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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).)

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 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.

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).)

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.)

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.)

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).)

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 (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.)

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.)

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.

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.)

Respiratory syncytial virus (RSV) is a significant cause of morbidity, particularly in infants. This study describes RSV genomic diversity and disease outcomes during the 2023-2024 season in the Johns Hopkins Hospital System (JHHS). Between August and December 2023, 406 patient samples were sequenced, showing that RSV-B GB5.0.5a was the dominant genotype detected. RSV-A genotype GA2.3.5 was detected less frequently. Metadata analysis of patient data revealed that, although RSV-B was more commonly detected, patients with RSV-A infections were more frequently hospitalized. Analysis of both the G- and F-genes revealed multiple amino acid substitutions in both RSV-A and RSV-B, with some positions within the F-protein that could be associated with evasion of antibody responses. Phylogenetic analysis revealed the genetic diversity of circulating GB5.0.5a and GA2.3.5 genotypes. This study serves as an important baseline for genomic surveillance of RSV within the JHHS and will assist in characterizing the impact of the newly approved RSV vaccines on RSV genomic evolution and the emergence of escape mutations., Competing Interests: The authors declare no relevant conflicts of interest.

The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait., Competing Interests: The authors declare no conflict of interest.

Background: Respiratory syncytial virus (RSV) is widely recognized as a cause of acute respiratory failure in infants and immunocompromised patients. However, RSV can also contribute to acute respiratory failure in adults, particularly among the elderly population. The objective of this study was to analyze the clinical characteristics and outcomes of immunocompetent adults hospitalized for RSV infection., Methods: This retrospective study included all immunocompetent adult patients consecutively admitted to a tertiary care hospital with RSV-related acute respiratory failure over a seven-year period (2016-2023). Diagnosis of RSV infection was made through nasal swabs or pulmonary samples, with multiplex reverse transcription polymerase chain reaction (RT-PCR). Patients were eligible for inclusion if they required supplemental oxygen therapy for at least 48 h., Results: One hundred and four patients met the inclusion criteria. Median age [IQR] was 77 years [67-85]. Ninety-seven patients had at least one comorbidity (97/104, 93%). At the time of RSV diagnosis, 67 patients (67/104, 64%) experienced acute decompensation of a pre-existing chronic comorbidity. Antibiotics were started in 80% (77/104) of patients; however, only 16 patients had a confirmed diagnosis of bacterial superinfection. Twenty-six patients needed ventilatory support (26/104, 25%) and 21 were admitted to the intensive care unit (21/104, 20%). The median duration of oxygen therapy [IQR] was 6 days [3-9], while the median hospital length of stay [IQR] was 11 days [6-15]. The overall mortality rate within 1 month of hospital admission was 13% (14/104). The sole variables associated with one-month mortality were age and maximum oxygen flow during hospitalization., Conclusion: RSV-associated acute respiratory failure affected elderly individuals with multiple comorbidities and was associated with prolonged hospitalization and a high mortality rate., 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. PL has received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Astrazeneca, GlaxoSmithKline, Janssen, Merck Sharp & Dohme, Moderna, Pfizer, Sanofi Pasteur and support for attending meetings and/or travel from Astrazeneca, Pfizer and Sanofi Pasteur. VB received support for attending meetings from LFB and worked with the advisory boards of Biotest, Novartis and Takeda., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nonpharmaceutical interventions (NPIs) implemented during the COVID-19 pandemic have disrupted the dynamics of respiratory syncytial virus (RSV) on a global scale; however, the cycling of RSV subtypes in the pre- and post-pandemic period remains poorly understood. Here, we used a two subtype RSV model supplemented with epidemiological data to study the impact of NPIs on the two circulating subtypes, RSV-A and RSV-B. The model is calibrated to historic RSV subtype data from the United Kingdom and Finland and predicts a tendency for RSV-A dominance over RSV-B immediately following the implementation of NPIs. Using a global genetic dataset, we confirm that RSV-A has prevailed over RSV-B in the post-pandemic period, consistent with a higher R 0 for RSV-A. With new RSV infant monoclonals and maternal and elderly vaccines becoming widely available, these results may have important implications for understanding intervention effectiveness in the context of disrupted subtype dynamics., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Although administrative claims data have a high degree of completeness, not all medically attended Respiratory Syncytial Virus-associated lower respiratory tract infections (RSV-LRTIs) are tested or coded for their causative agent. We sought to determine the attribution of RSV to LRTI in claims data via modeling of temporal changes in LRTI rates against surveillance data., Methods: We estimated the weekly incidence of LRTI (inpatient, outpatient, and total) for children 0-4 years using 2011-2019 commercial insurance claims, stratified by HHS region, matched to the corresponding weekly NREVSS RSV and influenza positivity data for each region, and modelled against RSV, influenza positivity rates, and harmonic functions of time assuming negative binomial distribution. LRTI events attributable to RSV were estimated as predicted events from the full model minus predicted events with RSV positivity rate set to 0., Results: Approximately 42% of predicted RSV cases were coded in claims data. Across all regions, the percentage of LRTI attributable to RSV were 15-43%, 10-31%, and 10-31% of inpatient, outpatient, and combined settings, respectively. However, when compared to coded inpatient RSV-LRTI, 9 of 10 regions had improbable corrected inpatient LRTI estimates (predicted RSV/coded RSV ratio < 1). Sensitivity analysis based on separate models for PCR and antigen-based positivity showed similar results., Conclusions: Underestimation based on coding in claims data may be addressed by NREVSS-based adjustment of claims-based RSV incidence. However, where setting-specific positivity rates is unavailable, we recommend modeling across settings to mirror NREVSS's positivity rates which are similarly aggregated, to avoid inaccurate adjustments., (© 2024. The Author(s).)

Background: Respiratory Syncytial Virus (RSV) disease in young children ranges from mild cold symptoms to severe symptoms that require hospitalization and sometimes result in death. Studies have shown a statistical association between RSV subtype or phylogenic lineage and RSV disease severity, although these results have been inconsistent. Associations between variation within RSV gene coding regions or residues and RSV disease severity has been largely unexplored., Methods: Nasal swabs from children (< 8 months-old) infected with RSV in Rochester, NY between 1977-1998 clinically presenting with either mild or severe disease during their first cold-season were used. Whole-genome RSV sequences were obtained using overlapping PCR and next-generation sequencing. Both whole-genome phylogenetic and non-phylogenetic statistical approaches were performed to associate RSV genotype with disease severity., Results: The RSVB subtype was statistically associated with disease severity. A significant association between phylogenetic clustering of mild/severe traits and disease severity was also found. GA1 clade sequences were associated with severe disease while GB1 was significantly associated with mild disease. Both G and M2-2 gene variation was significantly associated with disease severity. We identified 16 residues in the G gene and 3 in the M2-2 RSV gene associated with disease severity., Conclusion: These results suggest that phylogenetic lineage and the genetic variability in G or M2-2 genes of RSV may contribute to disease severity in young children undergoing their first infection., (© 2024. The Author(s).)

This study aimed to determine the incidence and etiological, seasonal, and genetic characteristics of respiratory viral coinfections involving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Between October 2020 and January 2024, nasopharyngeal samples were collected from 2277 SARS-CoV-2-positive patients. Two multiplex approaches were used to detect and sequence SARS-CoV-2, influenza A/B viruses, and other seasonal respiratory viruses: multiplex real-time polymerase chain reaction (PCR) and multiplex next-generation sequencing. Coinfections of SARS-CoV-2 with other respiratory viruses were detected in 164 (7.2%) patients. The most common co-infecting virus was respiratory syncytial virus (RSV) (38 cases, 1.7%), followed by bocavirus (BoV) (1.2%) and rhinovirus (RV) (1.1%). Patients ≤ 16 years of age had the highest rate (15%) of mixed infections. Whole-genome sequencing produced 19 complete genomes of seasonal respiratory viral co-pathogens, which were subjected to phylogenetic and amino acid analyses. The detected influenza viruses were classified into the genetic groups 6B.1A.5a.2a and 6B.1A.5a.2a.1 for A(H1N1)pdm09, 3C.2a1b.2a.2a.1 and 3C.2a.2b for A(H3N2), and V1A.3a.2 for the B/Victoria lineage. The RSV-B sequences belonged to the genetic group GB5.0.5a, with HAdV-C belonging to type 1, BoV to genotype VP1, and PIV3 to lineage 1a(i). Multiple amino acid substitutions were identified, including at the antibody-binding sites. This study provides insights into respiratory viral coinfections involving SARS-CoV-2 and reinforces the importance of genetic characterization of co-pathogens in the development of therapeutic and preventive strategies.

Following an interseasonal rise in mainly pediatric respiratory syncytial virus (RSV) cases in Germany in 2021, an exceptionally high number of adult cases was observed in the subsequent respiratory season of 2022/2023. The aim of this study was to compare the clinical presentation of RSV infections in the pre- and post-SARS-CoV-2 pandemic periods. Additionally, the local epidemiology of the RSV fusion protein was analyzed at a molecular genetic and amino acid level. RSV detections in adults peaked in calendar week 1 of 2023, 8 weeks earlier than the earliest peak observed in the three pre-pandemic seasons. Although the median age of the adult patients was not different (66.5 vs. 65 years), subtle differences between both periods regarding comorbidities and the clinical presentation of RSV cases were noted. High rates of comorbidities prevailed; however, significantly lower numbers of patients with a history of lung transplantation ( p = 0.009), chronic kidney disease ( p = 0.013), and immunosuppression ( p = 0.038) were observed in the 2022/2023 season. In contrast, significantly more lower respiratory tract infections ( p < 0.001), in particular in the form of pneumonia ( p = 0.015) and exacerbations of obstructive lung diseases ( p = 0.008), were detected. An ICU admission was noted for 23.7% of all patients throughout the study period. Sequence analysis of the fusion protein gene revealed a close phylogenetic relatedness, regardless of the season of origin. However, especially for RSV-B, an accumulation of amino acid point substitutions was noted, including in antigenic site Ø. The SARS-CoV-2 pandemic had a tremendous impact on the seasonality of RSV, and the introduction of new vaccination and immunization strategies against RSV warrants further epidemiologic studies of this important pathogen.

Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection., (© 2024. The Author(s).)

Background: The concurrent circulation of SARS-CoV-2 with other respiratory viruses is unstoppable and represents a new diagnostic reality for clinicians and clinical microbiology laboratories. Multiplexed molecular testing on automated platforms that focus on the simultaneous detection of multiple respiratory viruses in a single tube is a useful approach for current and future diagnosis of respiratory infections in the clinical setting., Methods: Two time periods were included in the study: from February to April 2022, an early 2022 period, during the gradual lifting of COVID-19 prevention measures in the country, and from October 2022 to April 2023, the 2022/23 respiratory infections season. We analysed a total of 1,918 samples in the first period and 18,131 respiratory samples in the second period using a multiplex molecular assay for the simultaneous detection of Influenza A (Flu-A), Influenza B (Flu-B), Human Respiratory Syncytial Virus (HRSV) and SARS-CoV-2., Results: The results from early 2022 showed a strong dominance of SARS-CoV-2 infections with 1,267/1,918 (66.1%) cases. Flu-A was detected in 30/1,918 (1.6%) samples, HRSV in 14/1,918 (0.7%) samples, and Flu-B in 2/1,918 (0.1%) samples. Flu-A/SARS-CoV-2 co-detections were observed in 11/1,267 (0.9%) samples, and HRSV/SARS-CoV-2 co-detection in 5/1,267 (0.4%) samples. During the 2022/23 winter respiratory season, SARS-CoV-2 was detected in 1,738/18,131 (9.6%), Flu-A in 628/18,131 (3.5%), Flu-B in 106/18,131 (0.6%), and HRSV in 505/18,131 (2.8%) samples. Interestingly, co-detections were present to a similar extent as in early 2022., Conclusion: The results show that the multiplex molecular approach is a valuable tool for the simultaneous laboratory diagnosis of SARS-CoV-2, Flu-A/B, and HRSV in hospitalized and outpatients. Infections with Flu-A/B, and HRSV occurred shortly after the COVID-19 control measures were lifted, so a strong reoccurrence of various respiratory infections and co-detections in the post COVID-19 period was to be expected., (© 2024. The Author(s).)

Monitoring the genetic variability of human respiratory syncytial virus (hRSV) is of paramount importance, especially for the potential implication of key antigenic mutations on the emergence of immune escape variants. Thus, to describe the genetic diversity and evolutionary dynamics of hRSV circulating in Sicily (Italy), a total of 153 hRSV whole-genome sequences collected from 770 hRSV-positive subjects between 2017 and 2023, before the introduction of expanded immunization programs into the population, were investigated. The phylogenetic analyses indicated that the genotypes GA.2.3.5 (ON1) for hRSV-A and GB.5.0.5a (BA9) for hRSV-B co-circulated in our region. Amino acid (AA) substitutions in the surface and internal proteins were evaluated, including the F protein antigenic sites, as the major targets of immunoprophylactic monoclonal antibodies and vaccines. Overall, the proportion of AA changes ranged between 1.5% and 22.6% among hRSV-A, whereas hRSV-B varied in the range 0.8-16.9%; the latter was more polymorphic than hRSV-A within the key antigenic sites. No AA substitutions were found at site III of both subgroups. Although several non-synonymous mutations were found, none of the polymorphisms known to potentially affect the efficacy of current preventive measures were documented. These findings provide new insights into the global hRSV molecular epidemiology and highlight the importance of defining a baseline genomic picture to monitor for future changes that might be induced by the selective pressures of immunological preventive measures, which will soon become widely available.

The social restriction measures implemented due to the COVID-19 pandemic have impacted the pattern of occurrences of respiratory viruses. According to surveillance results in the Gwangju region of South Korea, respiratory syncytial virus (RSV) did not occur during the 2020/2021 season. However, there was a delayed resurgence in the 2021/2022 season, peaking until January 2022. To analyze this, a total of 474 RSV positive samples were investigated before and after the COVID-19 pandemic. Among them, 73 samples were selected for whole-genome sequencing. The incidence rate of RSV in the 2021/2022 season after COVID-19 was found to be approximately three-fold higher compared to before the pandemic, with a significant increase observed in the age group from under 2 years old to under 5 years old. Phylogenetic analysis revealed that, for RSV-A, whereas four lineages were observed before COVID-19, only the A.D.3.1 lineage was observed during the 2021/2022 season post-pandemic. Additionally, during the 2022/2023 season, the A.D.1, A.D.3, and A.D.3.1 lineages co-circulated. For RSV-B, while the B.D.4.1.1 lineage existed before COVID-19, both the B.D.4.1.1 and B.D.E.1 lineages circulated after the pandemic. Although atypical RSV occurrences were not due to new lineages, there was an increase in the frequency of mutations in the F protein of RSV after COVID-19. These findings highlight the need to continue monitoring changes in RSV occurrence patterns in the aftermath of the COVID-19 pandemic to develop and manage strategies in response.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory tract disease worldwide, and a pediatric vaccine is not available. We generated a filamentous RSV-based virus-like particle (VLP) that presents the central conserved region of the attachment protein G. This was achieved by co-expressing the matrix protein, phosphoprotein, nucleoprotein, and a hybrid fusion protein in which the F ectodomain was replaced with the G central region (GCR). The latter is relatively conserved and contains a receptor binding site and hence is a logical vaccine target. The immunogenicity and efficacy of the resulting VLP, termed VLP-GCR, were examined in mice using intranasal application without adjuvant. VLP-GCR induced substantial anti-N antibody levels but very low anti-G antibody levels, even after three vaccinations. In contrast, a VLP presenting prefusion-stabilized fusion (preF) protein instead of GCR induced both high anti-F and anti-nucleoprotein antibody levels, suggesting that our GCR antigen was poorly immunogenic. Challenge of VLP-GCR-vaccinated mice caused increased weight loss and lung pathology, and both VLPs induced mucus in the lungs. Thus, neither VLP is suitable as a vaccine for RSV-naive individuals. However, VLP-preF enhanced the proportion of preF antibodies and could serve as a multi-antigen mucosal booster vaccine in the RSV-experienced population., Competing Interests: The authors declare no conflicts of interest.

Human parainfluenza virus type 3 (HPIV-3, human respirovirus 3) is the second most frequently detected virus in lower respiratory tract infections in children after human respiratory syncytial virus (HRSV). HPIV-3, similar to related respiratory viruses such as HRSV and influenza virus, may cause encephalopathy; however, the relevance of HPIV-3 as a pathogenic factor in encephalopathy is unknown. We attempted to detect HPIV-1, HPIV-2, HPIV-3, HPIV-4, HRSV, and human metapneumovirus (HMPV) in 136 patients with encephalitis/encephalopathy or suspected encephalitis/encephalopathy during a 6-year period from 2014 to 2019. HPIV-3 was detected in 6 patients, followed by HRSV in 3 patients. The HPIV-3 strains detected were closely related to those detected in a patient with respiratory disease during the same period. Although HPIV-3 is less widely recognized than HRSV as a triggering virus of encephalopathy, our results suggest that HPIV-3 is as important as HRSV. Surveillance of the causative viruses of encephalopathy, including HPIV-3, would help clarify the causes of encephalopathy in Japan, as the cause is currently reported in less than half of cases in Japan.

When designing live-attenuated respiratory syncytial virus (RSV) vaccine candidates, attenuating mutations can be developed through biologic selection or reverse-genetic manipulation and may include point mutations, codon and gene deletions, and genome rearrangements. Attenuation typically involves the reduction in virus replication, due to direct effects on viral structural and replicative machinery or viral factors that antagonize host defense or cause disease. However, attenuation must balance reduced replication and immunogenic antigen expression. In the present study, we explored a new approach in order to discover attenuating mutations. Specifically, we used protein structure modeling and computational methods to identify amino acid substitutions in the RSV nonstructural protein 1 (NS1) predicted to cause various levels of structural perturbation. Twelve different mutations predicted to alter the NS1 protein structure were introduced into infectious virus and analyzed in cell culture for effects on viral mRNA and protein expression, interferon and cytokine expression, and caspase activation. We found the use of structure-based machine learning to predict amino acid substitutions that reduce the thermodynamic stability of NS1 resulted in various levels of loss of NS1 function, exemplified by effects including reduced multi-cycle viral replication in cells competent for type I interferon, reduced expression of viral mRNAs and proteins, and increased interferon and apoptosis responses.

microRNAs (miRNAs) regulate nearly all physiological processes but our understanding of exactly how they function remains incomplete, particularly in the context of viral infections. Here, we adapt a biochemical method (CLEAR-CLIP) and analysis pipeline to identify targets of miRNAs in lung cells infected with Respiratory syncytial virus (RSV). We show that RSV binds directly to miR-26 and miR-27 through seed pairing and demonstrate that these miRNAs target distinct gene networks associated with cell cycle and metabolism (miR-27) and antiviral immunity (miR-26). Many of the targets are de-repressed upon infection and we show that the miR-27 targets most sensitive to miRNA inhibition are those associated with cell cycle. Finally, we demonstrate that high confidence chimeras map to long noncoding RNAs (lncRNAs) and pseudogenes in transcriptional regulatory regions. We validate that a proportion of miR-27 and Argonaute 2 (AGO2) is nuclear and identify a long non-coding RNA (lncRNA) as a miR-27 target that is linked to transcriptional regulation of nearby genes. This work expands the target networks of miR-26 and miR-27 to include direct interactions with RSV and lncRNAs and implicate these miRNAs in regulation of key genes that impact the viral life cycle associated with cell cycle, metabolism, and antiviral immunity., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M)., Results: The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance., Conclusions: Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring., (© 2024. The Author(s).)

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze-thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log 10 TCID 50 /ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze-thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities., (© 2024. The Author(s).)

Introduction: Acute respiratory infections (ARI) are the most common infections in the general population and are mainly caused by respiratory viruses. Detecting several viruses in a respiratory sample is common. To better understand these viral codetections and potential interferences, we tested for the presence of viruses and developed quantitative PCR (Polymerase Chain Reaction) for the viruses most prevalent in coinfections: human rhinovirus (HRV) and respiratory syncytial virus (RSV), and quantified their viral loads according to coinfections and health status, age, cellular abundance and other variables., Materials and Methods: Samples from two different cohorts were analyzed: one included hospitalized infants under 12 months of age with acute bronchiolitis (n=719) and the other primary care patients of all ages with symptoms of ARI (n=685). We performed Multiplex PCR on nasopharyngeal swabs, and quantitative PCR on samples positive for HRV or/and RSV to determine viral loads (VL). Cellular abundance (CA) was also estimated by qPCR targeting the GAPDH gene. Genotyping was performed either directly from first-line molecular panel or by PCR and sequencing for HRV., Results: The risks of viral codetection were 4.1 (IC 95 [1.8; 10.0]) and 93.9 1 (IC 95 [48.7; 190.7]) higher in infants hospitalized for bronchiolitis than in infants in primary care for RSV and HRV respectively (p<0.001). CA was higher in samples positive for multiple viruses than in mono-infected or negative samples (p<0.001), and higher in samples positive for RSV (p<0.001) and HRV (p<0.001) than in negative samples. We found a positive correlation between CA and VL for both RSV and HRV. HRV VL was higher in children than in the elderly (p<0.05), but not RSV VL. HRV VL was higher when detected alone than in samples coinfected with RSV-A and with RSV-B. There was a significant increase of RSV-A VL when codetecting with HRV (p=0.001) and when co-detecting with RSV-B+HRV versus RSV-A+ RSV-B (p=0.02)., Conclusions: Many parameters influence the natural history of respiratory viral infections, and quantifying respiratory viral loads can help disentangle their contributions to viral outcome., 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 Petat, Corbet, Leterrier, Vabret and Ar Gouilh.)

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory illness worldwide, but there is no approved pediatric vaccine. Here, we describe the development of the live-attenuated RSV vaccine candidate Min AL as well as engineered derivatives. Min AL was attenuated by codon-pair deoptimization (CPD) of seven of the 11 RSV open reading frames (ORFs) (NS1, NS2, N, P, M, SH and L; 2,073 silent nucleotide substitutions in total). Min AL replicated efficiently in vitro at the permissive temperature of 32°C but was highly temperature sensitive (shut-off temperature of 36°C). When serially passaged at increasing temperatures, Min AL retained greater temperature sensitivity compared to previous candidates with fewer CPD ORFs. However, whole-genome deep-sequencing of passaged Min AL revealed mutations throughout its genome, most commonly missense mutations in the polymerase cofactor P and anti-termination transcription factor M2-1 (the latter was not CPD). Reintroduction of selected mutations into Min AL partially rescued its replication in vitro at temperatures up to 40°C, confirming their compensatory effect. These mutations restored the accumulation of positive-sense RNAs to wild-type (wt) RSV levels, suggesting increased activity by the viral transcriptase, whereas viral protein expression, RNA replication, and virus production were only partly rescued. In hamsters, Min AL and derivatives remained highly restricted in replication in the upper and lower airways, but induced serum IgG and IgA responses to the prefusion form of F (pre F) that were comparable to those induced by wt RSV, as well as robust mucosal and systemic IgG and IgA responses against RSV G. Min AL and derivatives were fully protective against challenge virus replication. The derivatives had increased genetic stability compared to Min AL. Thus, Min AL and derivatives with selected mutations are stable, attenuated, yet highly-immunogenic RSV vaccine candidates that are available for further evaluation., Competing Interests: U.J.B and C.L.N are coinventors on a patent application for the development of respiratory syncytial virus vaccines by codon pair deoptimization., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Objectives: This study investigated the prevalence, genetic diversity, and evolution of human respiratory syncytial virus (HRSV) in Barcelona from 2013 to 2023., Methods: Respiratory specimens from patients with RTI suspicion at Hospital Universitari Vall d'Hebron were collected from October 2013 to May 2023 for laboratory-confirmation of respiratory viruses. Next-generation sequencing was performed in randomly-selected samples with Illumina technology. Phylogenetic analyses of whole genome sequences were performed with BEAST v1.10.4. Signals of selection and evolutionary pressures were inferred by population dynamics and evolutionary analyses. Mutations in major surface proteins were genetic and structurally characterised, emphasizing those within antigenic epitopes., Results: Analyzing 139,625 samples, 5.3% were HRSV-positive (3008 HRSV-A, 3882 HRSV-B, 56 HRSV-A and -B, and 495 unsubtyped HRSV), with a higher prevalence observed in the paediatric population. Pandemic-related shifts in seasonal patterns returned to normal in 2022-2023. A total of 198 whole-genome sequences were obtained for HRSV-A (6.6% of the HRSV-A positive samples) belonging to GA2.3.5 lineage. For HRSV-B, 167 samples were sequenced (4.3% of the HRSV-B positive samples), belonging to GB5.0.2, GB5.0.4a and GB5.0.5a. HRSV-B exhibited a higher evolution rate. Post-SARS-CoV-2 pandemic, both subtypes showed increased evolutionary rates and decreased effective population size initially, followed by a sharp increase. Analyses indicated negative selective pressure on HRSV. Mutations in antigenic epitopes, including S276N and M274I in palivizumab-targeted site II, and I206M, Q209R, and S211N in nirsevimab-targeted site Ø, were identified., Discussion: Particularly in the context of the large-scale use in 2023-2024 season of nirsevimab, continuous epidemiological and genomic surveillance is crucial., 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 Ltd.. All rights reserved.)

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-β, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

Background: Data available for RSV and influenza infections among children < 2 years in Mongolia are limited. We present data from four districts of Ulaanbaatar from April 2015 to June 2021., Methods: This study was nested in an enhanced surveillance project evaluating pneumococcal conjugate vaccine (PCV13) impact on the incidence of hospitalized lower respiratory tract infections (LRTIs). Our study was restricted to children aged < 2 years with arterial O 2 saturation < 93% and children with radiological pneumonia. Nasopharyngeal (NP) swabs collected at admission were tested for RSV and influenza using qRT-PCR. NP swabs of all patients with radiological pneumonia and of a subset of randomly selected NP swabs were tested for S. pneumoniae (S.p.) by qPCR and for serotypes by culture and DNA microarray., Results: Among 5705 patients, 2113 (37.0%) and 386 (6.8%) had RSV and influenza infections, respectively. Children aged 2-6 months had a higher percentage of very severe RSV infection compared to those older than 6 months (42.2% versus 31.4%, p-value Fisher's exact = 0.001). S.p. carriage was detected in 1073/2281 (47.0%) patients. Among S.p. carriage cases, 363/1073 (33.8%) had S.p. and RSV codetection, and 82/1073 (7.6%) had S.p. and influenza codetection. S.p. codetection with RSV/influenza was not associated with more severe LRTIs, compared to only RSV/influenza cases., Conclusion: In Mongolia, RSV is an important pathogen causing more severe LRTI in children under 6 months of age. Codetection of RSV or influenza virus and S.p. was not associated with increased severity., (© 2024 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with the greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially in the United States (US) following the implementation of COVID-19-related non-pharmaceutical interventions but later rebounded with abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study from 2009 to 2023 in Chicago, Illinois, US, we examined RSV epidemiology, clinical severity, and genetic diversity. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings post-2020 and that hospitalized adults infected with RSV-A were at higher risk of intensive care admission than those with RSV-B. While population structures of RSV-A remained unchanged, RSV-B exhibited a genetic shift into geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development., (© 2024. The Author(s).)