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Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) cause human respiratory diseases and are major targets for vaccine development. In this study, we design uncleaved prefusion-closed (UFC) trimers for the fusion protein (F) of both viruses by examining mutations critical to F metastability. For RSV, we assess four previous prefusion F designs, including the first and second generations of DS-Cav1, SC-TM, and 847A. We then identify key mutations that can maintain prefusion F in a native-like, closed trimeric form (up to 76%) without introducing any interprotomer disulfide bond. For hMPV, we develop a stable UFC trimer with a truncated F 2 -F 1 linkage and an interprotomer disulfide bond. Dozens of UFC constructs are characterized by negative-stain electron microscopy (nsEM), x-ray crystallography (11 RSV-F structures and one hMPV-F structure), and antigenic profiling. Using an optimized RSV-F UFC trimer as bait, we identify three potent RSV neutralizing antibodies (NAbs) from a phage-displayed human antibody library, with a public NAb lineage targeting sites Ø and V and two cross-pneumovirus NAbs recognizing site III. In mouse immunization, rationally designed RSV-F and hMPV-F UFC trimers induce robust antibody responses with high neutralizing titers. Our study provides a foundation for future prefusion F-based RSV and hMPV vaccine development., Competing Interests: Competing interests The authors declare the following competing interests: J.Z. serves as the Co-Founder, Interim Chief Scientific Officer, Consultant, and Scientific Advisory Board member of Uvax Bio, LLC, and holds associated financial interests. Other authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: The mRNA-1345 vaccine demonstrated efficacy against respiratory syncytial virus (RSV) disease with acceptable safety in adults aged ≥60 years in the ConquerRSV trial. Here, humoral immunogenicity results from the trial are presented., Methods: This phase 2/3 trial randomly assigned adults (≥60 years) to mRNA-1345 50-µg encoding prefusion F (preF) glycoprotein (n = 17 793) vaccine or placebo (n = 17 748). RSV-A and RSV-B neutralizing antibody (nAb) and preF binding antibody (bAb) levels at baseline and day 29 postvaccination were assessed in a per-protocol immunogenicity subset (PPIS; mRNA-1345, n = 1515; placebo, n = 333)., Results: Day 29 nAb geometric mean titers (GMTs) increased 8.4-fold against RSV-A and 5.1-fold against RSV-B from baseline. Seroresponses (4-fold rise from baseline) in the mRNA-1345 groups were 74.2% and 56.5% for RSV-A and RSV-B, respectively. Baseline GMTs were lower among participants who met the seroresponse criteria than those who did not. mRNA-1345 induced preF bAbs at day 29, with a pattern similar to nAbs. Day 29 antibody responses across demographic and risk subgroups were generally consistent with the overall PPIS., Conclusions: mRNA-1345 enhanced RSV-A and RSV-B nAbs and preF bAbs in adults (≥60 years) across various subgroups, including those at risk for severe disease, consistent with its demonstrated efficacy in the prevention of RSV disease., Clinical Trials Registration: NCT05127434., Competing Interests: Potential conflicts of interest. G. P. M. reports funding from Moderna, Merck, Sanofi, Pfizer, Universidad Nacional de San Martín, Medicago, GSK, and Cassara. C. J. A. D. reports receiving grants from the Medical Research Council and Wellcome, a consultancy for Synarigen, and is a member of the data and safety monitoring board at Oxford University. J. G., L. L., J. D., A. K., S. M., W. H., H. Z., S. K. S., F. P., N. L., C. A. S., E. W., J. M. M., and R. D. are employees of Moderna, Inc., and may hold stock/stock options in the company. N. L. C., K. S., and J. E. T. are consultants for Moderna, Inc. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Human respiratory syncytial virus (RSV) is a major cause of acute respiratory infection. In 2020, RSV was eliminated from New Zealand due to non-pharmaceutical interventions (NPI) used to control the spread of SARS-CoV-2. However, in 2021, following a brief quarantine-free travel agreement with Australia, there was a large-scale nationwide outbreak of RSV that led to reported cases more than five-times higher than typical seasonal patterns. We generated 1470 viral genomes of both RSV-A and RSV-B sampled between 2015-2022 from across New Zealand. Using a phylodynamics approach, we used these data to better understand RSV transmission patterns in New Zealand prior to 2020, and how RSV became re-established in the community following the relaxation of COVID-19 restrictions. We found that in 2021, there was a large epidemic of RSV due to an increase in importations, leading to several large genomic clusters of both RSV-A ON1 and RSV-B BA9 genotypes. However, while a number of viral importations were detected, there was also a major reduction in RSV genetic diversity compared to pre-pandemic years. These data reveal the impact of NPI used during the COVID-19 pandemic on other respiratory infections and highlight the important insights that can be gained from viral genomes., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

We looked at the interactions between viral loads, coinfection rates and disease severity for children infected with two pneumoviruses: human metapneumovirus (HMPV) and respiratory syncytial virus (RSV). The HMPV RNA load in nasopharyngeal swabs of hospitalized children was significantly higher in mono-infections compared to coinfections but this was not the case for RSV, which could be explained by different innate immune responses. Also, the viral load of neither of the two viruses was associated with disease severity although RSV-infected children had higher severity scores than HMPV-infected children., (© 2024 Wiley Periodicals LLC.)

We recently expanded the viral genomic surveillance program in Minnesota, USA, to include human respiratory syncytial virus. We performed whole-genome sequencing of 575 specimens collected at Minnesota healthcare facilities during July 2023-February 2024. Subgroups A and B differed in their genomic landscapes, and we identified 23 clusters of genetically identical genomes.

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43-25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Using a nasopharyngeal (NP) or anterior nasal (NS) swab from prospectively collected or retrospective specimens, we assessed the clinical performance of the BD Respiratory Viral Panel (BD RVP) for BD MAX System against FDA-cleared or authorized comparators. Across prospective and retrospective specimens, positive percent agreement (PPA) was ≥ 98.4% for SARS-CoV-2, ≥ 96.7% for influenza (flu) A, ≥ 91.7% for respiratory syncytial virus (RSV), and 100% for flu B (retrospective only) while negative percent agreement (NPA) was ≥ 97.7% across all targets, leading to the assay FDA clearance. A head-to-head comparison of NS versus NP results with BD RVP was also performed; PPA was ≥ 90% and NPA ≥ 98.2% for SARS-CoV-2, flu A and RSV. These findings confirm that the BD MAX RVP assay performs well for detection and differentiation of the three viruses in NP and NS specimens, with strong interrater agreements for NS versus NP comparisons., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Becton, Dickinson and Company reports financial support was provided by Biomedical Advanced Research and Development Authority. Nathan Ledeboer reports a relationship with Copan Italy that includes: consulting or advisory. Nathan Ledeboer reports a relationship with Thermo Fisher that includes: consulting or advisory. Barbara Van Der Pol reports a relationship with Abbott that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Becton, Dickinson and Company that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Biofire Diagnostics Inc that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Cue that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Cepheid that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Detect that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Hologic Inc that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Preventx and FIND that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Rheonix that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Roche that includes: consulting or advisory and funding grants. Barbara Van Der Pol reports a relationship with Visby that includes: consulting or advisory and funding grants. Matthew L. Faron reports a relationship with Becton, Dickinson and Company that includes: consulting or advisory. Sonia Paradis reports a relationship with Becton, Dickinson and Company that includes: employment and equity or stocks. William Laviers reports a relationship with Becton, Dickinson and Company that includes: employment and equity or stocks. Elizabeth Lockamy reports a relationship with Becton, Dickinson and Company that includes: employment and equity or stocks. Karen Yanson reports a relationship with Becton, Dickinson and Company that includes: employment and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Human respiratory syncytial virus (HRSV) is worldwide one of the leading causes of acute respiratory tract infections in young children and the elderly population. Two distinct subtypes of HRSV (A and B) and a multitude of genotypes have been described. The laboratory of Clinical and Epidemiological Virology (KU Leuven/University Hospitals Leuven) has a long-standing history of HRSV surveillance in Belgium., Methods: In this study, the seasonal circulation of HRSV in Belgium was monitored during 8 consecutive seasons prior to the SARS-CoV-2 pandemic (2011-2012 until 2018-2019). By use of a multiplex quantitative real time PCR panel, 27,386 respiratory samples were tested for HRSV. Further subtyping and sequencing of the HRSV positive samples was performed by PCR and Sanger sequencing. The prevalence and positivity rate were estimated in 4 distinct age groups and the circulating strains of each subtype were situated in a global context and in reference to the described genotypes in literature., Results: HRSV circulated in Belgium in a yearly re-occurring pattern during the winter months and both HRSV subtypes co-circulated simultaneously. All HRSV-B strains contained the 60 nt duplication in the HVR2 region of the G gene. Strains of subtype HRSV-A with a 72 nt duplication in the HVR2 region were first observed during the 2011-2012 season and replaced all other circulating strains from 2014 to 2015 onwards., (© 2024. The Author(s).)

Background: Nirsevimab is an extended half-life monoclonal antibody (mAb) licensed for the prevention of respiratory syncytial virus (RSV)-associated lower respiratory tract disease in neonates, infants and medically vulnerable children. We characterized RSV isolates recovered from participants enrolled in MEDLEY: a randomized, palivizumab-controlled phase 2/3 trial of nirsevimab in infants born preterm and/or with congenital heart disease or chronic lung disease of prematurity., Methods: Participants were assessed in two RSV seasons (Season 1 and 2). Season 1 participants were randomized (2:1) to receive a single dose of nirsevimab (50 mg if weight <5 kg or 100 mg if weight ≥5 kg in Season 1; 200 mg in Season 2) followed by four monthly doses of placebo, or five once-monthly doses of palivizumab (15 mg/kg weight per dose). Season 2 participants continued nirsevimab and placebo (nirsevimab/nirsevimab) or were re-randomized (1:1) to switch to nirsevimab (palivizumab/nirsevimab) or continue palivizumab (palivizumab/palivizumab). Cases of RSV infection were identified by central testing of nasal swabs from participants seeking medical attention for respiratory illnesses. Nirsevimab and palivizumab binding site substitutions were assessed via microneutralization assay., Results: Twenty-five cases of confirmed RSV infection were observed during the trial and sequenced: 12 in nirsevimab recipients and 10 in palivizumab recipients during Season 1, and 1 case in each Season 2 group. Molecular sequencing of RSV A (n = 14) isolates detected no nirsevimab binding site substitutions, and 3 palivizumab neutralization-resistant substitutions (Lys272Met, Lys272Thr, Ser275Leu). The nirsevimab binding site Ile206Met:Gln209Arg and Ile206Met:Gln209Arg:Ser211Asn substitutions were the only anti-RSV mAb binding site substitutions detected among RSV B isolates (n = 11). Nirsevimab neutralized all nirsevimab and palivizumab binding site substitutions in RSV A and B isolates recovered from MEDLEY participants., Conclusion: No binding site substitution detected during MEDLEY affected RSV susceptibility to nirsevimab neutralization., Competing Interests: Declaration of competing interest Kevin M. Tuffy, Bahar Ahani, Hong Ji, Vaishali S. Mankad, Ulrika Wählby Hamrén, Tonya Villafana, Yingyi Wang and Deidre Wilkins are current employees of AstraZeneca and may hold AstraZeneca stock or stock options. Elizabeth J. Kelly is a former employee of AstraZeneca and a current employee of Sanofi and may hold stock or stock options for both institutions. Ulrika Wählby Hamrén, Tonya Villafana and Deidre Wilkins are named inventors on patents planned, issued or pending related to nirsevimab. Joseph B. Domachowske has received funding to their institution for the conduct of clinical trials from AstraZeneca, Merck and Regeneron, honoraria from Sanofi and has participated in advisory boards for GSK and Sanofi. Kenji Furuno has received honoraria from AstraZeneca and AbbVie. Shabir A. Madhi has received funding from AstraZeneca, Novavax, Providence, Gritstone and ImmunityBio for the conduct of clinical trials; institutional grants from the Bill and Malinda Gates Foundation, GSK, Pfizer and Minervax, honoraria from GSK and MSD and is a member of Data Safety Monitoring Boards for PATH and CAPRISA., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescent-tagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release.IMPORTANCERespiratory syncytial virus is responsible for severe lower respiratory tract infections in young children under 5 years old, the elderly, and the immunosuppressed. The interaction of the respiratory syncytial virus matrix protein with the host actin cytoskeleton is important in infection but has not been investigated in depth. In this study, we show that the respiratory syncytial virus matrix protein associates with actin microfilaments and the actin-binding protein palladin, suggesting a role for palladin in respiratory syncytial virus release. This study provides new insight into the role of the actin cytoskeleton in respiratory syncytial virus infection, a key host-RSV interaction in assembly. Understanding the mechanism by which the RSV M protein and actin interact will ultimately provide a basis for the development of therapeutics targeted at RSV infections., Competing Interests: The authors declare no conflict of interest.

Respiratory syncytial virus (RSV), along with other prominent respiratory RNA viruses such as influenza and SARS-CoV-2, significantly contributes to the global incidence of respiratory tract infections. These pathogens induce the production of reactive oxygen species (ROS), which play a crucial role in the onset and progression of respiratory diseases. However, the mechanisms by which viral RNA manages ROS-induced base oxidation remain poorly understood. Here, we reveal that 8-oxo-7,8-dihydroguanine (8-oxoGua) is not merely an incidental byproduct of ROS activity but serves as a strategic adaptation of RSV RNA to maintain genetic fidelity by hijacking the 8-oxoguanine DNA glycosylase 1 (OGG1). Through RNA immunoprecipitation and next-generation sequencing, we discovered that OGG1 binding sites are predominantly found in the RSV antigenome, especially within guanine-rich sequences. Further investigation revealed that viral ribonucleoprotein complexes specifically exploit OGG1. Importantly, inhibiting OGG1's ability to recognize 8-oxoGua significantly decreases RSV progeny production. Our results underscore the viral replication machinery's adaptation to oxidative challenges, suggesting that inhibiting OGG1's reading function could be a novel strategy for antiviral intervention., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limit the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of severe acute respiratory syndrome coronavirus 2 in the community, but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying reverse-transcription quantitative polymerase chain reaction and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and nonpharmaceutical interventions., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Acute lower respiratory tract infections (ALRTIs) are a leading cause of mortality in young children worldwide due to human respiratory syncytial virus (RSV). The aim of this study was to monitor genetic variations in RSV and provide genomic data support for RSV prevention and control. A total of 105 complete RSV genome sequences were determined during 2017-2020. Phylogenetic analysis showed that all of the RSVA sequences were of genotype ON1, and all of the RSVB sequences were of genotype BA9. Notably, a phylogenetic tree based on the whole genome had more branches than a tree based on the G gene. In comparison to the RSV prototype sequences, 71.43% (50/70) of the ON1 sequences had five amino acid substitutions (T113I, V131N, N178G, H258Q, and H266L) that occurred simultaneously, and 68.57% (24/35) of the BA9 genotype sequences had 12 amino acid substitutions, four of which (A131T, T137I, T288I, and T310I) occurred simultaneously. In the F gene, there were 19 amino acid substitutions, which were mainly located in the antigenic sites Ø, II, V, and VII. Other amino acid substitutions were found in the NS1, NS2, P, SH, and L proteins. No significant evidence of recombination was found in any of the sequences. These findings provide important data that will be useful for prevention, control, and vaccine development against RSV., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Respiratory pathogen infections are seasonally prevalent and are likely to cause co-infections or serial infections during peak periods of infection. Since they often cause similar symptoms, simultaneous and on-site detection of respiratory pathogens is essential for accurate diagnosis and efficient treatment of these infectious diseases. However, molecular diagnostic techniques for multiple pathogens in this field are lacking. Herein, we developed a microfluidic LAMP and real-time fluorescence assay for rapid detection of multiple respiratory pathogens using a ten-channel microfluidic chip with pathogen primers pre-embedded in the chip reaction well. The microfluidic chip provided a closed reaction environment, effectively preventing aerosol contamination and improving the accuracy of the detection results. Its corresponding detection instrument could automatically collect and display the fluorescence curve in real time, which was more conducive to the interpretation of results. The results showed that the developed method could specifically recognize the nucleic acid of influenza A(H1N1), Mycoplasma pneumoniae , respiratory syncytial virus type A, and SARS-CoV-2 with low detection limits of 10 4 copies per mL or 10 3 copies per mL. The test results on clinical samples demonstrated that the developed method has high sensitivity (92.00%) and high specificity (100.00%) and even has the capability to differentiate mixed-infection samples. With simple operation and high detection efficiency, the present portable and simultaneous detection assay could significantly improve the efficiency of on-site detection of respiratory infectious diseases and promote the accurate treatment, efficient prevention and control of the diseases.

This study focuses on the cases(mainly characterized by respiratory symptoms such as cough, runny nose, fever, sore throat, and nasal congestion)of an outbreak of upper respiratory tract infections in a kindergarten in Jingyuan County, Baiyin City, Gansu Province, in May 2023. The epidemiological data were collected, and pharyngeal swab specimens were also obtained from the patients. The specimens of the research participants were subjected to respiratory multi-pathogen testing, and the positive specimens were further analyzed by sequencing the second hypervariable region (HRV2) of the G gene of respiratory syncytial virus (RSV) and constructing a phylogenetic tree. A total of 90 patients were collected, with an incidence rate of 22.84% (90/394), and the highest incidence was observed in the junior class group at 29.55%. Among the 17 pharyngeal swab specimens collected, 16 specimens were identified with the A subtype of respiratory syncytial virus. Sequencing analysis confirmed that it was the A subtype ON1 genotype. Based on the aforementioned testing results, it can be concluded that the current epidemic was primarily caused by infection with the A subtype of respiratory syncytial virus. Following the implementation of intervention measures, the epidemic has been effectively controlled.

Controlled human infection models (CHIMs) are a critical tool for the understanding of infectious disease progression, characterising immune responses to infection and rapid assessment of vaccines or drug treatments. There is increasing interest in using CHIMs for vaccine development and an obvious need for widely available and fit-for-purpose challenge agents. Inno4Vac is a large European consortium working towards accelerating and de-risking the development of new vaccines, including development of CHIMs for influenza, respiratory syncytial virus and Clostridium difficile. This report (in two parts) summarises a workshop held at the MHRA in 2021, focused on how to select CHIM candidate strains of influenza and respiratory syncytial virus (RSV) based on desirable virus characteristics and which immune assays would provide relevant information for assessing pre-existing and post-infection immune responses and defining correlates of protection. This manuscript (part 2) summarises presentations and discussions centred around RSV CHIMs and immune assays (an additional manuscript summarises influenza CHIM and immune assays: Inno4Vac workshop report Part 1: Controlled human influenza virus infection model (CHIVIM) strain selection and immune assays for CHIVIM studies, November 2021, MHRA, UK)., (© 2024 The Author(s). Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Respiratory syncytial virus (RSV) is a significant cause of acute lower respiratory tract infection (ALRTI) in children under five years of age. Between 2017 and 2021, 396 complete sequences of the RSV F gene were obtained from 500 RSV-positive throat swabs collected from ten hospitals across nine provinces in China. In addition, 151 sequences from China were sourced from GenBank and GISAID, making a total of 549 RSV F gene sequences subjected to analysis. Phylogenetic and genetic diversity analyses revealed that the RSV F genes circulating in China from 2017 to 2021 have remained relatively conserved, although some amino acids (AAs) have undergone changes. AA mutations with frequencies ≥ 10% were identified at six sites and the p27 region: V384I (site I), N276S (site II), R213S (site Ø), and K124N (p27) for RSV A; F45L (site I), M152I/L172Q/S173 ​L/I185V/K191R (site V), and R202Q/I206M/Q209R (site Ø) for RSV B. Comparing mutational frequencies in RSV-F before and after 2020 revealed minor changes for RSV A, while the K191R, I206M, and Q209R frequencies increased by over 10% in RSV B. Notably, the nirsevimab-resistant mutation, S211N in RSV B, increased in frequency from 0% to 1.15%. Both representative strains aligned with the predicted RSV-F structures of their respective prototypes exhibited similar conformations, with low root-mean-square deviation values. These results could provide foundational data from China for the development of RSV mAbs and vaccines., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

The severity of respiratory syncytial virus (RSV) may be linked to host genetic susceptibility. Surfactant protein (SP) genetic variants have been associated with RSV severity, but the impact of single-nucleotide polymorphism (SNP)-SNP interactions remains unexplored. Therefore, we used a novel statistical model to investigate the association of SNP-SNP interactions of SFTP genes with RSV severity in two- and three-interaction models. We analyzed available genotype and clinical data from prospectively enrolled 405 children diagnosed with RSV, categorizing them into moderate or severe RSV groups. Using Wang's statistical model, we studied significant associations of SNP-SNP interactions with RSV severity in a case-control design. We observed, first, association of three interactions with increased risk of severe RSV in a two-SNP model. One intragenic interaction was between SNPs of SFTPA2 , and the other two were intergenic, involving SNPs of hydrophilic and hydrophobic SPs alone. We also observed, second, association of 22 interactions with RSV severity in a three-SNP model. Among these, 20 were unique, with 12 and 10 interactions associated with increased or decreased risk of RSV severity, respectively, and included at least one SNP of either SFTPA1 or SFTPA2 . All interactions were intergenic except one, among SNPs of SFTPA1 . The remaining interactions were either among SNPs of hydrophilic SPs alone ( n = 8) or among SNPs of both hydrophilic or hydrophobic SPs ( n = 11). Our findings indicate that SNPs of all SFTP s may contribute to genetic susceptibility to RSV severity. However, the predominant involvement of SFTPA1 and/or SFTPA2 SNPs in these interactions underscores their significance in RSV severity. NEW & NOTEWORTHY Although surfactant protein (SP) genetic variants are associated with respiratory syncytial virus (RSV) severity, the impact of single-nucleotide polymorphism (SNP)-SNP interactions of SP genes remained unexplored. Using advanced statistical models, we uncovered 22 SNP-SNP interactions associated with RSV severity, with notable involvement of SFTPA1 and SFTPA2 SNPs. This highlights the comprehensive role of all SPs in genetic susceptibility to RSV severity, shedding light on potential avenues for targeted interventions.

Respiratory syncytial virus (RSV) is the main pathogen that causes hospitalization for acute lower respiratory tract infections (ALRIs) in children. With the reopening of communities and schools, the resurgence of RSV in the COVID-19 post-pandemic era has become a major concern. To understand the circulation patterns and genotype variability of RSV in Tianjin before and during the COVID-19 pandemic, a total of 19,531 nasopharyngeal aspirate samples from hospitalized children in Tianjin from July 2017 to June 2022 were evaluated. Direct immunofluorescence and polymerase chain reaction (PCR) were used for screening RSV-positive samples and subtyping, respectively. Further analysis of mutations in the second hypervariable region (HVR2) of the G gene was performed through Sanger sequencing. Our results showed that 16.46% (3215/19,531) samples were RSV positive and a delayed increase in the RSV infection rates occurred in the winter season from December 2020 to February 2021, with the average RSV-positive rate of 35.77% (519/1451). The ON1, with H258Q and H266L substitutions, and the BA9, with T290I and T312I substitutions, are dominant strains that alternately circulate every 1-2 years in Tianjin, China, from July 2017 to June 2022. In addition, novel substitutions, such as N296Y, K221T, N230K, V251A in the BA9 genotype, and L226I in the ON1 genotype, emerged during the COVID-19 pandemic. Analysis of clinical characteristics indicated no significant differences between RSV-A and RSV-B groups. This study provides a theoretical basis for clinical prevention and treatment. However, further studies are needed to explore the regulatory mechanism of host immune responses to different lineages of ON1 and BA9 in the future., Competing Interests: Conflict of interest All authors declare that there are no conflicts of interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Background/purpose: Recent large-scale epidemiological studies have revealed significant temporal associations between certain viral infections and the subsequent development of Kawasaki disease (KD). Despite these associations, definitive laboratory evidence linking acute or recent viral infections to KD cases remains elusive. The objective of this study is to employ a molecular epidemiological approach to investigate the temporal association between viral infections and the development of KD., Methods: We analyzed 2460 patients who underwent the FilmArray® Respiratory Panel test between April 2020 and September 2021., Results: Following the application of inclusion criteria, 2402 patients were categorized into KD (n = 148), respiratory tract infection (n = 1524), and control groups (n = 730). The KD group exhibited higher positive rates for respiratory syncytial virus (RSV), human rhinovirus/enterovirus (hRV/EV), parainfluenza virus (PIV) 3, and adenovirus (AdV) compared to the control group. Additionally, coinfections involving two or more viruses were significantly more prevalent in the KD group. Notably, RSV-positive, hRV/EV-positive, and PIV3-positive KD patients exhibited a one-month delay in peak occurrence compared to non-KD patients positive for corresponding viruses. In contrast, AdV-positive KD cases did not show a one-month delay in peak occurrence. Moreover, anti-RSV, anti-PIV3, and anti-AdV antibody-positive rates or antibody titers were higher in RSV-, PIV3-, and AdV-positive KD cases, respectively, compared to non-KD cases with the same viral infections., Conclusion: Recent infection with RSV, PIV3, or AdV, occasionally in conjunction with other viruses, may contribute to the pathogenesis of KD as infrequent complications., Competing Interests: Declaration of competing interest The authors declare no financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Background: Cell-penetrating peptides (CPPs) are effective for delivering therapeutic molecules with minimal toxicity. This study focuses on the use of penetratin, a well-characterized CPP, to deliver a DNA vector encoding short hairpin RNA (shRNA) targeting the respiratory syncytial virus (RSV) F gene into infected cells. RSV is known to cause severe lower respiratory infections in infants and poses significant risks to immunocompromised individuals and the elderly. We evaluated the antiviral efficacy of the penetratin-shRNA complex by comparing its ability to inhibit RSV replication and induce apoptosis with ribavirin treatment., Methods: Penetratin-shRNA complexes were prepared at different ratios and analyzed using gel retardation assays, dynamic light scattering, and zeta potential measurements. The complexes were tested in HEp-2 and A549 cells for transfection efficiency, cytotoxicity, viral load, and apoptosis using plaque assays, real-time reverse transcription-polymerase chain reaction (RT-PCR), DNA fragmentation, propidium iodide staining, and caspase 3/7 activation assays., Results: The gel shift assay determined that a 20:1 CPP-to-shRNA ratio was optimal for effective complexation, resulting in particles with a size of 164 nm and a zeta potential of 8.7 mV. Transfection efficiency in HEp-2 cells was highest at this ratio, reaching up to 93%. The penetratin-shRNA complex effectively silenced the RSV F gene, reduced viral titers, and decreased DNA fragmentation and apoptosis in infected cells., Conclusion: Penetratin effectively delivers shRNA targeting the RSV F gene, significantly reducing viral load and preventing apoptosis without toxicity. This approach surpasses Lipofectamine and shows potential for future therapeutic interventions, especially when combined with ribavirin, against RSV infection., (© 2024. The Author(s).)

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is highly immunogenic in its prefusion (pre-F) conformation. However, the protein is unstable, and its conformation must be stabilized for it to function effectively as an immunogen in vaccines. We present a mutagenesis strategy to arrest the RSV F protein in its pre-F state by blocking localized changes in protein structure that accompany large-scale conformational rearrangements. We generated a series of mutants and screened them in vitro to assess their potential for forming a stable pre-F. In animals, the immunogenicity of a representative mutant F protein, with a conformation confirmed by cryo-electron microscopy, elicited levels of neutralizing antibodies and protection against RSV-induced lung damage that were comparable to those of DS-Cav1, a pre-F used in a licensed vaccine.

Background: Respiratory syncytial virus (RSV) presents a global health concern. A lipid nanoparticle-encapsulated mRNA-based RSV vaccine (mRNA-1345) that encodes the membrane-anchored RSV prefusion-stabilized F glycoprotein is under clinical investigation., Methods: This phase 1 dose escalation study was based on a randomized, observer-blind, placebo-controlled design, and it assessed the safety and immunogenicity of mRNA-1345 in healthy adults aged 18 to 49 years. Participants were randomized to receive 1 dose of mRNA-1345 (50, 100, or 200 µg) or placebo or 3 doses of mRNA-1345 (100 µg) or placebo 56 days apart., Results: mRNA-1345 was well tolerated at all dose levels. The most common solicited adverse reactions were pain, headache, fatigue, myalgia, or chills, which were all generally mild to moderate. At 1 month postinjection, a single injection of mRNA-1345 boosted RSV neutralizing antibody titers (geometric mean fold rise: RSV-A, 20.0-23.5; RSV-B, 11.7-16.0) and RSV prefusion binding antibody concentrations (geometric mean fold rise, 16.1-21.8), with no apparent dose response. Antibody levels remained above baseline through 6 months. Sequential doses of 100 µg were well tolerated but did not further boost antibody levels., Conclusions: A single mRNA-1345 injection demonstrated an acceptable safety profile in younger adults and induced a durable neutralizing antibody response, supporting its continued development., Clinical Trials Registration: ClinicalTrials.gov NCT04528719., Competing Interests: Potential conflicts of interest. C. A. S., R. M., A. K., R. D., L. W., L. E. A., S. S.-G., K. S., and S. K. S. are employees of Moderna, Inc, and may hold stock/stock options in the company. A. A. and G. L. C. were Moderna, Inc, employees at the time of study conduct. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: An mRNA-based respiratory syncytial virus (RSV) vaccine, mRNA-1345, is under clinical investigation to address RSV disease burden in older adults., Methods: Based on a randomized, observer-blind, placebo-controlled design, this phase 1 dose-ranging study evaluated the safety, reactogenicity, and immunogenicity of mRNA-1345 in adults aged 65 to 79 years. Participants were randomized to receive 1 dose of mRNA-1345 (12.5, 25, 50, 100, or 200 µg) or placebo and matched mRNA-1345 booster or placebo at 12 months., Results: Overall, 298 participants received the first injection and 247 received the 12-month booster injection. mRNA-1345 was generally well tolerated after both injections, with the most frequently reported solicited adverse reactions being injection site pain, fatigue, headache, arthralgia, and myalgia. Reactogenicity was higher after the booster injection but with severity, time to onset, and duration similar to the first injection. A single mRNA-1345 injection boosted RSV-A and RSV-B neutralizing antibody titers and prefusion F binding antibody (preF bAb) concentrations at 1 month (geometric mean fold rises: RSV-A, 10.2-16.5; RSV-B, 5.3-12.5; preF bAb, 7.2-12.1). RSV antibody levels remained above baseline through 12 months, indicating immune persistence. A 12-month booster injection also increased RSV-A and RSV-B neutralizing antibody titers and preF bAb concentrations; titers after booster injection were numerically lower than those after the first dose, with overlapping 95% CIs., Conclusions: mRNA-1345 was well tolerated and immunogenic following a single injection and a 12-month booster., Clinical Trials Registration: NCT04528719 (ClinicalTrials.gov)., Competing Interests: Potential conflicts of interest. C. A. S., R. M., A. K., R. D., L. W., C. A. P., J. G., E. W., R. G., A. K. S., C. R., S. K. S., and G. L. C. are employees of Moderna, Inc, and may hold stock/stock options in the company. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Infection with respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract disease in young children and older people. Despite intensive efforts over the past few decades, no direct-acting small-molecule agents against RSV are available. Most small-molecule candidates targeting the RSV fusion (F) protein pose a considerable risk of inducing drug-resistant mutations. Here, we explored the in vitro and in vivo virological properties of the K394R variant, a cross-resistant mutant capable of evading multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in vitro and more pathogenic than the parental strain in vivo . The small molecule (2 E ,2' E )- N , N '-((1 R ,2 S ,3 S )-3-hydroxycyclohexane-1,2-diyl)bis(3-(2-bromo-4-fluorophenyl) acrylamide) (CL-A3-7), a structurally optimized compound derived from a natural caffeoylquinic acid derivative, substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant. Mechanistically, CL-A3-7 significantly inhibited virus-cell fusion during RSV entry by blocking the interaction between the viral F protein and the cellular insulin-like growth factor 1 receptor (IGF1R). Collectively, these results indicate severe disease risks caused by the K394R variant and reveal a new anti-RSV mechanism to overcome K394R-associated resistance., Importance: Respiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance., Competing Interests: The authors declare no conflict of interest.

Objectives: We aim to investigate the prevalence of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) among pediatric patients with acute respiratory tract illness (ARTI) in southern China both pre- and post-COVID-19 pandemic, as well as identify associated risk factors for severe infections., Methods: The study conducted a real-time PCR analysis on hospitalized children with ARTI from 2012 to 2023, specifically targeting RSV, hMPV, and other respiratory pathogens. Additionally, demographic data was collected during this analysis., Results: The prevalence of RSV occurs triennially, and likewise, the temporal pattern of hMPV outbreaks mirrors that of RSV. The peak infection rates of RSV and hMPV occurred during and following the implementation of COVID-19 epidemic prevention and control measures. The incidence of RSV infection exhibited bimodal peaks in 2022, while hMPV demonstrated seasonal peaks during the spring, fall, and winter periods post-COVID-19 pandemic. After the COVID-19 outbreak, there has been an upward trend in the proportion of female patients and patients aged one year and older presenting with ARTI, RSV infections, and hMPV infections. Infant (OR = 4.767, 95%CI: [3.888-5.846], p < 0.0001), presence of co-infection (OR = 0.540, 95%CI: [0.404-0.722], p < 0.0001), and existence of comorbidities (OR = 1.582, 95%CI: [1.285-1.949], p < 0.0001) was the risk ratio for the severity of RSV infection. Children infected with hMPV under the age of 1 year (OR = 0.322, 95%CI: [0.180 - 0.575], p < 0.0001), as well as those with comorbidities (OR = 8.809, 95%CI: [4.493 - 17.272], p < 0.0001), have a higher risk of developing severe illness., Conclusion: The changing epidemiological patterns have the potential to lead to widespread severe outbreaks among children, particularly those with underlying medical conditions who may experience more severe symptoms. Conducting surveillance for pneumoviridae viruses in children is an imperative measure to establish a robust foundation for future epidemic prevention and treatment strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kuang, Xu, Wang, Xie, Zhang, Guo, Liang and Zhu.)

Respiratory syncytial virus is a major causative agent of lower respiratory tract infection in children, especially infants with substantial morbidity and mortality implications. The virus undergoes continuous evolution documented by accumulation of mutations in the glycoprotein gene necessitating vigilant surveillance to provide essential data to epidemiologists and researchers involved in development of vaccines. This study was aimed to perform molecular characterization of respiratory syncytial virus (RSV) among children ≤ 5 years admitted in hospital. In the current study we observed RSV-A (2019 (n = 95) and 2021 (n = 61) seasons) and RSV-B (2022 season (n = 68)). Phylogenetic analysis revealed all RSV-A strains (n = 47) to be GA.2.3.5 and RSV-B (n = 22) were classified as GB.5.0.5a. Selection pressure analysis identified one positive (P274L/V) and one negative site (P230T) in RSV-A, while in RSV-B there was only one negatively selected site (S295). This study spanning over three seasons contributes to RSV evolutionary dynamics in India emphasizing the importance of on-going surveillance to inform effective public health strategies and vaccine development efforts., (© 2024 Wiley Periodicals LLC.)

Respiratory syncytial virus (RSV) is the most predominant viral pathogen worldwide in children with lower respiratory tract infections. The Coronavirus disease 2019 (COVID-19) pandemic and resulting nonpharmaceutical interventions perturbed the transmission pattern of respiratory pathogens in South Africa. A seasonality shift and RSV resurgence was observed in 2020 and 2021, with several infected children observed. Conventional RSV-positive nasopharyngeal swabs were collected from various hospitals in the Free State province, Bloemfontein, South Africa, from children suffering from respiratory distress and severe acute respiratory infection between 2020 to 2021. Overlapping genome fragments were amplified and complete genomes were sequenced using the Illumina MiSeq platform. Maximum likelihood phylogenetic and evolutionary analysis were performed on both RSV-A/-B G-genes with published reference sequences from GISAID and GenBank. Our study strains belonged to the RSV-A GA2.3.2 and RSV-B GB5.0.5a clades. The upsurge of RSV was due to pre-existing strains that predominated in South Africa and circulating globally also driving these off-season RSV outbreaks during the COVID-19 pandemic. The variants responsible for the resurgence were phylogenetically related to pre-pandemic strains and could have contributed to the immune debt resulting from pandemic imposed restrictions. The deviation of the RSV season from the usual pattern affected by the COVID-19 pandemic highlights the need for ongoing genomic surveillance and the identification of genetic variants to prevent unforeseen outbreaks in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Respiratory syncytial virus (RSV) causes respiratory disease and complications in infants, the elderly and the immunocompromised. While three vaccines and two prophylactic monoclonal antibodies are now available, only one antiviral, ribavirin, is currently approved for treatment. This review aims to summarize the current state of treatments directly targeting RSV. Two major viral processes are attractive for RSV-specific antiviral drug discovery and development as they play essential roles in the viral cycle: the entry/fusion process carried out by the fusion protein and the replication/transcription process carried out by the polymerase complex constituted of the L, P, N and M2-1 proteins. For each viral target resistance mutations to small molecules of different chemotypes seem to delineate definite binding pockets in the fusion proteins and in the large proteins. Elucidating the mechanism of action of these inhibitors thus helps to understand how the fusion and polymerase complexes execute their functions. While many inhibitors have been studied, few are currently in clinical development for RSV treatment: one is in phase III, three in phase II and two in phase I. Progression was halted for many others because of strategic decisions, low enrollment, safety, but also lack of efficacy. Lessons can be learnt from the halted programs to increase the success rate of the treatments currently in development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human Respiratory Syncytial Virus (hRSV), a prevalent respiratory pathogen affecting various age groups, can trigger prolonged and intense inflammation in humans. The severity and outcome of hRSV infection correlate with elevated levels of pro-inflammatory agents, yet the underlying reasons for this immune system overstimulation remain elusive. We focused on the impact of hRSV non-structural proteins, NS1 and NS2, on immune response within epithelial cells. Available data indicates that these proteins impair the interferon pathway. We reinforce that NS1 and NS2 induce heightened secretion of the pro-inflammatory cytokines IL-6 and CXCL8. We also indicate that hRSV non-structural proteins provoke differential gene expression of human host FosB and long non-coding RNAs (MALAT1, RP11-510N19.5). It suggests an impact of NS molecules beyond IFN pathways. Thus, new light is shed on the interplay between hRSV and host cells, uncovering unexplored avenues of viral interference, especially the NS2 role in cytokine expression and immune modulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Respiratory syncytial virus is the major cause of respiratory viral infections, particularly in infants, immunocompromised populations, and the elderly (over 65 years old), the prevention of RSV infection has become a priority. In this study, we generated a chimeric influenza virus, termed LAIV/RSV/HA-3F, using reverse genetics technology which contained three repeats of the RSV fusion protein neutralizing epitope site II to the N terminal in the background of the hemagglutinin (HA) gene of cold adapted influenza vaccine A/California/7/2009 ca. LAIV/RSV/HA-3F exhibited cold-adapted (ca) and attenuated (att) phenotype. BALB/c mice immunized intranasally with LAIV/RSV/HA-3F showed robust immunogenicity, inducing viral-specific antibody responses against both influenza and RSV, eliciting RSV-specific humoral, cellular and mucosal immune responses. LAIV/RSV/HA-3F also conferred protection as indicated by reduced viral titers and improved lung histopathological alterations against live RSV virus challenge. Mechanismly, single-cell RNA sequencing (scRNA-seq) and single-cell T cell antigen receptor (TCR) sequencing were employed to characterize the immune responses triggered by chimeric RSV vaccine, displaying that LAIV/RSV/HA-3F provided protection mainly via interferon-γ (IFN-γ). Moreover, we found that LAIV/RSV/HA-3F significantly inhibited viral replication in the challenged lung and protected against subsequent RSV challenge in cotton rats without causing lung disease. Taken together, our findings demonstrated that LAIV/RSV/HA-3F has potential as a promising bivalent vaccine with dual purpose candidate for the prevention of influenza and RSV, and preclinical and clinical studies warrant further investigations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Recent studies have documented prolonged expression of viral antigens and RNA and associated inflammation after infection with SARS-CoV-2 in a substantial proportion of infected patients. The persisting SARS-CoV-2 effects and findings, with inflammation associated with continued detection of viral antigens, especially resemble those previously reported for influenza virus, as well as respiratory syncytial virus (RSV). The reports indicate the need for improved insight into the mechanisms whereby post-SARS-CoV-2 infection-related illness is apparently more common and perhaps even more persistent after infection than observed for other respiratory viruses.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo . Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment., Competing Interests: The authors declare no conflict of interest.

Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.IMPORTANCEGiven the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value., Competing Interests: The authors declare no conflict of interest.

The human respiratory syncytial virus (RSV) is a significant health concern, particularly for infants, young children, and the elderly. This virus is known to evolve continuously due to environmental factors and herd immunity. In light of this, our study aimed to analyze the genetic variability of the G protein in RSV-A and RSV-B genotypes in Kuwait from 2020 to 2022. Between January 2020 and September 2022, we collected 490 respiratory samples from hospitalized patients with acute respiratory tract infections. These samples were tested and confirmed positive for RSV using multiplex Real-Time PCR. Subsequently, the samples underwent nucleic acid sequencing using the advanced Nanopore sequencing technology to analyze the full-length G gene. Sequence analysis showed that 64 isolates (76%) were RSV-A, and 20 isolates (24%) were RSV-B. The G genes of RSV-A belonged to genotype GA2.3.5, while all the RSV-B genotypes belonged to GB5.0.5a. New lineages and sub-lineages of RSV-A and RSV-B were detected, indicating the circulation of new strains in Kuwait. Many unique and new amino acid changes, including insertions, were found in the G proteins of Kuwaiti isolates, with the highest variability in the second hypervariable region. An increased number of N and O-linked glycosylation sites were also identified in the G protein, which could speculate to alter the antigenicity of RSV. The identified changes in the G protein of RSV-A and RSV-B genotypes might result from immune pressure and could affect the antigenic characteristics of circulating strains in Kuwait. This could potentially lead to new RSV variants that can evade the immune response. Our in-depth analysis of the G proteins of both RSV-A and RSV-B could aid in the development of more potent treatments and vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Madi, Sadeq, Safar, Al-Adwani and Al-Turab.)

Background: Worldwide, respiratory syncytial virus (RSV) infections are among the most common causes of infant hospitalization. Host genetic factors influencing the risk and severity of RSV infection are not well known., Methods: We conducted a genome-wide association study (GWAS) to investigate single-nucleotide polymorphisms (SNPs) associated with severe RSV infections using a nested case-control design based on 2 Danish cohorts. We compared SNPs from 1786 children hospitalized with RSV to 45 060 controls without an RSV-coded hospitalization. We performed gene-based testing, tissue enrichment, gene-set enrichment, and a meta-analysis of the 2 cohorts. Finally, an analysis of potential associations between the severity of RSV infection and genetic markers was performed., Results: We did not detect any significant genome-wide associations between SNPs and RSV infection or the severity of RSV. We did find potential loci associated with RSV infections on chromosome 5 in 1 cohort but failed to replicate any signals in both cohorts., Conclusions: Despite being the largest GWAS of severe RSV infection, we did not detect any genome-wide significant loci. This may be an indication of a lack of power or an absence of signal. Future studies might include mild illness and need to be larger to detect any significant associations., Competing Interests: Potential conflicts of interest . T. F. K. served as principal investigator (unpaid) in a Pfizer RSV vaccine trial, outside the submitted work. A. J. P. partnered with AstraZeneca for development of a COVID-19 vaccine. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The study focused on individuals with influenza-like symptoms (fever, cough, sore throat, runny nose, and other respiratory symptoms) in three kindergartens in Tongzhou District, Beijing City, in April 2023. Nasopharyngeal swab specimens were collected, and real-time fluorescent quantitative PCR was used to detect common respiratory pathogens in the collected specimens. Positive specimens were subjected to sequencing analysis of the highly variable region of human respiratory syncytial virus (HRSV) G protein, homology analysis and phylogenetic tree analysis. A total of 25 fever cases were collected from 3 kindergartens, aged 3-8 years old, with an age M ( Q 1 , Q 3 ) of 4 (3.5, 5) years old. Ten confirmed cases of HRSV positive were screened and detected using the fluorescent quantitative PCR method, with a total detection rate of 40% (10/25). Typing identification and sequencing analysis confirmed that the main epidemic type was HRSV subtype B, which was highly homologous and closely related to previous epidemic strains in the region. Through pathogen investigation and analysis, it was preliminarily determined that this epidemic was dominated by HRSV subtype B.

Objective: To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among cases presenting with influenza-like illness (ILI) in Shenzhen City from 2019 to 2023. Methods: Respiratory specimens were collected from two national sentinel hospitals in Shenzhen from March 2019 to December 2023, specifically targeting cases of ILI. The real-time PCR method was used for the detection and genotyping of HRSV. Basic demographic information was collected and used for the epidemiological analysis. Results: A total of 9 278 respiratory specimens of influenza-like cases were collected and detected, with a total positive rate of 4.77% (443/9 278) for HRSV. In 2021 (8.48%, 167/1 970), the positive rate of HRSV was significantly higher than in 2019 (3.35%, 52/1 552), 2022 (1.80%, 39/2 169), and 2023 (4.49%, 133/2 960), and the difference was statistically significant ( χ 2 =102.395, P <0.001). The prevalence of HRSV was mainly in summer and early autumn (September), and there was an abnormal increase in the positive rate of HRSV in winter 2022. The highest positive rate of HRSV was in children under five years old (9.84%, 330/335). The typing results showed that in 2022, the prevalence of HRSV-A was predominant (71.79%, 28/39), and in 2023, HRSV-A and HRSV-B subtypes coexisted. Conclusions: The prevalence of HRSV in Shenzhen from 2019 to 2023 has obvious seasonality, mainly in summer and early autumn. Children under five years old are the main population of HRSV infections.

Seasonal increase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV) require rapid diagnostic test methods for the management of respiratory tract infections. In this study, we compared the diagnostic accuracy of Savanna RVP4 (RVP4, QuidelOrtho) with Xpert Xpress Plus SARS-CoV-2/Flu/RSV (Xpert, Cepheid). Nasopharyngeal swabs from patients treated at a tertiary care hospital (Germany) were tested for SARS-CoV-2, Flu A/B, and RSV by RVP4 to assess diagnostic accuracy (reference standard: Xpert). The intra and inter assay precision of Ct-values was assessed by repeated test in triplicates (on day 1) and duplicates (days 2-3). All patients with a physician's order for a multiplex test for SARS-CoV-2, Flu, and RSV test were included. Duplicate swabs from the same patient, samples with a total volume ≤1 mL, or inappropriate shipment/storage were excluded. In total, 229 swabs were included between September 2023 and February 2024. The concordance between both tests was 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.6% (RSV). Flu B was not detected by both tests. The RVP4 test had a sensitivity of 85%-95% and a specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV. The intra and inter assay precision of Ct-values from RVP4 was 3% and 2% (SARS-CoV-2), 5% and 4% (Flu A), and 0% and 3% (RSV), respectively. The Savanna RVP4 has a favorable diagnostic accuracy for the detection of SARS-CoV-2, Flu A, and RSV., Importance: We assessed the diagnostic accuracy of a new point-of-care test for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus A/B (Flu A/B), and respiratory syncytial virus (RSV). The new test has a concordance with the reference standard of 96.5% (SARS-CoV-2), 98.7% (Flu A), and 99.1% (RSV). The sensitivity of 85%-95% and specificity of 100% for the detection of SARS-CoV-2, Flu A, and RSV is comparable with similar nucleic acid amplification-based point of care tests but at lower costs., Competing Interests: The authors declare no conflict of interest.

Non-pharmaceutical interventions (NPIs) implemented to control SARS-CoV-2 have significantly influenced the activity of respiratory pathogens. This study investigated epidemiological changes among hospitalized patients with respiratory syncytial virus (RSV) before (2017-2019) and during (2020-2022) the COVID-19 pandemic in Hangzhou, China. We also examined viral load distribution across demographic and temporal variables. Nasopharyngeal swabs were collected and RSV loads were quantified using reverse transcriptase polymerase chain reaction (RT-qPCR). RSV epidemic characteristics, seasonal dynamics, and viral load distributions were compared between pre- and pandemic years. General linear models were employed to assess associations between viral loads and age. Among 19 742 cases, 1576 and 2092 tested positive during the pre- and pandemic years, respectively. From February to July 2020, the implementation of NPIs led to the cessation of RSV circulation. However, after these measures were relaxed, RSV cases resurged over two consecutive seasons during the pandemic, notably affecting older children compared to those in the pre-pandemic years (1.00 years, IQR: 0.50-2.00 vs. 0.58 years, IQR: 0.27-1.00, p < 0.001). Specifically, in 2021-2022, an off-season resurgence of RSV began earlier (mid-June), lasted longer (40 weeks), and involved more positive cases (1238 cases) than both 2020-2021 and pre-pandemic years. Viral load distribution demonstrated a clear age-related relationship in both pre- and pandemic years, with younger children consistently showing higher viral loads, independently of gender and season (all p-values for trends <0.001). These findings highlight the impact of NPIs on RSV epidemiology and underscore the need to prioritize RSV infection prevention in younger children from the perspective of viral load., (© 2024 Wiley Periodicals LLC.)

Background: Following the pandemic restrictions, the epidemiology of respiratory syncytial virus (RSV) has changed, leading to intense hospitalization peaks., Objectives: This study, conducted at multiple sites in Italy, aimed to describe the temporal dynamics of two post-COVID-19 RSV epidemics. Additionally, the circulating RSV-A and -B lineages were characterized and compared to those found in 2018 and 2019., Study Design: Respiratory specimens and data were collected from RSV-positive patients, both inpatients, and outpatients, of all ages at three sites in north-central Italy. To analyze these samples, roughly one-sixth were sequenced in the attachment glycoprotein G gene and subjected to phylogenetic and mutational analyses, including pre-pandemic sequences from north-central Italy., Results: The first post-pandemic surge of RSV cases was quite intense, occurring from October 2021 to early January 2022. The subsequent RSV epidemic (from November 2022 to early March 2023) also had a high impact, characterized by a rise in elderly patient cases. Post-pandemic cases of RSV-A were caused by various strains present in Italy prior to COVID-19. In contrast, a distinct RSV-B lineage, which was concurrently spreading in other countries, was identified as the main cause of the surge in 2022-2023 but remained undetected in Italy before the pandemic., Conclusions: This study describes the temporal dynamics of post-pandemic RSV subgroups and uncovers a lineage of RSV-B with high genetic divergence that may have increased the impact of decreased population immunity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

BackgroundHealthcare personnel (HCP) are at high risk for respiratory infections through occupational exposure to respiratory viruses.AimWe used data from a prospective influenza vaccine effectiveness study in HCP to quantify the incidence of acute respiratory infections (ARI) and their associated presenteeism and absenteeism.MethodsAt the start and end of each season, HCP at two Israeli hospitals provided serum to screen for antibodies to influenza virus using the haemagglutination inhibition assay. During the season, active monitoring for the development of ARI symptoms was conducted twice a week by RT-PCR testing of nasal swabs for influenza and respiratory syncytial virus (RSV). Workplace presenteeism and absenteeism were documented. We calculated incidences of influenza- and RSV-associated ARI and applied sampling weights to make estimates representative of the source population.ResultsThe median age of 2,505 participating HCP was 41 years, and 70% were female. Incidence was 9.1 per 100 person-seasons (95% CI: 5.8-14.2) for RT-PCR-confirmed influenza and 2.5 per 100 person-seasons (95% CI: 0.9-7.1) for RSV illness. Each season, 18-23% of unvaccinated and influenza-negative HCP seroconverted. The incidence of seroconversion or RT-PCR-confirmed influenza was 27.5 per 100 person-seasons (95% CI: 17.8-42.5). Work during illness occurred in 92% (95% CI: 91-93) of ARI episodes, absence from work in 38% (95% CI: 36-40).ConclusionInfluenza virus and RSV infections and associated presenteeism and absenteeism were common among HCP. Improving vaccination uptake among HCP, infection control, and encouraging sick HCP to stay home are important strategies to reduce ARI incidence and decrease the risk of in-hospital transmission.

Background: Respiratory syncytial virus (RSV) infection imposes substantial health burden and disproportionally affects young infants, elderly, and immunocompromised hosts. RSV harbors key surface glycoproteins F and G, both crucial for viral infection and evolution., Methods: In this study, we examined the genetic characteaistics of 179 RSV isolates collected between 2017 and 2021 in Taiwan. G ectodomain and whole F gene were sequenced and aligned with available references from GenBank., Results: RSV ON1 and BA9 were two predominant genotypes throughout the study period. Genetic variations of G protein accumulated over time. New ON1 strains containing E257K and K204R-V225A-T238I-Y280H in combination emerged in 2019 and contributed to a local endemic in 2020. RSV-B strain with A131T and T137I substitution in G protein emerged in 2018. On the other hand, F protein of both RSV genotypes was generally conserved but some feature changes should be noted: RSV-B in Taiwan harbored 100% of I206M and Q209R in site Ø, and L172Q and S173L in site V. These amino acid changes do not affect the susceptibility of Nirsevimab but imply no effectiveness of Suptavumab., Conclusion: RSV continuously evolves in Taiwan and accumulated signature genetic changes over time. Vigilant RSV genomic surveillance is important to monitor the viral evolution in the upcoming future of new RSV vaccines and prophylaxis., (Copyright © 2024. Published by Elsevier B.V.)

A globally implemented unified phylogenetic classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. We formulated global consensus of HRSV classification on the basis of the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality curated dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to GenBank and GISAID (https://www.gisaid.org) public sequence databases through March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B and provided guidelines for defining prospective lineages. Our classification demonstrated robustness in its applicability to both complete and partial genomes. We envision that this unified HRSV classification proposal will strengthen HRSV molecular epidemiology on a global scale.

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Respiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. Using molecular methods, we quantified RSV A and RSV B RNA in wastewater solids across multiple seasons and metropolitan areas to gain insight into the predominance of RSV subtypes. We determined the predominant subtype for each group using the proportion of RSV A to total RSV (RSV A + RSV B) in each wastewater sample ( P A,WW ) and conducted a comparative analysis temporally, spatially, and against clinical specimens. A median P A,WW of 0.00 in the first season and 0.58 in the second season indicated a temporal shift in the predominant subtype. Spatially, while we observed dominance of the same subtype, P A,WW was higher in some areas ( P A,WW = 0.58-0.88). The same subtype predominated in wastewater and clinical samples, but clinical samples showed higher levels of RSV A (RSV A positivity in clinical samples = 0.79, median P A,WW = 0.58). These results suggest that wastewater, alongside clinical data, holds promise for enhanced subtype surveillance.IMPORTANCERespiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. The study illustrates that information on subtype predominance can be gleaned from wastewater. As a biological composite sample from the entire contributing population, wastewater monitoring of RSV A and B can complement clinical surveillance of RSV., Competing Interests: The authors declare no conflict of interest.

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSV A ) and B (HRSV B ) of HRSV. Next, the Pyrococcus furiosus Argonaute ( PfAgo ) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/μL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSV A and HRSV B infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liao, Feng, Zhang, Yang, Zhan, Zeng, Huang, Lian, Ke, Cai, Zhang, Fang, Cai, Chen, Lin, Lin, Chen, Liu, Huang, Lin and Lin.)

Background: Respiratory syncytial virus (RSV), a highly transmissible virus, is the leading cause of lower respiratory tract infections. We examined molecular changes in the RSV genome before and after the coronavirus disease 2019 (COVID-19) pandemic in Korea, and investigated whether drug-resistant mutations were present., Methods: In this prospective, single-center study, RSV-positive respiratory samples were collected between September 2019 and December 2022. Long-read whole-genome sequencing (WGS) was performed, and the presence of known drug-resistant substitutions for palivizumab, nirsevimab, and suptavumab was investigated., Results: Overall, 288 respiratory samples were collected from 276 children. WGS data were available for 133 samples (71 and 62 samples from the pre- and post-pandemic periods, respectively). All RSV-A strains (n = 56) belonged to the GA2.3.5 (ON1) genotype, whereas all RSV-B strains (n = 77) belonged to the GB5.0.5a (BA) genotype. No significant differences in genotypes were observed between the pre- and post-pandemic periods. In addition, no notable mutations related to nirsevimab or palivizumab resistance were detected in the F gene. However, the L172Q and S173L substitutions, which are known to confer resistance to suptavumab, were present in all RSV-B samples., Conclusion: Despite the unprecedented interruption of RSV seasonality, there were no significant molecular changes in circulating RSV strains in Korea related to nirsevimab or palivizumab resistance before and after the COVID-19 pandemic. However, RSV-specific drug-resistance substitutions for suptavumab were identified., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2024 The Korean Academy of Medical Sciences.)