Structural characterization of M8C10, a neutralizing antibody targeting a highly conserved prefusion-specific epitope on the metapneumovirus fusion trimerization interface.

IMPORTANCE: Human metapneumovirus (hMPV) is a common pathogen causing lower respiratory tract infections worldwide and can develop severe symptoms in high-risk populations such as infants, the elderly, and immunocompromised patients. There are no approved hMPV vaccines or neutralizing antibodies available for therapeutic or prophylactic use. The trimeric hMPV fusion F protein is the major target of neutralizing antibodies in human sera. Understanding the immune recognition of antibodies to hMPV-F antigen will provide critical insights into developing efficacious hMPV monoclonal antibodies and vaccines.

Epidemiological and clinical characteristics of hospitalized human metapneumovirus patients in Israel, 2015-2021: A retrospective cohort study.

This study evaluated the epidemiological and clinical characteristics of human metapneumovirus (hMPV) infection among hospitalized patients with acute respiratory infections during 2015-2021 and assessed the impact of the coronavirus disease 2019 pandemic on hMPV infection. A single-center, retrospective cohort study was performed, including pediatric and adult patients with laboratory-confirmed hMPV. Of a total of 990 patients, 253 (25.6%), 105 (10.6%), 121 (12.2%), and 511 (51.6%) belonged to age groups 0-2, 3-17, 18-59, and ≥60 years, respectively. The highest percentage (23.0%) of patients were hospitalized during 2019 and the lowest (4.7%) during 2020. Patients < 18 years experienced high rates of comorbidities (immunodeficiencies: 14.4% and malignancies: 29.9%). Here, 37/39 (94.9%) of all bronchiolitis cases were diagnosed in patients < 2 years, whereas more patients in older age groups were diagnosed with pneumonia. A greater proportion of hMPV patients diagnosed with viral coinfection (mostly respiratory syncytial virus and adenovirus) were <18 years. The highest percentages of intensive care unit admissions were recorded among patients < 18 years. Our findings demonstrate that hMPV is an important cause of morbidity in young children and a possibly underestimated cause of morbidity among older adults.

Evidence against the Human Metapneumovirus G, SH, and M2-2 Proteins as Bona Fide Interferon Antagonists.

The production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies. IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.

Characterization of the Interaction Domains between the Phosphoprotein and the Nucleoprotein of Human Metapneumovirus.

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein (N), forming an RNA-N complex (NNuc), which serves as the template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping, and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the NNuc template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between HMPV P and NNuc, which has not been demonstrated yet. Here, we finely characterized the binding P-NNuc interaction domains by using recombinant proteins, combined with a functional assay for the polymerase complex activity, and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc and that P binds to the N-terminal domain of N (NNTD), and we identified conserved N residues critical for the interaction. Our results allowed us to propose a structural model for the HMPV P-NNuc interaction. IMPORTANCE Human metapneumovirus (HMPV) is a leading cause of severe respiratory infections in children but also affects human populations of all ages worldwide. Currently, no vaccine or efficient antiviral treatments are available for this pneumovirus. A better understanding of the molecular mechanisms involved in viral replication could help the design or discovery of specific antiviral compounds. In this work, we have investigated the interaction between two major viral proteins involved in HMPV RNA synthesis, the N and P proteins. We finely characterized their domains of interaction and identified a pocket on the surface of the N protein, a potential target of choice for the design of compounds interfering with N-P complexes and inhibiting viral replication.

Molecular epidemiological surveillance of viral agents of acute lower respiratory tract infections in children in Accra, Ghana.

BACKGROUND: Acute lower respiratory tract infection (ALRTI) in children under 5 years is known to be predominantly caused by respiratory syncytial virus (RSV). In recent times, however, human metapneumovirus (HMPV) has also been implicated. This study sought to investigate and genotype respiratory syncytial virus and human metapneumovirus in children presenting with ALRTIs infection at the Princess Marie Louis Children's Hospital in Accra, Ghana. METHODS: Children below 5 years who were clinically diagnosed of ALRTI and on admission at the study site were recruited between September 2015 and November 2016 for this study. Demographic data information was obtained by means of a standardized questionnaire; and relevant clinical information was obtained from medical records. Nasopharyngeal swabs were collected from 176 children recruited for the study. Ribonucleic acid was extracted from swabs and cDNA syntheses were performed by RT-PCR. RSV-positive amplicons were sequenced and analyzed for genotype assignment. RESULTS: RSV and HMPV prevalence among the sampled subjects were 11.4 and 1.7% respectively. Of the RSV positives, 8/20 (40%) were RSV-A and 12/20 (60%) were RSV-B. The highest prevalence was observed in children less than 12 months old. Phylogenetic analysis of the second hypervariable region of the RSV G-gene revealed that all RSV group A viruses belonged to the "novel" ON1 genotype containing the 72-nucleotide duplication; and RSV group B viruses belong to the BA IX genotype. CONCLUSION: RSV is frequently detected in children aged under 5 years admitted with ALRTI in Ghana. Continued surveillance of viral aetiological agents is warranted to elucidate the prevalence and transmission patterns of viral pathogens that cause respiratory tract infections among children. This will help inform appropriate intervention approaches.

Feasibility and Performance of Self-Collected Nasal Swabs for Detection of Influenza Virus, Respiratory Syncytial Virus, and Human Metapneumovirus.

BACKGROUND: We assessed performance of participant-collected midturbinate nasal swabs compared to study staff-collected midturbinate nasal swabs for the detection of respiratory viruses among pregnant women in Bangkok, Thailand. METHODS: We enrolled pregnant women aged ≥18 years and followed them throughout the 2018 influenza season. Women with acute respiratory illness self-collected midturbinate nasal swabs at home for influenza viruses, respiratory syncytial viruses (RSV), and human metapneumoviruses (hMPV) real-time RT-PCR testing and the study nurse collected a second midturbinate nasal swab during home visits. Paired specimens were processed and tested on the same day. RESULTS: The majority (109, 60%) of 182 participants were 20-30 years old. All 200 paired swabs had optimal specimen quality. The median time from symptom onsets to participant-collected swabs was 2 days and to staff-collected swabs was also 2 days. The median time interval between the 2 swabs was 2 hours. Compared to staff-collected swabs, the participant-collected swabs were 93% sensitive and 99% specific for influenza virus detection, 94% sensitive and 99% specific for RSV detection, and 100% sensitive and 100% specific for hMPV detection. CONCLUSIONS: Participant-collected midturbinate nasal swabs were a valid alternative approach for laboratory confirmation of influenza-, RSV-, and hMPV-associated illnesses among pregnant women in a community setting.

Respiratory Syncytial Virus and Human Metapneumovirus Infections in Three-Dimensional Human Airway Tissues Expose an Interesting Dichotomy in Viral Replication, Spread, and Inhibition by Neutralizing Antibodies.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are two of the leading causes of respiratory infections in children and elderly and immunocompromised patients worldwide. There is no approved treatment for HMPV and only one prophylactic treatment against RSV, palivizumab, for high-risk infants. Better understanding of the viral lifecycles in a more relevant model system may help identify novel therapeutic targets. By utilizing three-dimensional (3-D) human airway tissues to examine viral infection in a physiologically relevant model system, we showed that RSV infects and spreads more efficiently than HMPV, with the latter requiring higher multiplicities of infection (MOIs) to yield similar levels of infection. Apical ciliated cells were the target for both viruses, but RSV apical release was significantly more efficient than HMPV. In RSV- or HMPV-infected cells, cytosolic inclusion bodies containing the nucleoprotein, phosphoprotein, and respective viral genomic RNA were clearly observed in human airway epithelial (HAE) culture. In HMPV-infected cells, actin-based filamentous extensions were more common (35.8%) than those found in RSV-infected cells (4.4%). Interestingly, neither RSV nor HMPV formed syncytia in HAE tissues. Palivizumab and nirsevimab effectively inhibited entry and spread of RSV in HAE tissues, with nirsevimab displaying significantly higher potency than palivizumab. In contrast, 54G10 completely inhibited HMPV entry but only modestly reduced viral spread, suggesting HMPV may use alternative mechanisms for spread. These results represent the first comparative analysis of infection by the two pneumoviruses in a physiologically relevant model, demonstrating an interesting dichotomy in the mechanisms of infection, spread, and consequent inhibition of the viral lifecycles by neutralizing monoclonal antibodies.IMPORTANCE Respiratory syncytial virus and human metapneumovirus are leading causes of respiratory illness worldwide, but limited treatment options are available. To better target these viruses, we examined key aspects of the viral life cycle in three-dimensional (3-D) human airway tissues. Both viruses establish efficient infection through the apical surface, but efficient spread and apical release were seen for respiratory syncytial virus (RSV) but not human metapneumovirus (HMPV). Both viruses form inclusion bodies, minimally composed of nucleoprotein (N), phosphoprotein (P), and viral RNA (vRNA), indicating that these structures are critical for replication in this more physiological model. HMPV formed significantly more long, filamentous actin-based extensions in human airway epithelial (HAE) tissues than RSV, suggesting HMPV may promote cell-to-cell spread via these extensions. Lastly, RSV entry and spread were fully inhibited by neutralizing antibodies palivizumab and the novel nirsevimab. In contrast, while HMPV entry was fully inhibited by 54G10, a neutralizing antibody, spread was only modestly reduced, further supporting a cell-to-cell spread mechanism.

Structures of the Mononegavirales Polymerases.

Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5' methylated cap and 3' poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.

The role of M2-2 PDZ-binding motifs in pulmonary innate immune responses to human metapneumovirus.

Human metapneumovirus (HMPV) is a leading cause of lower respiratory tract infection (LRTI) in pediatric and geriatric populations. We recently found that two PDZ-binding motifs of the M2-2 protein, 29-DEMI-32 and 39-KEALSDGI-46, play a significant role in mediating HMPV immune evasion in airway epithelial cells (AECs). However, their role in the overall pulmonary responses to HMPV infection has not been investigated. In this study, we found that two recombinant HMPVs (rHMPV) lacking the individual M2-2 PDZ-binding motif are attenuated in mouse lungs. Mice infected with mutants produce more cytokines/chemokines in bronchoalveolar lavage (BAL) fluid compared to mice infected with wild-type rHMPV. In addition, both mutants are able to enhance the pulmonary recruitment of dendritic cells (DCs) and T cells and induce effective protections against the HMPV challenge. The DC maturation is also significantly improved by the motif mutation. Taken together, our data provide proof-of-principle for two live-attenuated M2-2 mutants to be promising HMPV vaccine candidates that are effective in inducing higher pulmonary innate immunity and generating protection against HMPV infection.

Etiology of acute viral respiratory infections common in Pakistan: A review.

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.

Infant Pneumococcal Carriage During Influenza, RSV, and hMPV Respiratory Illness Within a Maternal Influenza Immunization Trial.

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.

Severe Respiratory Illness Associated with Human Metapneumovirus in Nursing Home, New Mexico, USA.

Human metapneumovirus is an emerging pathogen that causes upper and lower respiratory illness. Nursing home outbreaks of infection with this virus can cause severe illness and lead to poor patient outcomes. We report an outbreak investigation in a nursing home during 2018 and infection control guidelines to assist in disease control.

Genetic characterization of human metapneumovirus identified through community and facility-based surveillance of infants in Dhaka, Bangladesh.

BACKGROUND: Acute respiratory infection (ARI) is a leading cause of morbidity and mortality in children in low and middle-income countries. Human metapneumovirus (hMPV) is one of the most common viral etiological agents for ARIs in children. OBJECTIVES: In this study, we explored the genotypic diversity and the epidemiology of hMPV among infants in Dhaka, Bangladesh. STUDY DESIGN: Between December 2014 and August 2016, a total of 3810 mid-turbinate nasal swab samples were collected from infants (0 to 6 months of age) who met clinical ARI criteria, as a part of a prospective ARI cohort study. hMPV was detected using polymerase chain reaction, and genotyped by sequencing and phylogenetic analysis. RESULTS: hMPV was identified in 206 (5.4%) nasal swab specimens. One-tenth of the hMPV-positive swabs (n = 19) were also positive for other respiratory viruses. hMPV activity peaked in January and September in 2015; however, no seasonal pattern of hMPV infection was detected. Phylogenetic analyses of the N and F gene-fragments revealed that the hMPV strains circulating in Dhaka, Bangladesh, belonged to three genotypes: A2b, A2c, and B1. Genotype A (57%) was the predominant hMPV genotype circulating in Bangladesh during the study period. CONCLUSION: This study describes both the epidemiology of hMPV infection and its genotypic strain diversity in Dhaka, Bangladesh.

High antibiotic prescription rates in hospitalized children with human metapneumovirus infection in comparison to RSV infection emphasize the value of point-of-care diagnostics.

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.

Time series non-Gaussian Bayesian bivariate model applied to data on HMPV and RSV: a case of Dadaab in Kenya.

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.

Evaluation of anticoagulant agents for the treatment of human metapneumovirus infection in mice.

Thrombin has been demonstrated to be involved in several viral diseases including human metapneumovirus (hMPV) infections. We previously showed that immediate administration of thrombin inhibitor argatroban post-infection protected mice against hMPV disease. This current work aims at determining whether warfarin and heparin, two other anticoagulants inhibiting thrombin formation and activities, may also be used for treatment against hMPV in vivo. We found that immediate injections of argatroban, warfarin or heparin after virus challenge protected mice against hMPV infection, as evidenced by decreased or no mortality, less weight loss, reduced viral load and attenuated inflammation. However, delayed treatments starting 1 day post-infection with argatroban or warfarin almost did not impact the survival whereas delayed treatment with heparin induced an increased mortality during infection. Moreover, these treatments also did not reduce weight loss, viral replication and inflammation. In agreement with these results, thrombin generation was decreased upon immediate anticoagulant treatments but was unaltered upon delayed treatments. Thus, thrombin generation occurs at the onset of hMPV infection and thrombin inhibition may be only useful for the treatment of this disease when initiated in the early stage. In this case, heparin is not recommended because of its reduced efficacy on mortality in infected mice whereas argatroban and warfarin appear as safe and effective drugs for the treatment of hMPV disease. The antiviral and anti-inflammatory effects of argatroban occur via thrombin-dependent pathways whereas the mechanisms by which warfarin exerts its beneficial effects against hMPV infection were not elucidated and need to be further studied.

NK-cell receptors NKp46 and NCR1 control human metapneumovirus infection.

Natural killer (NK) cells are capable of killing various pathogens upon stimulation of activating receptors. Human metapneumovirus (HMPV) is a respiratory virus, which was discovered in 2001 and is responsible for acute respiratory tract infection in infants and children worldwide. HMPV infection is very common, infecting around 70% of all children under the age of five. Under immune suppressive conditions, HMPV infection can be fatal. Not much is known on how NK cells respond to HMPV. In this study, using reporter assays and NK-cell cytotoxicity assays performed with human and mouse NK cells, we demonstrated that the NKp46-activating receptor and its mouse orthologue Ncr1, both members of the natural cytotoxicity receptor (NCR) family, recognized an unknown ligand expressed by HMPV-infected human cells. We demonstrated that MHC class I is upregulated and MICA is downregulated upon HMPV infection. We also characterized mouse NK-cell phenotype in the blood and the lungs of HMPV-infected mice and found that lung NK cells are more activated and expressing NKG2D, CD43, CD27, KLRG1, and CD69 compared to blood NK cells regardless of HMPV infection. Finally, we demonstrated, using Ncr1-deficient mice, that NCR1 plays a critical role in controlling HMPV infection.

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications.IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets.

Skewed balance of regulatory T cell and inflammatory T cell in IL-17 defect with human metapneumovirus infection.

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. However, the precise mechanisms underlying the development of hMPV-induced pulmonary pathology remain unknown. Studies show that IL-17 plays an important role in some inflammatory diseases of the airways, including asthma and chronic obstructive pulmonary disease. Here, we generated an IL-17 KO murine model of hMPV infection and used it to characterize the role of IL-17 hMPV-induced pulmonary inflammation. The results demonstrated that the defect in IL-17 resulted in less neutrophil influx into the lungs, along with reduced ventilatory function. Meanwhile, viral infection in IL-17 KO mice increased regulatory T cells (Tregs) and reduced Th1 and Th2 cells in the lung, suggesting that lack of IL-17 skews the immune response in the lung toward an anti-inflammatory profile, as exhibited by a greater number of Treg cells and fewer Th1 and Th2 cells.

Human metapneumovirus in hospitalized children less than 5 years of age in Pakistan.

Acute respiratory infection (ARI) is the second leading cause of death in children less than 5 years of age worldwide. Human Metapneumovirus (hMPV) is associated with around 5-7% of the total pneumonia admissions in children. The aim of this study was to determine the magnitude of hMPV associated hospitalizations among children, in Karachi, Pakistan. A 3 years prospective study was conducted at the Aga Khan University Hospital (AKUH), from August 2009 to June 2012. Children less than 5 years of age, admitted with ARIs, were enrolled. Throat swabs were collected and tested for hMPV using real-time PCR. Multivariable log binomial regression analysis was performed. Out of 1150 children enrolled, hMPV was detected among 84/1150 (7%). About 87% of the enrolled children presented with cough, followed by fever (73%), nasal congestion (69%) and shortness of breath (68%). Of the hMPV positive subjects, most (56/84, 67%) were less than 12 months of age. The most common diagnosis in hMPV positive infants was pneumonia, followed by asthma and bronchiolitis. HMPV was identified year round, with peaks during February and August. Sore throat was found to be significantly associated with the hMPV infection (Adjusted RR 2.23; 95%CI 1.42-3.52). The proportion of hMPV was higher among hospitalized infants with ARI. Pneumonia was the primary discharge diagnoses of patients who tested positive for hMPV. hMPV could be a target for future vaccine to further decrease the burden of ARI morbidity and possibly mortality in developing countries.