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Human metapneumovirus (HMPV) and human respiratory virus (HRSV) are two leading causes of acute respiratory tract infection in young children. While there is no licensed drug against HMPV, the monoclonal antibody (mAb) Palivizumab is approved against HRSV for prophylaxis use only. Novel therapeutics against both viruses are therefore needed. Here, we describe the identification of human mAbs targeting these viruses by using flow cytometry-based cell sorting. One hundred and two antibodies were initially identified from flow cytometry-based cell sorting as binding to the fusion protein from HRSV, HMPV or both. Of those, 95 were successfully produced in plants, purified and characterized for binding activity by ELISA and neutralization assays as well as by inhibition of virus replication in mice. Twenty-two highly reactive mAbs targeting either HRSV or HMPV were isolated. Of these, three mAbs inhibited replication in vivo of a single virus while one mAb could reduce both HRSV and HMPV titers in the lung. Overall, this study identifies several human mAbs with virus-specific therapeutic potential and a unique mAb with inhibitory activities against both HRSV and HMPV.

Most viral vaccines are based on inducing neutralizing antibodies (NAbs) against the virus envelope or spike glycoproteins. Many viral surface proteins exist as trimers that transition from a pre-fusion state when key NAb epitopes are exposed to a post-fusion form in which the potential for virus-cell fusion no longer exists. For optimal vaccine performance, these viral proteins are often engineered to enhance stability and presentation of these NAb epitopes. The method involves the structure-guided introduction of proline residues at key positions that maintain the trimer in the pre-fusion configuration. We review how this technique emerged during HIV-1 Env vaccine development and its subsequent wider application to other viral vaccines including SARS-CoV-2., The rapid development of spike protein-based COVID-19 vaccines was facilitated by various lessons learned over the past 20 years. Sanders and Moore review a method devised for HIV-1 vaccine development that was utilized in most COVID-19 vaccines: the stabilization of the spike protein by appropriately positioned proline amino acid substitutions.

Background The hospitalized acute respiratory tract infection (HARTI) study used the Respiratory Intensity and Impact Questionnaire (RiiQ™) Symptom Scale, derived from FluiiQ™, to assess and compare the burden of respiratory infection symptoms for patients with influenza, respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) infection, with or without core risk factors (CRF) (age ≥65; chronic heart, renal, obstructive pulmonary disease; asthma). Methods This was a prospective cohort study in adult patients hospitalized with acute respiratory tract infection (40 centers, 12 countries) during two consecutive influenza/RSV/hMPV seasons (2017–2019). The RiiQ™ Symptom Scale and EuroQol 5‐Dimensions 5‐Levels (EQ‐5D‐5L) were assessed by interview at two timepoints during hospitalization and at 1, 2, and 3 months post‐discharge. Results Mean lower respiratory tract (LRT) symptom scores were higher for RSV and hMPV participants compared to influenza at 48 h after enrollment/early discharge (p = 0.001) and 3 months post‐discharge (p = 0.007). This was driven by LRT symptoms, including shortness of breath (SOB) (p

Human metapneumovirus (hMPV) is an emergent virus, which mainly infects the upper and lower respiratory tract epithelium. This pathogen is responsible for a significant portion of hospitalizations due to bronchitis and pneumonia in infants and the elderly worldwide. hMPV infection induces a pro-inflammatory immune response upon infection of the host, which is not adequate for the clearance of this pathogen. The lack of knowledge regarding the different molecular mechanisms of infection of this virus has delayed the licensing of effective treatments or vaccines. As part of this work, we evaluated whether a single and low dose of a recombinant Mycobacterium bovis Bacillus Calmette-Guérin (BCG) expressing the phosphoprotein of hMPV (rBCG-P) can induce a protective immune response in mice. Immunization with the rBCG-P significantly decreased neutrophil counts and viral loads in the lungs of infected mice at different time points. This immune response was also associated with a modulated infiltration of innate cells into the lungs, such as interstitial macrophages (IM) and alveolar macrophages (AM), activated CD4+ and CD8+ T cells, and changes in the population of differentiated subsets of B cells, such as marginal zone B cells and plasma cells. The humoral immune response induced by the rBCG-P led to an early and robust IgA response and a late and constant IgG response. Finally, we determined that the transfer of cells or sera from immunized and infected mice to naïve mice promoted an efficient viral clearance. Therefore, a single and low dose of rBCG-P can protect mice from the disease caused by hMPV, and this vaccine could be a promising candidate for future clinical trials.

The human respiratory syncytial virus (hRSV) is one of the leading causes of acute lower respiratory tract infections in children under five years old. Notably, hRSV infections can give way to pneumonia and predispose to other respiratory complications later in life, such as asthma. Even though the social and economic burden associated with hRSV infections is tremendous, there are no approved vaccines to date to prevent the disease caused by this pathogen. Recently, coinfections and superinfections have turned into an active field of study, and interactions between many viral and bacterial pathogens have been studied. hRSV is not an exception since polymicrobial infections involving this virus are common, especially when illness has evolved into pneumonia. Here, we review the epidemiology and recent findings regarding the main polymicrobial infections involving hRSV and several prevalent bacterial and viral respiratory pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Klebsiella pneumoniae, human rhinoviruses, influenza A virus, human metapneumovirus, and human parainfluenza viruses. As reports of most polymicrobial infections involving hRSV lack a molecular basis explaining the interaction between hRSV and these pathogens, we believe this review article can serve as a starting point to interesting and very much needed research in this area.

Human metapneumovirus (HMPV) is a major etiological agent of acute respiratory infections in humans. HMPV has been circulating worldwide for more than six decades and is currently divided into five agreed-upon subtypes: A1, A2a, A2b, B1, and B2. Recently, the novel HMPV subtypes A2c, A2b1, and A2b2 have been proposed. However, the phylogenetic and evolutionary relationships between these recently proposed HMPV subtypes are unclear. Here, we report a genome-wide phylogenetic and evolutionary analysis of 161 HMPV strains, including unique HMPV subtype A2b strains with a 180- or 111-nucleotide duplication in the G gene (nt-dup). Our data demonstrate that the HMPV A2b subtype contains two distinct subtypes, A2b1 and A2b2, and that the HMPV subtypes A2c and A2b2 may be different names for the same subtype. HMPV A2b strains with a nt-dup also belong to subtype A2b2. Molecular evolutionary analyses indicate that subtypes A2b1 and A2b2 diverged from subtype A2b around a decade after the subtype A2 was divided into the subtypes A2a and A2b. These data support the A2b1 and A2b2 subtypes proposed in 2012 and are essential for the unified classification of HMPV subtype A2 strains, which is important for future HMPV surveillance and epidemiological studies.

Background Respiratory infections are the main causes for hospitalization in children and a common reason for the initiation of antibiotic treatment. Rapid antigen detection tests and point-of-care mPCR-based assays provide a fast detection of viral pathogens. Nonetheless, the prescription rate of antibiotics for respiratory infections is exceedingly high. In particular, human metapneumovirus (hMPV) infections frequently cause antibiotic treatment. Methods Children hospitalized in our clinic with an acute respiratory infection between January 2008 and January 2013 were included in the present study. Data of 3799 children were analyzed retrospectively for clinical symptoms, laboratory findings, and antibiotic and inhalation treatment. We performed an in-house m-RT-PCR-ELISA method for pathogen detection. Results Pathogen detection was possible in 2464 patients. In 6.3%, hMPV and, in 24.0%, RSV were detected. Patients positively tested for hMPV received inhalation therapy in 62.9%; patients positive for RSV in 73.8%. Patients positive for hMPV were treated with antibiotics in 62.3%. Patients with RSV infection received antibiotic treatment in 44.4%; all others in 43.5%. Notably, a positive result in RSV-RADT was associated with reduced number of antibiotic treatment. Conclusion hMPV infections inherit a two times higher probability of antibiotic treatment. There was no significant difference in laboratory findings or body temperature between hMPV infection and infections caused by other pathogens. Clinical symptoms seem not to differ from those in RSV illness. Nonetheless, RSV infections triggered significantly lower antibiotic prescription rates. A considerate application of a POC-mPCR for patients with RSV-like symptoms and age of 1 year and older with a negative RSV-RADT might lead to higher detection rates of hMPV and a reduction in prescription of antibiotics.

Viruses possessing class I fusion proteins require proteolytic activation by host cell proteases to mediate fusion with the host cell membrane. The mammalian SPINT2 gene encodes a protease inhibitor that targets trypsin-like serine proteases. Here we show the protease inhibitor, SPINT2, restricts cleavage-activation efficiently for a range of influenza viruses and for human metapneumovirus (HMPV). SPINT2 treatment resulted in the cleavage and fusion inhibition of full-length influenza A/CA/04/09 (H1N1) HA, A/Aichi/68 (H3N2) HA, A/Shanghai/2/2013 (H7N9) HA and HMPV F when activated by trypsin, recombinant matriptase or KLK5. We also demonstrate that SPINT2 was able to reduce viral growth of influenza A/CA/04/09 H1N1 and A/X31 H3N2 in cell culture by inhibiting matriptase or TMPRSS2. Moreover, inhibition efficacy did not differ whether SPINT2 was added at the time of infection or 24 hours post-infection. Our data suggest that the SPINT2 inhibitor has a strong potential to serve as a novel broad-spectrum antiviral.

Background Human metapneumovirus (HMPV) have similar symptoms to those caused by the respiratory syncytial virus (RSV). The modes of transmission and dynamics of time series data still remain poorly understood. Climatic factors have long been suspected to be implicated in impacting on the number of cases for these epidemics. Currently, only a few models satisfactorily capture the dynamics of time series data of these two viruses. Our objective was to assess the presence of influence of high incidences between the viruses and to ascertain whether higher incidences of one virus are influenced by the other. Methods In this study, we used a negative binomial model to investigate the relationship between RSV and HMPV while adjusting for climatic factors. We specifically aimed at establishing the heterogeneity in the autoregressive effect to account for the influence between these viruses. Results In this study, our findings showed that RSV incidence contributed to the severity of HMPV incidence. This was achieved through comparison of 12 models with different structures, including those with and without interaction between climatic factors. The models with climatic factors out-performed those without. Conclusions The study has improved our understanding of the dynamics of RSV and HMPV in relation to climatic cofactors thereby setting a platform to devise better intervention measures to combat the epidemics. We conclude that preventing and controlling RSV infection subsequently reduces the incidence of HMPV.

In this post-hoc analysis of midnasal pneumococcal carriage in a community-based, randomized prenatal influenza vaccination trial in Nepal with weekly infant respiratory illness surveillance, 457 of 605 (75.5%) infants with influenza, respiratory syncytial virus (RSV), or human metapneumovirus (hMPV) illness had pneumococcus detected. Pneumococcal carriage did not impact rates of lower respiratory tract disease for these 3 viruses. Influenza-positive infants born to mothers given influenza vaccine had lower pneumococcal carriage rates compared to influenza-positive infants born to mothers receiving placebo (58.1% versus 71.6%, P = 0.03). Maternal influenza immunization may impact infant acquisition of pneumococcus during influenza infection. Clinical Trials Registration. NCT01034254., Among influenza-positive infants, those born to mothers given influenza vaccine had lower pneumococcal carriage rates. Maternal influenza immunization may impact infant pneumococcal acquisition during influenza infection. Pneumococcal carriage did not impact lower respiratory disease rates in RSV, hMPV, or influenza.

The family Paramyxoviridae includes many viruses that significantly affect human and animal health. An essential step in the paramyxovirus life cycle is viral entry into host cells, mediated by virus-cell membrane fusion. Upon viral entry, infection results in expression of the paramyxoviral glycoproteins on the infected cell surface. This can lead to cell-cell fusion (syncytia formation), often linked to pathogenesis. Thus membrane fusion is essential for both viral entry and cell-cell fusion and an attractive target for therapeutic development. While there are important differences between viral-cell and cell-cell membrane fusion, many aspects are conserved. The paramyxoviruses generally utilize two envelope glycoproteins to orchestrate membrane fusion. Here, we discuss the roles of these glycoproteins in distinct steps of the membrane fusion process. These findings can offer insights into evolutionary relationships among Paramyxoviridae genera and offer future targets for prophylactic and therapeutic development.

Human Metapneumovirus (HMPV) remains one of the most common viral infections causing acute respiratory tract infections, especially in young children, elderly, and immunocompromised populations. Clinical symptoms can range from mild respiratory symptoms to severe bronchiolitis and pneumonia. The production of mucus is a common feature during HMPV infection, but its contribution to HMPV-induced pathogenesis and immune response is largely unknown. Mucins are a major component of mucus and they could have an impact on how the host responds to infections. Using an in vitro system and a mouse model of infection, we identified that Mucin 19 is predominantly expressed in the respiratory tract upon HMPV infection. Moreover, the lack of Muc19 led to an improved disease, lower lung viral titers and a decrease in the number of CD4+ T cells. These data indicate that mucin 19 contributes to the activation of the immune response to HMPV and to HMPV-induced pathogenesis.

The human respiratory syncytial virus (hRSV) and human Metapneumovirus (hMPV) are two of the leading etiological agents of acute lower respiratory tract infections, which constitute the main cause of mortality in infants. However, there are currently approved vaccines for neither hRSV nor hMPV. Moreover, despite the similarity between the pathology caused by both viruses, the immune response elicited by the host is different in each case. In this review, we discuss how dendritic cells, alveolar macrophages, neutrophils, eosinophils, natural killer cells, innate lymphoid cells, and the complement system regulate both pathogenesis and the resolution of hRSV and hMPV infections. The roles that these cells play during infections by either of these viruses will help us to better understand the illnesses they cause. We also discuss several controversial findings, relative to some of these innate immune components. To better understand the inflammation in the lungs, the role of the respiratory epithelium in the recruitment of innate immune cells is briefly discussed. Finally, we review the main prophylactic strategies and current vaccine candidates against both hRSV and hMPV.

The Human Respiratory Syncytial Virus (hRSV) and the Human Metapneumovirus (hMPV) are two pneumoviruses that are leading agents causing acute lower respiratory tract infections (ALRTIs) affecting young infants, the elderly, and immunocompromised patients worldwide. Since these pathogens were first discovered, many approaches for the licensing of safe and effective vaccines have been explored being unsuccessful to date. We have previously described that immunization with recombinant strains of Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing the hRSV nucleoprotein (rBCG-N) or the hMPV phosphoprotein (rBCG-P) induced immune protection against each respective virus. These vaccines efficiently promoted viral clearance without significant lung damage, mainly through the induction of a T helper 1 cellular immunity. Here we show that upon viral challenge, rBCG-immunized mice developed a protective humoral immunity, characterized by production of antibodies specific for most hRSV and hMPV proteins. Further, isotype switching from IgG1 to IgG2a was observed in mice immunized with rBCG vaccines and correlated with an increased viral clearance, as compared to unimmunized animals. Finally, sera obtained from animals immunized with rBCG vaccines and infected with their respective viruses exhibited virus neutralizing capacity and protected naïve mice from viral replication and pulmonary disease. These results support the notion that the use of rBCG strains could be considered as an effective vaccination approach against other respiratory viruses with similar biology as hRSV and hMPV.

Summary Respiratory infections, especially those of the lower respiratory tract, remain a foremost cause of mortality and morbidity of children greater than 5 years in developing countries including Pakistan. Ignoring these acute‐level infections may lead to complications. Particularly in Pakistan, respiratory infections account for 20% to 30% of all deaths of children. Even though these infections are common, insufficiency of accessible data hinders development of a comprehensive summary of the problem. The purpose of this study was to determine the prevalence rate in various regions of Pakistan and also to recognize the existing viral strains responsible for viral respiratory infections through published data. Respiratory viruses are detected more frequently among rural dwellers in Pakistan. Lower tract infections are found to be more lethal. The associated pathogens comprise respiratory syncytial virus (RSV), human metapneumovirus (HMPV), coronavirus, enterovirus/rhinovirus, influenza virus, parainfluenza virus, adenovirus, and human bocavirus. RSV is more dominant and can be subtyped as RSV‐A and RSV‐B (BA‐9, BA‐10, and BA‐13). Influenza A (H1N1, H5N1, H3N2, and H1N1pdm09) and Influenza B are common among the Pakistani population. Generally, these strains are detected in a seasonal pattern with a high incidence during spring and winter time. The data presented include pneumonia, bronchiolitis, and influenza. This paper aims to emphasise the need for standard methods to record the incidence and etiology of associated pathogens in order to provide effective treatment against viral infections of the respiratory tract and to reduce death rates.

Objective Human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are responsible for respiratory diseases, mostly in children. Despite the clinical and epidemiological similarities between these two pneumoviruses, they elicit different immune responses. This work aims to further our understanding of the differential immune response induced by these respiratory viruses by determining the changes of small non-coding RNAs (miRNAs), which regulate gene expression and are involved in numerous cellular processes including the immune system. Results In the present study, we analyzed the expression of miRNA transcripts of human dendritic cells infected with RSV or HMPV by high throughput sequencing using Illumina sequencing technology. Further validation of miRNA expression by quantitative polymerase chain reaction indicated that HMPV infection up-regulated the expression of 2 miRNAs (hsa-miR-182-5p and hsa-miR-4634), while RSV infection induced significant expression of 3 miRNAs (hsa-miR-4448, hsa-miR-30a-5p and hsa-miR-4634). The predominant miRNA induced by both viruses was hsa-miR-4634.

Human metapneumovirus (HMPV) is a relative newly described virus. It was first isolated in 2001 and currently appears to be one of the most significant and common human viral infections. Retrospective serologic studies demonstrated the presence of HMPV antibodies in humans more than 50 years earlier. Although the virus was primarily known as causative agent of respiratory tract infections in children, HMPV is an important cause of respiratory infections in adults as well. Almost all children are infected by HMPV below the age of five; the repeated infections throughout life indicate transient immunity. HMPV infections usually are mild and self-limiting, but in the frail elderly and the immunocompromised patients, the clinical course can be complicated. Since culturing the virus is relatively difficult, diagnosis is mostly based on a nucleic acid amplification test, such as reverse transcriptase polymerase chain reaction. To date, no vaccine is available and treatment is supportive. However, ongoing research shows encouraging results. The aim of this paper is to review the current literature concerning HMPV infections in adults, and discuss recent development in treatment and vaccination.

// Mohammad Diab 1,* , Alon Vitenshtein 1,* , Yaron Drori 2,3 , Rachel Yamin 1 , Oded Danziger 4 , Rachel Zamostiano 4 , Michal Mandelboim 2,3 , Eran Bacharach 4 and Ofer Mandelboim 1 1 The Lautenberg Center for General and Tumor Immunology, The BioMedical Research Institute Israel Canada of the Faculty of Medicine, The Hebrew University Hadassah Medical School, Jerusalem, Israel 2 Central Virology Laboratory, Ministry of Health, Public Health Services, Chaim, Sheba Medical Center, Tel Hashomer, Ramat-Gan, Israel 3 Department of Epidemiology and Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel 4 Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel * These authors have equally contributed to this work Correspondence to: Ofer Mandelboim, email: // Keywords : HMPV, CEACAM1, PRRs, PAMPs, RLRs, Immunology and Microbiology Section, Immune response, Immunity Received : July 18, 2016 Accepted : September 01, 2016 Published : September 12, 2016 Abstract The innate sensing system is equipped with PRRs specialized in recognizing molecular structures (PAMPs) of various pathogens. This leads to the induction of anti-viral genes and inhibition of virus growth. Human Metapneumovirus (HMPV) is a major respiratory virus that causes an upper and lower respiratory tract infection in children. In this study we show that upon HMPV infection, the innate sensing system detects the viral RNA through the RIG-I sensor leading to induction of CEACAM1 expression. We further show that CEACAM1 is induced via binding of IRF3 to the CEACAM1 promoter. We demonstrate that induction of CEACAM1 suppresses the viral loads via inhibition of the translation machinery in the infected cells in an SHP2-dependent manner. In summary, we show here that HMPV-infected cells upregulates CEACAM1 to restrict HMPV infection.

We investigated the proportions of mono vs. mixed infections for human metapneumovirus (hMPV) as compared to adenovirus (ADV), four types of coronavirus (CRV), parainfluenza virus (PIV), RSV, and enterovirus/rhinovirus (ERV) in Alberta, Canada. Using the Data Integration for Alberta Laboratories (DIAL) platform, 26,226 respiratory specimens at ProvLab between 1 July 2009 and 30 June 2012 were selected and included in the study. Using the Respiratory Virus Panel these specimens tested positive for one or more respiratory virus and negative for influenza A and B. From our subset hMPV was the fourth most common virus (n=2,561) with 373 (15%) identified as mixed infection using DIAL. Mixed infection with hMPV was most commonly found in infants less than 6 months old and ERV was most commonly found in mixed infection with hMPV (230/373, 56%) across all age groups. The proportion of mixed-infection vs. mono-infection was highest for ADV (46%), followed by CRV 229E (32%), CRV HKU1 (31%), CRV NL63 (28%), CRV OC43 (23%), PIV (20%), RSV (17%), hMPV (15%) and ERV (13%). hMPV was significantly more likely to be identified in mono infection as compared with ADV, CRV, PIV, and RSV with the exception of ERV [p < 0.05].

Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. Here we show that infection of plasmacytoid dendritic cells (pDCs) with a recombinant virus lacking SH expression (rhMPV-ΔSH) enhanced the secretion of type I interferons (IFNs), which required TLR7 and MyD88 expression. HMPV SH protein inhibited TLR7/MyD88/TRAF6 signaling leading to IFN gene transcription, identifying a novel mechanism by which paramyxovirus SH proteins modulate innate immune responses.

Human metapneumovirus (hMPV) is a newly identified paramyxovirus that has been associated with respiratory tract illness in children aged < 5 years, the elderly, and immunocompromised patients. This study determined the frequency of respiratory tract infections (RTIs) associated with hMPV in the Republic of Ireland. Bronchoalveolar lavage (BAL) samples from 168 adult patients and respiratory specimens from 122 children aged < 5 years were collected between September 2003 and May 2004. The virus was detected by reverse-transcription (RT)-PCR using hMPV polymerase (L) and matrix (M)-specific primers in four (2.4%) of 171 BAL specimens obtained from 168 adults. No other respiratory virus was detected in these specimens, and no hMPV RNA was detected in respiratory specimens from children during the same time period. In all four adult cases, two of whom had underlying disease, hMPV was associated with mild, self-limiting upper RTIs. The most common clinical findings included fever (3/4 patients), cough (4/4) and rhinorrhoea (3/4). No patient died as a result of these RTI episodes. Phylogenetic analysis was performed using the amplified regions of the Mand fusion (F) genes of hMPV. The Irish isolates belonged to cluster 1B, and did not show a separate Irish sub-lineage.

International audience; 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. Keywords: Dendritic cells r hMPV r Inflammation r Neutrophils r OX40L r TSLP r Viral replication.

Respiratory Syncytial Virus (RSV), Human metapneumovirus (HMPV), and Rhinoviruses (RV) are frequent causes of respiratory tract infections in young children. We compared laboratory and clinical findings in children with comparable age distribution and hospitalized due to RSV, HMPV or RV infections. Viral pathogens were detected by a quantitative real time PCR from nasopharyngeal aspirates. No significant differences in the admission diagnosis, laboratory parameters, patient demographics and treatment measures between the three viral causes of respiratory illness were found. No correlation between viral load and disease severity was observed however, there was a significantly lower concentration of the nasopharyngeal interleukin 8 (IL‐8) in children with RV compared to HMPV and RSV, indicating a milder proinflammatory reaction. Moreover, RV‐infected children had significantly lower body temperature, higher leucocyte counts in peripheral blood, and a tendency to have a shorter stay in hospital than children with either HMPV or RSV infection. Taken together, clinical presentation of the infections with RSV, HMPV, and RV is similar among children of the same age group and not clearly distinguishable by standard clinical or laboratory findings. Therefore, virus specific testing should be included regularly for routine diagnosis of children with respiratory tract infections. J. Med. Virol. 87:275–280, 2015. © 2014 Wiley Periodicals, Inc.

To the Editor: Respiratory viral infections can be associated with a wide range of clinical manifestations from self-limiting upper respiratory tract diseases to pneumonia (1). However, in general, respiratory viral infections are more likely to progress to more severe diseases in immmunocompromised patients. Human metapneumovirus (hMPV) has been reported in most parts of the world as a cause of acute respiratory tract infections in persons of all age groups (2). Fatal hMPV infections have been reported in immunocompromised patients, including a 17-month-old girl who had acute lymphoblastic leukemia (3) and a 33-year-old woman who had received a hematopoietic stem cell transplant (HSCT) (4). In adult HSCT recipients, fatal pneumonia (5) and persistent hMPV infection without respiratory symptoms have been described (6). In addition, adult lung transplant recipients have been able to clear hMPV infection despite high levels of immunosuppression (7). We report a case of persistent hMPV infection in a child with severe combined immunodeficiency disorder (SCID) who shed hMPV during an 11-month period. The child, a girl who was born in January 2002, received an allogeneic haploidentical stem cell transplant from her father in May 2002 after her diagnosis of SCID. Infection with influenza A virus (H3N2) was diagnosed on April 2005 and progressed to a chronic pneumonitis of the lingula. She received successive courses of anti-influenza agents (amantadine, oseltamivir, and zanamivir) for 1 year during which time several positive influenza cultures were obtained (8). Four years after the transplant, she was still lymphopenic (800 × 109/L, mostly T cells) and had chronic graft-versus-host disease, which had been treated with steroids (prednisone 2.5 mg twice a day for many months). She also had a mild chronic cough but did not need supplemental oxygen while she was receiving nebulized zanamivir (10–20 mg twice a day). Her 2 nasopharyngeal aspirate (NPA) specimens from June and July 2006 were negative for influenza virus. However, positive cultures for hMPV were obtained from NPA and bronchoalveolar lavage specimens collected on July 2006. After receiving this result, we performed retrospective and prospective molecular detection studies for hMPV for this patient. HMPV was detected by reverse transcription–PCR for the F and G genes (9) in 6 and 7 NPA samples, respectively, collected during an 11-month period from November 4, 2005, through October 4, 2006. These samples were obtained for surveillance of influenza infection in this child with persistent cough. Amplified hMPV G sequences were aligned by using the ClustalW program (www.molecularevolution.org/cdc/software/clustalw). A phylogenetic tree was constructed with MEGA 3.1 software (www.megasoftware.net) by using the neighbor-joining algorithm with Kimura-2 parameters. Sequence analysis of the hMPV G gene showed that all strains belonged to the B2 genotype, which clustered with hMPV Can98–75 and NL1/94 reference strains (Figure, panel A). Amplified hMPV G gene sequences of the 6 samples collected in 2006 were identical, but they had 96.7% and 92.8% nucleotide and amino acid identities, respectively, with the initial strain from November 2005, which clearly indicates 2 viral strains (Figure, panel B). Similar results were obtained with the F gene (data not shown). Inoculation of the respiratory samples on a panel of 10 cell lines as previously described (10) showed that only 2 of 7 NPA samples were positive for hMPV by culture; 2 of the 5 remaining samples were positive for influenza A, which may have masked the cytopathic effects of hMPV on rhesus monkey kidney (LLC-MK2) cells. Figure A) Phylogenetic analysis of human metapneumovirus (hMPV) strains isolated during an 11-month period based on nucleotide sequences of the G gene. Multiple nucleotide sequence alignments were performed by using the ClustalW program (www.molecularevolution.org/cdc/software/clustalw ... Persistent hMPV infection in asymptomatic adult HSCT recipients has been described (6). In that study, hMPV was isolated from 2 patients in 2 consecutive samples collected 12–56 days apart. However, virus evolution was not adequately investigated because it was based on sequence analysis of a 150-bp fragment from the highly conserved nucleoprotein gene (6). Unlike in previous reports (6,7), characterization of hMPV strains in our study was performed by sequence analysis of a 633-bp fragment from the most variable hMPV G gene. Our findings showed 2 distinct hMPV variants of the same genotype (B2). These variants might represent a viral drift after immune pressure but most likely was the result of 2 different infections in the immunocompromised child. The latter hypothesis is suggested by the considerable amino acid variability (15 aa differences in the 211-aa region of the G protein) between strains collected on November, 4, 2005, and January, 20, 2006, compared with identical sequences for the strains recovered over the next 10 months. Debiaggi et al. (6) previously suggested that persistent hMPV infection in HSCT patients was attributable to their inability to clear the virus because of impaired immune response. By contrast, adult lung transplant recipients were found to be able to achieve hMPV clearance despite their severe immunosuppression status (7). Because both fatal and mild or asymptomatic hMPV infections have been reported in immunocompromised hosts, additional studies are needed to determine whether such differing outcomes are due to viral, host, or environmental factors. In conclusion, this case of persistent hMPV infection associated with relatively mild respiratory symptoms in an immunocompromised child suggests that the host’s immune response may play a key role in disease pathogenesis.

Summary We describe a case of bronchiolitis associated with the newly detected human bocavirus (hBoV) in a child with a suspected Noonan syndrome. This is the first report of a bronchiolitis probably linked to hBoV that required intensive care while being accompanied by a congenital heart disease and a history of several episodes of severe respiratory symptoms.

Background Human metapneumovirus (hMPV) is a significant viral respiratory pathogen of infants and children, the elderly and immunocompromised individuals. Disease associated with hMPV infection resembles that of human respiratory syncytial virus (RSV) and includes bronchiolitis and pneumonia. The glycosylated G attachment protein of hMPV is required for viral entry in vivo and has also been identified as an inhibitor of innate immune responses. Findings We designed and validated two siRNA molecules against the G gene using A549 cells and demonstrated consistent 88-92% knock-down for one siRNA molecule, which was used in subsequent experiments. Significant reduction of G mRNA in A549 cells infected with hMPV did not result in a reduction in viral growth, nor did it significantly increase the production of type I interferon (α/β) in response to infection. However, there was a moderate increase in IFN-β mRNA expression in response to infection in siG-transfected cells compared to untransfected and si-mismatch-transfected cells. Expression of G by recombinant adenovirus did not affect type I IFN expression. Conclusion G has been previously described as a type I interferon antagonist, although our findings suggest it may not be a significant antagonist.

Background Human metapneumovirus (HMPV) has been shown to cause respiratory infection, accounting for approximately 7% of all such disease, and contributes to the development of asthma in humans. HMPV has a worldwide distribution with infectivity rates approaching 100%, and immunocompromised patients are particularly at risk from viral exposure. No anti-HMPV vaccine is available and diagnosis is primarily based on in-house molecular or serological tests, in part due to limited availability of recombinant HMPV antigens. Objective To generate a panel of HMPV-derived recombinant antigens, develop standardised ELISA systems for HMPV IgG detection and explore the nature of B cell memory against HMPV to underpin future vaccine studies. Study design HMPV viral RNA was isolated from a clinical specimen and RT-PCR was conducted. The HMPV M and P genes were cloned and expressed in Escherichia coli. The HMPV N gene was cloned and expressed in insect cells using the baculovirus expression system. Each purified recombinant antigens was subsequently employed in HMPV-specific ELISA. Results High-level expression, and purification, of both HMPV matrix (M) (10 mg/g cells) and phosphoprotein (P) (3.82 mg/g cells) were achieved in an E. coli expression system. Recombinant HMPV (N) was successfully expressed in, and purified from the baculovirus expression system. Overall, a 99% HMPV IgG seroprevalence was observed (n = 96) using HMPV M-, N- and P-ELISA, respectively. The M antigen proved to be the most diagnostically useful with 99% of specimens tested exhibiting anti-M protein reactivity. A high correlation was observed between anti-M and N IgG reactivity (r = 0.96), with significant correlation also evident for anti-N and P IgG reactivity (r = 0.74). Lowest correlation was evident for anti-M and P IgG reactivity (r = 0.57). Finally, the first demonstration of HMPV-specific B cell memory (ranging 1–15 spot forming cells (SFC)/million cells) was achieved against M and P antigens in 40% of individuals tested. Conclusion This work describes robust diagnostic systems for HMPV and new insight into antigen-specific B cell memory against HMPV.

Background: Respiratory virus infections in children are a leading cause of morbidity and mortality worldwide. Methods: A total of 897 clinical specimens were collected from February 2007 to January 2008 and transported to the National Influenza Center. Two hundred and two samples belonged to children under the age of six from 897 specimens, described above, were selected. Then they were tested for influenza virus types and subtypes by real time PCR assay subsequently, the specimens were tested for RSV and hMPV by hemi-nested multiplex PCR and parainfluenza viruses type 1–4 by hemi-nested multiplex PCR and adenovirus by hemi-nested PCR. Results: The throat swab was taken from the Kawasaki case with the history of chicken’s contact. The specimen was tested for all influenza subtypes especially H5N1 and the results were negative. Meanwhile PCR was done for screening of other respiratory viruses that results came out positive for RSV and hMPV. Conclusion: In the present study, we demonstrated the possibility to detect dual infection caused by RSV and hMPV, but because of the extravagant pattern of this case, more investigation is suggested specially on Kawasaki patients.

A prospective 2‐year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co‐pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV‐OC43, 35.7% hCoV‐NL63, 21.4% hCoV‐HKU1, and 3.6% hCoV‐229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co‐infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P

The performances of four multiplex PCR (m‐PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA‐positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR‐positive. The m‐PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m‐PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m‐PCR, the prevalence of each virus was compared between in‐patient and out‐patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in‐patient group than in the out‐patient group, and influenza viruses were detected more frequently in the out‐patient group than in the in‐patient group. Moreover, the use of m‐PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m‐PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children. J. Med. Virol. 78:1498–1504, 2006. © 2006 Wiley‐Liss, Inc.

In a pediatric surveillance network, 287 (5.1%) of 5,580 specimens from patients with acute respiratory infections tested positive for human metapneumovirus (HMPV). Phylogenetic analysis of N- and F-gene sequences of identified HMPV showed that 30% belonged to a novel phylogenetic cluster.

วารสารสถาบันบำราศนราดูร, 16, 1, 44-52

Background: Respiratory virus infections in children are a leading cause of morbidity and mortality worldwide. Methods: A total of 897 clinical specimens were collected from February 2007 to January 2008 and transported to the Na­tional Influenza Center. Two hundred and two samples belonged to children under the age of six from 897 specimens, de­scribed above, were selected. Then they were tested for influenza virus types and subtypes by real time PCR assay subse­quently, the specimens were tested for RSV and hMPV by hemi-nested multiplex PCR and parainfluenza viruses type 1-4 by hemi-nested multiplex PCR and adenovirus by hemi-nested PCR. Results: The throat swab was taken from the Kawasaki case with the history of chicken`s contact. The specimen was tested for all influenza subtypes especially H5N1 and the results were negative. Meanwhile PCR was done for screening of other respira­tory viruses that results came out positive for RSV and hMPV. Conclusion: In the present study, we demonstrated the possibility to detect dual infection caused by RSV and hMPV, but be­cause of the extravagant pattern of this case, more investigation is suggested specially on Kawasaki patients.