An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

[Genotype and epidemiological characteristics of human metapneumovirus among hospitalized cases of acute respiratory infection in children in Changchun City, Jilin Province from 2019 to 2023].

Objective: To investigate the genotype and epidemiological characteristics of human metapneumovirus (HMPV) among hospitalized cases with acute respiratory infections (ARI) in children in Changchun City, Jilin Province, China. Methods: From June 2019 to June 2023, throat swabs of ARI inpatients in Changchun Children's Hospital were collected, and their epidemiological and clinical information were also collected. Quantitative reverse transcription-PCR was used to identify HMPV-positive cases, followed by the amplification of the G gene and genetic analysis in the HMPV-positive cases. Results: A total of 3 311 children hospitalized with ARI were included in this study. Their age ranged from 0 to 17 years old, and the M (Q1, Q3) of age was 2 (1, 3) years. About 1 811 (54.70%) cases were males. A total of 167 HMPV-positive cases were detected with a positive rate of 5.04%, of which 92.81% (155/167) were children under 5 years old. The positive rate of HMPV in 2019 was 6.37% (30/471), which dropped to the lowest in 2020 (2.31%, 10/432). The HMPV-positive rate was then rebounded in 2021 (4.70%, 60/1 277) and 2022 (4.56%, 21/461), which increased to 6.87% (46/670) in 2023. The difference in HMPV-positive rate among each year was statistically significant (P<0.05). The prevalence peak of HMPV varied in different years, showing either a unimodal or bimodal distribution in one year. A total of 79 HMPV G gene sequences were obtained, of which subtype A and subtype B accounted for 48.10% and 51.90%, respectively. All of the subtype A sequences were clarified as A2c duplicated variants, and subtype B was mainly B2 genotype. Besides, subtypes A and B were prevalent alone or co-circulated in different years, and there was a subtype replacement pattern in HMPV. Conclusion: The positive rate of HMPV in hospitalized ARI cases in children is significantly different from 2019 to 2023 in Changchun City. Notably, there are certain switch patterns of HMPV subtypes A and B in different years.

Discovery and characterization of novel jeilongviruses in wild rodents from Hubei, China.

The genus Jeilongvirus comprises non-segmented negative-stranded RNA viruses that are classified within the Paramyxoviridae family by phylogeny. Jeilongviruses are found in various reservoirs, including rodents and bats. Rodents are typical viral reservoirs with diverse spectra and zoonotic potential. Little is currently known about jeilongviruses in rodents from central China. The study utilized high-throughput and Sanger sequencing to obtain jeilongvirus genomes, including those of two novel strains (HBJZ120/CHN/2021 (17,468 nt) and HBJZ157/CHN/2021 (19,143 nt)) and three known viruses (HBXN18/CHN/2021 (19,212 nt), HBJZ10/CHN/2021 (19,700 nt), HBJM106/CHN/2021 (18,871 nt)), which were characterized by genome structure, identity matrix, and phylogenetic analysis. Jeilongviruses were classified into three subclades based on their topology, phylogeny, and hosts. Based on the amino acid sequence identities and phylogenetic analysis of the L protein, HBJZ120/CHN/2021 and HBJZ157/CHN/2021 were found to be strains rather than novel species. Additionally, according to specific polymerase chain reaction screening, the positive percentage of Beilong virus in Hubei was 6.38%, suggesting that Beilong virus, belonging to the Jeilongvirus genus, is likely to be widespread in wild rodents. The identification of novel strains further elucidated the genomic diversity of jeilongviruses. Additionally, the prevalence of jeilongviruses in Hubei, China, was profiled, establishing a foundation for the surveillance and early warning of emerging paramyxoviruses.

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.

Assessment of Illness Severity in Adults Hospitalized With Acute Respiratory Tract Infection due to Influenza, Respiratory Syncytial Virus, or Human Metapneumovirus.

BACKGROUND: Influenza, respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) are common respiratory viruses causing similar symptoms. Optimal tools to assess illness severity for these viruses have not been defined. Using the Hospitalized Acute Respiratory Tract Infection (HARTI) study data, we report symptom severity by clinician-rated clinical severity scores (CSS) in adults with influenza, RSV, or hMPV and correlations between CSS and patient-reported outcomes (PROs). METHODS: HARTI was a global epidemiologic study in adults hospitalized with acute respiratory tract infections. Patients were assessed at enrollment within 24 h of admission with CSS and twice during hospitalization with CSS, Respiratory Infection Intensity and Impact Questionnaire™ (RiiQ™), and EQ-5D-5L. Data were summarized descriptively, stratified by pathogen and baseline and hospitalization characteristics. Domain (general, upper respiratory, and lower respiratory) and sign/symptom subscores are presented for CSS; sign/symptom subscores are presented for RiiQ™ results. RESULTS: Data from 635 patients with influenza, 248 with RSV, and 107 with hMPV were included. At enrollment, total CSS and general and lower respiratory signs/symptoms (LRS) scores were higher for RSV and hMPV than influenza. Between-pathogen differences were greatest for LRS scores. Dyspnea, rales/rhonchi, wheezing, and shortness of breath scores trended higher for RSV and hMPV than influenza. RiiQ™ scores for cough, fatigue, and short of breath were strongly correlated with corresponding clinician-rated symptoms. CONCLUSIONS: These findings support the use of PROs (e.g., the RiiQ™) correlating with clinician assessments to gauge patient well-being and aid patient management by accurately assessing respiratory illness severity due to RSV, hMPV, or influenza.

Prevalence of Parainfluenza Viruses in Seoul, 2021 - 2022.

BACKGROUND: Parainfluenza virus (PIV) is a significant etiological agent of acute lower respiratory tract infections (ALRIs) in infants and young children. The present study has been conducted to investigate the prevalence of recently identified respiratory viruses. METHODS: In total, 543 oropharyngeal or nasopharyngeal swab samples collected from hospitalized patients with acute respiratory symptoms (ARS) between January and December 2021 (5,653 females and 4,950 males) were tested for respiratory viruses using RT-PCR. RESULTS: At least one respiratory virus was detected by RT-PCR in 119 out of 175 samples (68%). The most frequently detected virus was human rhinovirus (HRV) (34, 6.5%), followed by human parainfluenza viruses (HPIVs) (19, 3.6%), human bocavirus (HBoV) (8, 1.5%), human adenovirus (HAdV) (7, 1.3%), and human respiratory syncytial virus (HRSV) (4, 0.8%). HPIV-3 accounted for 3.6% (19/175) of all viral pathogens and was the second most frequently detected viral pathogen in our study. HPIV-3 infections peaked in the fall (November) of 2021. Phylogenetic analysis of the coding region of the viral protein HA revealed that all 35 (100%) of 35 HPIV-infected patients were infected with HPIV-3. CONCLUSIONS: HPIV was an important causative pathogen associated with ALRI in children hospitalized in Korea in the late fall of 2021, as the social distancing rules for COVID-19 were relaxed. These findings highlight the im-portance of HPIV as a cause of ALRI.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galß1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Universal paramyxovirus vaccine design by stabilizing regions involved in structural transformation of the fusion protein.

The Paramyxoviridae family encompasses medically significant RNA viruses, including human respiroviruses 1 and 3 (RV1, RV3), and zoonotic pathogens like Nipah virus (NiV). RV3, previously known as parainfluenza type 3, for which no vaccines or antivirals have been approved, causes respiratory tract infections in vulnerable populations. The RV3 fusion (F) protein is inherently metastable and will likely require prefusion (preF) stabilization for vaccine effectiveness. Here we used structure-based design to stabilize regions involved in structural transformation to generate a preF protein vaccine antigen with high expression and stability, and which, by stabilizing the coiled-coil stem region, does not require a heterologous trimerization domain. The preF candidate induces strong neutralizing antibody responses in both female naïve and pre-exposed mice and provides protection in a cotton rat challenge model (female). Despite the evolutionary distance of paramyxovirus F proteins, their structural transformation and local regions of instability are conserved, which allows successful transfer of stabilizing substitutions to the distant preF proteins of RV1 and NiV. This work presents a successful vaccine antigen design for RV3 and provides a toolbox for future paramyxovirus vaccine design and pandemic preparedness.

Clinical and epidemiological characteristics of 96 pediatric human metapneumovirus infections in Henan, China after COVID-19 pandemic: a retrospective analysis.

BACKGROUND: In the aftermath of the COVID-19 pandemic, there has been a surge in human metapneumovirus (HMPV) transmission, surpassing pre-epidemic levels. We aim to elucidate the clinical and epidemiological characteristics of HMPV infections in the post-COVID-19 pandemic era. METHODS: In this retrospective single-center study, participants diagnosed with laboratory confirmed HMPV infection through Targeted Next Generation Sequencing were included. The study encompassed individuals admitted to Henan Children's Hospital between April 29 and June 5, 2023. Demographic information, clinical records, and laboratory indicators were analyzed. RESULTS: Between April 29 and June 5, 2023, 96 pediatric patients were identified as infected with HMPV with a median age of 33.5 months (interquartile range, 12 ~ 48 months). The majority (87.5%) of infected children were under 5 years old. Notably, severe cases were statistically younger. Predominant symptoms included fever (81.3%) and cough (92.7%), with wheezing more prevalent in the severe group (56% vs 21.1%). Coinfection with other viruses was observed in 43 patients, with Epstein-Barr virus (EBV) (15.6%) or human rhinovirus A (HRV type A) (12.5%) being the most common. Human respiratory syncytial virus (HRSV) coinfection rate was significantly higher in the severe group (20% vs 1.4%). Bacterial coinfection occurred in 74 patients, with Haemophilus influenzae (Hin) and Streptococcus pneumoniae (SNP) being the most prevalent (52.1% and 41.7%, respectively). Severe patients demonstrated evidence of multi-organ damage. Noteworthy alterations included lower concentration of IL-12p70, decreased lymphocytes percentages, and elevated B lymphocyte percentages in severe cases, with statistical significance. Moreover, most laboratory indicators exhibited significant changes approximately 4 to 5 days after onset. CONCLUSIONS: Our data systemically elucidated the clinical and epidemiological characteristics of pediatric patients with HMPV infection, which might be instructive to policy development for the prevention and control of HMPV infection and might provide important clues for future HMPV research endeavors.

Geographical Expansion of Avian Metapneumovirus Subtype B: First Detection and Molecular Characterization of Avian Metapneumovirus Subtype B in US Poultry.

Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

Molecular Epidemiology and Clinical Characteristics of an Outbreak on Respiratory Virus Coinfection in Gansu, China.

This study aims to analyze the epidemiological and pathogenic characteristics of an outbreak primarily caused by respiratory syncytial virus (RSV), human rhinovirus (HRV), and human metapneumovirus (HMPV) in a kindergarten and primary school. The outbreak was investigated by field epidemiological investigation, and the common respiratory pathogens were screened by RT-PCR detection technology. The attack rate of this outbreak was 63.95% (110/172). Main symptoms included cough (85.45%), sore throat (60.91%), and sneezing (60.00%). Multifactorial logistic regression analysis revealed that continuous handwashing and mouth and nose covering when sneezing were protective factors. All 15 collected throat swab specimens tested positive for viruses, with HMPV as the predominant pathogen (80.00%), followed by HRV (53.33%), and two cases of positive respiratory syncytial virus (13.33%). Among them, six samples showed coinfections of HMPV and HRV, and one had coinfections of HMPV and RSV, resulting in a coinfection rate of 46.67%. Genetic sequencing indicated that the HMPV genotype in this outbreak was A2c, and the HRV genotype was type A, resulting in a coinfection outbreak of HMPV, HRV, and RSV in schools and kindergartens, suggesting that multi-pathogen surveillance of respiratory tract infections should be strengthened.

Sosuga Virus Detected in Egyptian Rousette Bats (Rousettus aegyptiacus) in Sierra Leone.

Sosuga virus (SOSV), a rare human pathogenic paramyxovirus, was first discovered in 2012 when a person became ill after working in South Sudan and Uganda. During an ecological investigation, several species of bats were sampled and tested for SOSV RNA and only one species, the Egyptian rousette bat (ERBs; Rousettus aegyptiacus), tested positive. Since that time, multiple other species have been sampled and ERBs in Uganda have continued to be the only species of bat positive for SOSV infection. Subsequent studies of ERBs with SOSV demonstrated that ERBs are a competent host for SOSV and shed this infectious virus while exhibiting only minor infection-associated pathology. Following the 2014 Ebola outbreak in West Africa, surveillance efforts focused on discovering reservoirs for zoonotic pathogens resulted in the capture and testing of many bat species. Here, SOSV RNA was detected by qRT-PCR only in ERBs captured in the Moyamba District of Sierra Leone in the central region of the country. These findings represent a substantial range extension from East Africa to West Africa for SOSV, suggesting that this paramyxovirus may occur in ERB populations throughout its sub-Saharan African range.

SQSTM1 downregulates avian metapneumovirus subgroup C replication via mediating selective autophagic degradation of viral M2-2 protein.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.

IFN-λ drives distinct lung immune landscape changes and antiviral responses in human metapneumovirus infection.

Human metapneumovirus (HMPV) is a primary cause of acute respiratory infection, yet there are no approved vaccines or antiviral therapies for HMPV. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways. Type III interferon (IFN-λ) displays potent antiviral activity against respiratory viruses and is being investigated for therapeutic use. However, its role in HMPV infection remains largely unknown. Here, we show that IFN-λ is highly upregulated during HMPV infection in vitro in human and mouse airway epithelial cells and in vivo in mice. We found through several immunological and molecular assays that type II alveolar cells are the primary producers of IFN-λ. Using mouse models, we show that IFN-λ limits lung HMPV replication and restricts virus spread from upper to lower airways but does not contribute to clinical disease. Moreover, we show that IFN-λ signaling is predominantly mediated by CD45- non-immune cells. Mice lacking IFN-λ signaling showed diminished loss of ciliated epithelial cells and decreased recruitment of lung macrophages in early HMPV infection along with higher inflammatory cytokine and interferon-stimulated gene expression, suggesting that IFN-λ may maintain immunomodulatory responses. Administration of IFN-λ for prophylaxis or post-infection treatment in mice reduced viral load without inflammation-driven weight loss or clinical disease. These data offer clinical promise for IFN-λ in HMPV treatment. IMPORTANCE: Human metapneumovirus (HMPV) is a common respiratory pathogen and often contributes to severe disease, particularly in children, immunocompromised people, and the elderly. There are currently no licensed HMPV antiviral treatments or vaccines. Here, we report novel roles of host factor IFN-λ in HMPV disease that highlight therapeutic potential. We show that IFN-λ promotes lung antiviral responses by restricting lung HMPV replication and spread from upper to lower airways but does so without inducing lung immunopathology. Our data uncover recruitment of lung macrophages, regulation of ciliated epithelial cells, and modulation of inflammatory cytokines and interferon-stimulated genes as likely contributors. Moreover, we found these roles to be distinct and non-redundant, as they are not observed with knockout of, or treatment with, type I IFN. These data elucidate unique antiviral functions of IFN-λ and suggest IFN-λ augmentation as a promising therapeutic for treating HMPV disease and promoting effective vaccine responses.

Molecular Epidemiology of Human Metapneumovirus in East Japan before and after COVID-19, 2017-2022.

Human metapneumovirus (hMPV) is genetically classified into two major subgroups, A and B, based on attachment glycoprotein (G protein) gene sequences. The A2 subgroup is further separated into three subdivisions, A2a, A2b (A2b1), and A2c (A2b2). Subgroup A2c viruses carrying 180- or 111-nucleotide duplications in the G gene (A2c 180nt-dup or A2c 111nt-dup ) have been reported in Japan and Spain. The coronavirus disease 2019 (COVID-19) pandemic disrupted the epidemiological kinetics of other respiratory viruses, including hMPV. In this study, we analyzed the sequences of hMPV isolates in Tokyo and Fukushima obtained from 2017 to 2022, i.e., before and after the COVID-19 pandemic. Subgroup A hMPV strains were detected from 2017 to 2019, and most cases were A2c 111nt-dup, suggesting ongoing transmission of this clade, consistent with global transmission dynamics. Subgroup B viruses, but not subgroup A viruses, were detected in 2022 after the COVID-19 peak. Phylogenetic analysis showed that the subgroup B viruses were closely related to strains detected in Yokohama from 2013 to 2016, and strains detected in Fukushima in 2019, suggesting the reappearance of local endemic viruses in East Japan.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Clinical factors associated with extended hospitalization in pediatric patients ≥3 years of age with respiratory syncytial virus or human metapneumovirus infection: A Japanese single-center, retrospective study.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections are common in children worldwide. However, the clinical factors related to extended hospitalization in Japanese patients aged ≥3 years remain elusive. We aimed to elucidate the clinical risk factors contributing to hospital stays ≥7 days in patients with RSV and hMPV infections. Patients ≥3 years of age who were hospitalized due to RSV or hMPV infection between 2014 to 2020 were included. Twenty-one RSV- and 27 hMPV-infected patients were enrolled. Patients were divided into 2 groups: hospitalization for ≥ and <7 days. Univariate and multivariate analyses determined the clinical risk factors contributing to hospital stay ≥7 days. The RSV- and hMPV-infected patients had similar clinical characteristics. The clinical risk factors contributing to extended hospitalization were analyzed in the 48 infected patients of the 2 groups. The presence of prophylactic antibiotics usage, co-bacterial colonization, and underlying diseases were extracted by univariate analysis (P < .05). In multivariate analysis, underlying diseases were determined as an independent clinical risk factor (odds ratio 8.09, P = .005). Underlying diseases contributed to extended hospitalization in RSV- or hMPV-infected patients ≥3 years of age.

Whole genome sequencing and evolution analyses of Human metapneumovirus.

Human metapneumovirus (HMPV) is a major pathogen of acute respiratory tract infections (ARTIs) in children. Whole genome sequence analyses could help understand the evolution and transmission events of this virus. In this study, we sequenced HMPV whole genomes to improve the identification of molecular epidemiology in Beijing, China. Nasopharyngeal aspirates of hospitalized children aged < 14 years old with ARTIs were screened for HMPV infection using qPCR. Fourteen pairs of overlapping primers were used to amplify whole genome sequences of HMPV from positive samples with high viral loads. The epidemiology of HMPV was analysed and 27 HMPV whole genome sequences were obtained. Sequence identity and the positional entropy analyses showed that most regions of HMPV genome are conserved, whereas the G gene contained many variations. Phylogenetic analysis identified 25 HMPV sequences that belonged to a newly defined subtype A2b1; G gene sequences from 24 of these contained a 111-nucleotide duplication. HMPV is an important respiratory pathogen in paediatric patients. The new subtype A2b1 with a 111-nucleotide duplication has become predominate in Beijing, China.

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

Human Metapneumovirus (hMPV) Infection and MPV467 Treatment in Immunocompromised Cotton Rats Sigmodon hispidus.

Human metapneumovirus (hMPV) is an important cause of respiratory disease in immunocompromised individuals, yet hMPV infection has not been modeled before in immunocompromised animals. In this work, cotton rats S. hispidus immunosuppressed by cyclophosphamide were infected with hMPV, and viral replication and pulmonary inflammation in these animals were compared to those in normal hMPV-infected S. hispidus. The efficacy of prophylactic and therapeutic administration of the anti-hMPV antibody MPV467 was also evaluated. Immunosuppressed animals had higher pulmonary and nasal titers of hMPV on day 5 post-infection compared to normal animals, and large amounts of hMPV were still present in the respiratory tract of immunosuppressed animals on days 7 and 9 post-infection, indicating prolonged viral replication. Immunosuppression was accompanied by reduced pulmonary histopathology in hMPV-infected cotton rats compared to normal animals; however, a delayed increase in pathology and pulmonary chemokine expression was seen in immunosuppressed cotton rats. Prophylactic and therapeutic MPV467 treatments protected both upper and lower respiratory tracts against hMPV infection. The lung pathology and pulmonary expression of IP-10 and MIP-1α mRNA were reduced by therapeutic MPV467 administration. These results indicate that immunosuppressed cotton rats represent a useful model for studying hMPV pathogenesis and for evaluating therapeutics that could alleviate hMPV-induced disease in immunocompromised subjects.