adenovirus infections. Thus, access to multiplex molecular assays improves the diagnostic spectrum and accuracy over conventional methods, increasing the frequency of identification of the respiratory pathogens involved in community-acquired acute respiratory illness.

Differential mucin expression by respiratory syncytial virus and human metapneumovirus infection in human epithelial cells.

Mucins (MUC) constitute an important component of the inflammatory and innate immune response. However, the expression of these molecules by respiratory viral infections is still largely unknown. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two close-related paramyxoviruses that can cause severe low respiratory tract disease in infants and young children worldwide. Currently, there is not vaccine available for neither virus. In this work, we explored the differential expression of MUC by RSV and hMPV in human epithelial cells. Our data indicate that the MUC expression by RSV and hMPV differs significantly, as we observed a stronger induction of MUC8, MUC15, MUC20, MUC21, and MUC22 by RSV infection while the expression of MUC1, MUC2, and MUC5B was dominated by the infection with hMPV. These results may contribute to the different immune response induced by these two respiratory viruses.

Design and Evaluation of a Multi-Epitope Peptide of Human Metapneumovirus

Objectives: No licensed vaccines or therapeutic agents for human metapneumovirus (hMPV) infection exist to date. We aimed to construct a multi-epitope peptide (MEP) of hMPV to show promising results for epitope-based vaccine development. Methods: Six independent algorithms were screened to predict B-cell epitopes of hMPV, and three algorithms were used to predict cytotoxic T lymphocyte and T helper (Th) lymphocyte epitopes. Predicted epitopes were assembled in series with the spacers GPGPG and KK introduced, termed MEP. Recombinant mep genes were inserted into pET32a(+) plasmid and expressed in Escherichia coli strain BL21 (DE3). BALB/c mice were immunized with MEP with different adjuvants. Antibody titer, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) activity and splenocyte cytokines were detected 2 weeks later after the last immunization. Microneutralization assay was used to detect neutralizing antibodies. Results: Six B-cell epitopes, four CTL epitopes and two Th epitopes were screened to construct the mep gene. Expressed MEP induced >10⁴ antibodies in BALB/c mice, and produced anti-MEP antibody reacting with hMPV strains specifically as detected in indirect fluorescent assay (the titer was 160). The lymphocyte proliferation index, CTL activity and splenocyte cytokines of the MEP immunization groups were higher than in the control group (p < 0.05). Both IgG1 and IgG2a antibodies could be detected in the different groups, and balanced Th1/Th2 cytokines were secreted by splenocytes in these groups. The mean neutralizing titers of the MEP+CpG ODN, MEP+Alum and MEP+Alum+ CpG ODN groups were 87 (95% CI 50-126), 93 (95% CI 67-121) and 96 (95% CI 69-147), respectively. Conclusion: MEP of hMPV elicited both strong humoral immunity and cell-mediated immunity in mice. The anti-MEP serum could neutralize hMPV infection in vitro. Joint use of CpG ODN and aluminum hydroxide adjuvants obtained the best immune effects. This study may contribute to hMPV epitope-based vaccine development.

Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice

Event Abstract Back to Event Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice Pablo F. Céspedes1, Margarita K. Lay1*, Christian E. Palavecino1, Miguel Á. León1, Rodrigo A. Díaz1, Francisco J. Salazar1, Gonzalo P. Méndez2, Susan M. Bueno3, 4 and Alexis M. Kalergis1, 4, 5* 1 Pontificia Universidad Católica de Chile, Molecular Genetics and Microbiology Department, Chile 2 Pontificia Universidad Católica de Chile, Pathology, Medicine, Chile 3 Pontificia Universidad Católica de Chile, Molecular Genetics and Microbiology Department, Chile 4 INSERM U1064, France 5 Pontificia Universidad Católica de Chile, Rheumatology, Medicine, Chile Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in children and the elderly. The mechanism by which this virus triggers an inflammatory response still remains unknown. Here, we evaluated whether the thymic stromal lymphopoietin (TSLP) pathway contributes to lung inflammation upon hMPV infection. We found that hMPV infection promotes TSLP expression both in human airway epithelial cells and in the mouse lung. hMPV infection induced lung infiltration of OX40L+CD11b+ DCs. Mice lacking the TSLP receptor deficient mice (tslpr−/−) showed reduced lung inflammation and hMPV replication. These mice displayed a decreased number of neutrophils as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection. Furthermore, a higher frequency of CD4+ and CD8+ T cells was found in tslpr−/− mice compared to WT mice, which could contribute to controlling viral spread. Depletion of neutrophils in WT and tslpr−/− mice decreased inflammation and hMPV replication. Remarkably, blockage of TSLP or OX40L with specific Abs reduced lung inflammation and viral replication following hMPV challenge in mice. Altogether, these results suggest that activation of the TSLP pathway is pivotal in the development of pulmonary pathology and pulmonary hMPV replication. Acknowledgements This research was supported by CONICYT grants: FONDECYT POSTDOCTORADO N° 3120019; N° 3140455, and FONDECYT grant 1070352; FONDEF D061008; and the Millennium Institute on Immunology and Immunotherapy (P07/088-F). Keywords: TSLP, HMPV, OX40 Ligand, Lung, Inflammation Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Oral Presentation Topic: Infectious and parasitic diseases Citation: Céspedes PF, Lay MK, Palavecino CE, León MÁ, Díaz RA, Salazar FJ, Méndez GP, Bueno SM and Kalergis AM (2015). Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00316 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 14 May 2015; Published Online: 15 Sep 2015. * Correspondence: Dr. Margarita K Lay, Pontificia Universidad Católica de Chile, Molecular Genetics and Microbiology Department, Santiago, Chile, margarita.lay@uantof.cl Dr. Alexis M Kalergis, Pontificia Universidad Católica de Chile, Molecular Genetics and Microbiology Department, Santiago, Chile, akalergis@bio.puc.cl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Pablo F Céspedes Margarita K Lay Christian E Palavecino Miguel Á León Rodrigo A Díaz Francisco J Salazar Gonzalo P Méndez Susan M Bueno Alexis M Kalergis Google Pablo F Céspedes Margarita K Lay Christian E Palavecino Miguel Á León Rodrigo A Díaz Francisco J Salazar Gonzalo P Méndez Susan M Bueno Alexis M Kalergis Google Scholar Pablo F Céspedes Margarita K Lay Christian E Palavecino Miguel Á León Rodrigo A Díaz Francisco J Salazar Gonzalo P Méndez Susan M Bueno Alexis M Kalergis PubMed Pablo F Céspedes Margarita K Lay Christian E Palavecino Miguel Á León Rodrigo A Díaz Francisco J Salazar Gonzalo P Méndez Susan M Bueno Alexis M Kalergis Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Human metapneumovirus infection in chimpanzees, United States.

Zoonotic disease transmission and infections are of particular concern for humans and closely related great apes. In 2009, an outbreak of human metapneumovirus infection was associated with the death of a captive chimpanzee in Chicago, Illinois, USA. Biosecurity and surveillance for this virus in captive great ape populations should be considered.

Blood MxA protein as a marker for respiratory virus infections in young children

BackgroundType I interferon induced MxA response can differentiate viral from bacterial infections, but MxA responses in rhinovirus or asymptomatic virus infections are not known. ObjectiveTo study MxA protein levels in healthy state and during respiratory virus infection of young children in an observational prospective cohort. Study designBlood samples and nasal swabs were collected from 153 and 77 children with and without symptoms of respiratory infections, respectively. Blood MxA protein levels were measured by an enzyme immunoassay and PCR methods were used for the detection of respiratory viruses in nasal swabs. ResultsRespiratory viruses were detected in 81% of symptomatic children. They had higher blood MxA protein levels (median [interquartile range]) than asymptomatic virus-negative children (695 [345–1370] μg/L vs. 110 [55–170] μg/L; p<0.001). Within asymptomatic children, no significant difference was observed in MxA responses between virus-positive and virus-negative groups. A cut-off level of 175μg/L had 92% sensitivity and 77% specificity for a symptomatic respiratory virus infection. Rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, coronavirus, and human metapneumovirus infections were associated with elevated MxA responses. Asymptomatic virus-negative children vaccinated with a live virus vaccine had elevated MxA protein levels (240 [120–540] μg/L), but significantly lower than children with an acute respiratory infection, who had not received vaccinations (740 [350–1425] μg/L; p<0.001). ConclusionBlood MxA protein levels are increased in young children with symptomatic respiratory virus infections, including rhinovirus infections. MxA is an informative general marker for the most common acute virus infections.

Human metapneumovirus infection among outpatient children in Dibrugarh.

We describe the prevalence of human metapneumovirus infection in children visiting outpatient department with symptoms of respiratory illness in rural areas of Dibrugarh District of Assam. Human metapneumovirus was observed in 7.2% (20/276) of children aged =5 years with detection of genotypes A2b and B2.

Impact and regulation of lambda interferon response in human metapneumovirus infection.

Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-λ1), IFN-λ2, IFN-λ3, and IFN-λ4. The induction of IFN-λ expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN-λ reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN-λ response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-λs) play a critical protective role in hMPV infection. Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-λ, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN-λ exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

Respiratory Tract Infections Due to Human Metapneumovirus in Immunocompromised Children.

BackgroundThe clinical presentation and management of human metapneumovirus (hMPV) infections in immunocompromised children is not well understood.MethodsWe performed a retrospective evaluation of pediatric patients with laboratory-confirmed hMPV infections and underlying hematologic malignancy, solid tumors, solid organ transplant, rheumatologic disease, and/or receipt of chronic immunosuppressants. Data were analyzed using t tests and Fisher’s exact tests.ResultsOverall, 55 patients (median age: 5 years; range: 5 months–19 years) with hMPV infection documented between 2006 and 2010 were identified, including 24 (44%) with hematologic malignancy, 9 (16%) undergoing hematopoietic stem cell transplant, 9 (16%) with solid tumors, and 8 (15%) with solid organ transplants. Three (5%) presented with fever alone, 35 (64%) presented with upper respiratory tract infections, and 16 (29%) presented with lower respiratory tract infections (LRTI). Twelve (23%) patients required intensive care unit admission and/or supplemental oxygen ≥28% FiO2. Those with severe disease were more likely to be neutropenic (P = .02), but otherwise did not differ by age (P = .27), hematopoietic stem cell transplant recipient status (P = .19), or presence of lymphopenia (P = .09). Nine (16%) patients received treatment with ribavirin, intravenous immunoglobulin, or both. Three children (5%) died of hMPV pneumonia.ConclusionsImmunocompromised pediatric patients with hMPV infection have high rates of LRTI and mortality. The benefits of treatment with ribavirin and intravenous immunoglobulin in this patient population require further evaluation.

117: IRF-3, IRF-7, and IPS-1 promote host defense against acute human metapneumovirus infection in neonatal mice

Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-β promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1 −/− > IRF3/7 −/− > IRF3 −/− , but not IRF7 −/− , mice compared with wild-type mice. IFN-β and IFN-λ2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-λ2/3 was greater in IRF3/7 −/− mice at 5 days after infection. IRF7 −/− and IRF3/7 −/− mice presented with airway eosinophilia, whereas only IRF3/7 −/− mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1 −/− mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-β, but not IFN-λ2/3, produced via an IPS-1-IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-α/β response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence.

Preceding human metapneumovirus infection increases adherence of Streptococcus pneumoniae and severity of murine pneumococcal pneumonia.

Coinfection with respiratory virus and Streptococcus pneumoniae has been frequently reported in several epidemiologic studies. The aim of this study was to explore the effect of preceding human metapneumovirus (hMPV) inoculation on subsequent pneumococcal infection. Hep-2 and A549 cells were infected with hMPV then inoculated with S. pneumoniae. Bacterial adhesion was measured using colony forming unit and cytometric-fluorescence assays. In vivo bacterial adhesion was examined in hMPV-infected mice after inoculation of fluorescence-conjugated S. pneumoniae. Pulmonary inflammation (bacterial titers, cytokine levels, and histopathology) of hMPV-infected mice was investigated after inoculation with S. pneumoniae. In vitro results of bacterial infection with S. pneumoniae on A549 and Hep-2 monolayer cells showed that even though cellular adherence was variable among different serotypes, there was significantly enhanced bacterial adherence in A549 cells with preceding hMPV infection. In addition, in vivo study of hMPV-infected mice showed increased adhesion of S. pneumoniae on the bronchial epithelium with delayed bacterial clearance and exacerbated histopathology. Furthermore, mice with preceding hMPV infection showed repressed recruitment of airway neutrophils with decreased expression of neutrophil chemoattractants during pneumococcal infection. These results suggest that hMPV-infected airway cells, especially the lower airway epithelium, express increased adherence with S. pneumoniae. Furthermore, hMPV-infected mice showed impaired recruitment of airway neutrophils, possibly leading to delayed bacterial clearance and exacerbated pulmonary inflammation, after secondary infection with pneumococcal isolates.

A duplex recombinant viral nucleoprotein microbead immunoassay for simultaneous detection of seroresponses to human respiratory syncytial virus and metapneumovirus infections

Serologic diagnosis of human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) infections has been shown to complement virus detection methods in epidemiologic studies. Enzyme immunoassays (EIAs) using cultured virus lysate antigens are often used to diagnose infection by demonstration of a ≥4-fold rises in antibody titer between acute and convalescent serum pairs. In this study, hRSV and hMPV nucleocapsid (recN) proteins were expressed in a baculovirus system and their performance compared with virus culture lysate antigen in EIAs using paired serum specimens collected from symptomatic children. The recN proteins were also used to develop a duplex assay based on the Luminex microbead-based suspension array technology, where diagnostic rises in antibody levels could be determined simultaneously at a single serum dilution. Antibody levels measured by the recN and viral lysate EIAs correlated moderately (hRSV, r2=0.72; hMPV, r2=0.76); the recN EIAs identified correctly 35 of 37 (94.6%) and 48 of 50 (96%) serum pairs showing diagnostic antibody rises by viral lysate EIAs. Purified recN proteins were then coupled to microbeads and serum pairs were tested at a single dilution on a Luminex MAGPIX® analyzer. The duplex recN assay identified correctly 33 of 39 (85%) and 41 of 47 (86.7%) serum pairs showing diagnostic rises to hRSV and hMPV, respectively. The recN assay permits simultaneous testing for acute hRSV and hMPV infections and offers a platform for expanded multiplexing of other respiratory virus assays.

Human Metapneumovirus and Status Epilepticus

Investigators at Cincinnati Children's Hospital, OH, report 2 toddlers, ages 15 and 18 months, with human metapneumovirus (hMPV) infection who presented in status epilepticus and later developed respiratory failure.

IRF-3, IRF-7, and IPS-1 Promote Host Defense against Acute Human Metapneumovirus Infection in Neonatal Mice

Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-β promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1(-/-)>IRF3/7(-/-)>IRF3(-/-), but not IRF7(-/-), mice compared with wild-type mice. IFN-β and IFN-λ2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-λ2/3 was greater in IRF3/7(-/-) mice at 5 days after infection. IRF7(-/-) and IRF3/7(-/-) mice presented with airway eosinophilia, whereas only IRF3/7(-/-) mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1(-/-) mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-β, but not IFN-λ2/3, produced via an IPS-1-IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-α/β response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence.

Pretreatment of Epithelial Cells with Live Streptococcus pneumoniae Has No Detectable Effect on Influenza A Virus Replication In Vitro

Influenza A virus (IAV) and Streptococcus pneumoniae (pneumococcus) are two major upper respiratory tract pathogens responsible for exacerbated disease in coinfected individuals. Despite several studies showing increased susceptibility to secondary bacterial infections following IAV infection, information on the direct effect of S. pneumoniae on IAV in vitro is unknown. This is an important area of investigation as S. pneumoniae is a common commensal of the human upper respiratory tract, present as an important coinfecting pathogen with IAV infection. A recent study showed that S. pneumoniae enhances human metapneumovirus infection in polarized bronchial epithelial cells in vitro. The aim of the current study was to determine whether treatment of epithelial cells with S. pneumoniae affects IAV replication using a standard immunofluorescence assay (IFA). For this study we used four IAV permissive epithelial cell lines including two human-derived cell lines, 12 pneumococcal strains including recent human clinical isolates which represent different genetic backgrounds and serotypes, and six IAV strains of varying genetic nature and pathogenic potential including the pandemic 2009 H1N1 virus. Our results suggested that pretreatment of MDCK cells with 7.5×106 colony-forming units (CFUs) of live S. pneumoniae resulted in gradual cell-death in a time-dependent manner (0.5 to 4 hr). But, pretreatment of cell lines with 7.5×105 and lower CFUs of S. pneumoniae had no detectable effect on either the morphology of cells or on the IAV replication. However, unlike in epithelial cell lines, due to influence of secreted host factors the effect of pneumococci on IAV replication may be different during coinfections in vivo in the human upper respiratory tract, and in vitro with primary human polarized bronchial epithelial cells.