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Background: Most observational population-based studies identify respiratory syncytial virus (RSV) by nasal/nasopharyngeal swab reverse transcriptase real-time PCR (RT-PCR) only. We conducted a systematic review and meta-analyses to quantify specimen and diagnostic testing-based underascertainment of adult RSV infection., Methods: EMBASE, PubMed, and Web of Science were searched (January 2000-December 2021) for studies including adults using/comparing >1 RSV testing approach. We quantified test performance and RSV detection increase associated with using multiple specimen types., Results: Among 8066 references identified, 154 met inclusion. Compared to RT-PCR, other methods were less sensitive: rapid antigen detection test (RADT; pooled sensitivity, 64%), direct fluorescent antibody (DFA; 83%), and viral culture (86%). Compared to singleplex PCR, multiplex PCR's sensitivity was lower (93%). Compared to nasal/nasopharyngeal swab RT-PCR alone, adding another specimen type increased detection: sputum RT-PCR, 52%; 4-fold rise in paired serology, 44%; and oropharyngeal swab RT-PCR, 28%. Sensitivity was lower in estimates limited to only adults (for RADT, DFA, and viral culture), and detection rate increases were largely comparable., Conclusions: RT-PCR, particularly singleplex testing, is the most sensitive RSV diagnostic test in adults. Adding additional specimen types to nasopharyngeal swab RT-PCR testing increased RSV detection. Synergistic effects of using ≥3 specimen types should be assessed, as this approach may improve the accuracy of adult RSV burden estimates., Competing Interests: Potential conflicts of interest. P95 was contracted by Pfizer to conduct this study. C. O., L. M. M., S. M., B. M., M. S., N. A., M. R., Z. A.-T., and H. V. are employees of P95. D. C., W. K., J. E. A., B. D. G., M. R., and E. B. are employees of Pfizer and may hold stock or stock options. H. N. declares no conflicts of interest. 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.)

Respiratory syncytial virus (RSV) infection can cause life-threatening pneumonia and bronchiolitis, posing a significant threat to human health worldwide, especially to children and the elderly. Currently, there is no specific treatment for RSV infection. The most effective measures for preventing RSV infection are vaccines and prophylactic medications. However, not all population groups are eligible for the approved vaccines or antibody-based preventive medications. Therefore, there is an urgent need to develop novel vaccines and prophylactic drugs available for people of all ages. High-throughput assays that evaluate the efficacy of viral entry inhibitors or vaccine-induced neutralizing antibodies in blocking RSV entry are crucial for evaluating vaccine and prophylactic drug candidates. We developed an efficient entry assay using a lentiviral pseudovirus carrying the fusion (F) protein of type A or B RSV. In addition, the essential parameters were systematically optimized, including the number of transfected plasmids, storage conditions of the pseudovirus, cell types, cell numbers, virus inoculum, and time point of detection. Furthermore, the convalescent sera exhibited comparable inhibitory activity in this assay as in the authentic RSV virus neutralization assay. We established a robust pseudovirus-based entry assay for RSV, which holds excellent promise for studying entry mechanisms, evaluating viral entry inhibitors, and assessing vaccine-elicited neutralizing antibodies against RSV.

Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.

The validation of a bioanalytical method allows us to determine its validity for a designated purpose and to guarantee the reliability of its analytical results. The virus neutralization assay has proved to be suitable for the detection and quantification of specific serum-neutralizing antibodies against respiratory syncytial virus subtypes A and B. Respiratory syncytial virus is a negative-sense RNA virus and is responsible for the majority of acute lower respiratory tract infections in infants and older adults worldwide. Owing to its widespread infection, the WHO considers it a target for the development of preventive vaccines. Despite the high impact of its infections, however, only one vaccine has been recently approved. The aim of this paper is to provide a detailed validation process for the microneutralization assay and to demonstrate that this method can effectively support the efficacy assessment of candidate vaccines and the definition of correlates of protection., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Historically, the diagnosis of viral infections has been accomplished using a combination of laboratory-based methods, including culture, serology, antigen-based tests, and molecular (e.g., real-time PCR) assays. Although these methods provide an accurate way to detect viral pathogens, testing in a centralized laboratory may delay results, which could impact patient diagnosis and management. Point-of-care tests, including antigen- and molecular-based assays, have been developed to assist with the timely diagnosis of several viral infections, such as influenza, respiratory syncytial virus, and COVID-19. Despite the ability of point-of-care tests to provide rapid results (i.e., <30 min), there are issues to consider prior to their routine use, including test performance and specific regulatory requirements. This review will provide a summary of the regulatory landscape of point-of-care tests for viral infections in the United States, and address important considerations such as site certification, training and inspection readiness., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MJB is on the scientific advisory board for DiaSorin Molecular and Mammoth Biosciences. MJB has received honoraria for educational webinars sponsored by DiaSorin, Roche Diagnostics, Quidel, Abbott Molecular, and Seegene., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021., Methods: We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank., Findings: We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40·0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1·0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins., Interpretation: The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time., Funding: AstraZeneca and Sanofi., Competing Interests: Declaration of interests DW, CM, BA, AAA, TB, VG, EJK, KMT, and MTE are current employees of and hold stock or stock options in AstraZeneca. MEA and DET are former employees of and hold stock or stock options in AstraZeneca. EB has received honoraria from AstraZeneca and Sanofi for lectures, presentations, speaker bureaus, manuscript writing, or educational events. TH has participated in Data Safety Monitoring Boards and ad-hoc advisory boards for Sanofi, Data Safety Monitoring Boards for Enanta, and ad-hoc advisory boards for Janssen and has received honoraria from Janssen and Merck, Sharp & Dohme (MSD) for lectures at academic meetings. CK has received honoraria from F Hoffmann-La Roche for lectures. FM-T's research activities are supported by grants from Instituto de Salud Carlos III (grant numbers: PI16/01569, PI19/01090, PI22/00406, and CB21/06/00103), GEN-COVID (grant number: IN845D 2020/23), and Grupos de Referencia Competitiva (grant number: IIN607A2021/05), and he has received additional grants to his institution from AstraZeneca and Sanofi; he also received honoraria from GlaxoSmithKline, Pfizer, Sanofi Pasteur, MSD, Seqirus, Biofabri, and Janssen for taking part in advisory boards and expert meetings and for acting as speaker in congresses outside the scope of the submitted work, in addition to travel support from GlaxoSmithKline, MSD, Pfizer, and Sanofi. FM-T has also participated in advisory boards for Pfizer, MSD, Sanofi, and GlaxoSmithKline, and in Data Safety Monitoring Boards for Biofabri; is a member of The European Technical Advisory Group of Experts—WHO Europe and the Spanish Paediatric Infectious Diseases Society; and has also acted as principal investigator in randomised controlled trials for AstraZeneca, Biofabri Seqirus, GlaxoSmithKline, Janssen, MSD, Novavax, Novartis, Pfizer, Roche, Regeneron, and Sanofi Pasteur with honoraria paid to his institution. MCN has received grants from the Bill & Melinda Gates Foundation, European and Developing Countries Clinical Trials, Pfizer, and Sanofi Pasteur; honoraria from Sanofi for lectures presentations, speakers' bureaus, manuscript writing or educational events; payment for expert testimony from Pfizer and Sanofi Pasteur; and is a board member for Gavi vaccine alliance. JMP has received payments to his institution from the Canadian Paediatric Review and Yearly RSV Coordinators Workshop and is an unpaid co-chair of the Ontario Immunization Advisory Committee. PR has received investigator-initiated research grants to his institution from MSD and has received institutional funding from GlaxoSmithKline for local and international lectures and from AstraZeneca, GlaxoSmithKline, MSD, Sanofi, and Pfizer for participation in advisory boards. RTS has received payment or honoraria for lectures for AstraZeneca, Pfizer, and Sanofi Pasteur. CV has received payment or honoraria for lectures from AstraZeneca. LJB has not received personal fees or other personal benefits from pharmaceutical companies. His institution, University Medical Center Utrecht (UMCU), has received major funding (>€100 000 per industrial partner) from AbbVie, AstraZeneca, Sanofi, Janssen, Pfizer, MSD, and MeMed Diagnostics for investigator-initiated studies. UMCU has received major funding for the RSV-GOLD study from the Bill & Melinda Gates Foundation. UMCU has received major funding as part of the public private partnership IMI-funded RESCEU and PROMISE projects with partners GlaxoSmithKline, Novavax, Janssen, AstraZeneca, Pfizer, and Sanofi Pasteur. UMCU has received major funding by Julius Clinical for participating in clinical studies sponsored by AstraZeneca and Pfizer. UMCU has received minor funding (€1 000–25 000 per industrial partner) for consultation and invited lectures by AbbVie, AstraZeneca, Ablynx, Bavaria Nordic, MabXience, GlaxoSmithKline, Novavax, Pfizer, Moderna, AstraZeneca, MSD, Sanofi, Genzyme, and Janssen. LJB is the founding chairman of the ReSViNET Foundation. All other authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Since the COVID-19 pandemic, a decrease in the prevalence of Influenza A virus (IAV) and respiratory syncytial virus (RSV) has been suggested by clinical surveillance. However, there may be potential biases in obtaining an accurate overview of infectious diseases in a community. To elucidate the impact of the COVID-19 on the prevalence of IAV and RSV, we quantified IAV and RSV RNA in wastewater collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan, between October 2018 and January 2023, using highly sensitive EPISENS™ method. From October 2018 to April 2020, the IAV M gene concentrations were positively correlated with the confirmed cases in the corresponding area (Spearman's r = 0.61). Subtype-specific HA genes of IAV were also detected, and their concentrations showed trends that were consistent with clinically reported cases. RSV A and B serotypes were also detected in wastewater, and their concentrations were positively correlated with the confirmed clinical cases (Spearman's r = 0.36-0.52). The detection ratios of IAV and RSV in wastewater decreased from 66.7 % (22/33) and 42.4 % (14/33) to 4.56 % (12/263) and 32.7 % (86/263), respectively in the city after the COVID-19 prevalence. The present study demonstrates the potential usefulness of wastewater-based epidemiology combined with the preservation of wastewater (wastewater banking) as a tool for better management of respiratory viral diseases., Competing Interests: Declaration of competing interest Ryo Iwamoto and Yoshinori Ando are employees of SHIONOGI & Co., Ltd. Masaaki Kitajima who received research funding from SHIONOGI & Co., Ltd. and AdvanSentinel, Inc. and patent royalties from SHIONOGI Co., Ltd. Satoshi Okabe received research funding from SHIONOGI Co., Ltd. and AdvanSentinel, Inc. Hiroki Ando and Warish Ahmed have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Nucleic acid testing is a powerful tool for the detection of various pathogens. Respiratory syncytial virus (RSV) is a major cause of acute respiratory infection, especially in young children and infants. To improve the confidence and reliability of nucleic acid testing results for RSV, reference materials (RMs) of both type A and B of RSV were developed by the National Institute of Metrology, China, code numbers NIM-RM 4057 and 4058. The reference material was composed of in vitro transcribed RNA containing the nucleocapsid (N) gene, matrix (M) gene, and partial polymerase (L) gene of RSV. A duplex reverse transcription digital PCR method was established with limit of blank (LoB), limit of detection (LoD) and limit of quantification (LoQ) of 2, 5, and 23 copies per reaction for RSV-A and 4, 8, and 20 copies per reaction for RSV-B. The certified value and expanded uncertainty (U, k = 2) of the two RMs were determined to be (6.1 ± 1.4) × 10 4 copies/μL for RSV-A and (5.3 ± 1.2) × 10 4 copies/μL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Respiratory syncytial virus (RSV) can cause lower respiratory tract disease in infants and elderly populations. Despite decades of research, there remains no safe and approved RSV vaccine. Previously, we showed that an RSV G glycoprotein subunit vaccine candidate with a single point mutation within the central conserved domain (CCD), i.e. S177Q, considerably improved immunogenicity., Methods: Here, we examine the development of nanoparticle (NP) vaccines having either an RSV G protein CCD with wild-type sequence (NPWT) or an S177Q mutation (NP-S177Q). The NP vaccine immunogens were adjuvanted with monophosphoryl lipid A (MPLA), a TLR4 agonist to improve Th1- type responses. BALB/c mice were primed with 10 μg of NP-WT vaccine, NPS177Q, or vehicle, rested, and then boosted with a high (25 μg) or low (10 μg) dose of the NP-WT or NP-S177Q homologous candidate and subsequently challenged with RSV A2., Results: The results showed that mice boosted with NP-S177Q developed superior immunogenicity and neutralizing antibodies compared to NP-WT boosting. IgG from either NP-S177Q or NP-WT vaccinated mice did not interfere with fractalkine (CX3CL1) binding to CX3CR1 and effectively blocked G protein CX3C-CX3CR1 binding. Both NP-WT and NP-S177Q vaccination induced similar neutralizing antibodies to RSV in challenged mice compared to vehicle control. NP-S177Q boosting improved correlates of protection including reduced BAL cell infiltration following RSV challenge. However, the NP vaccine platform will require improvement due to the poor solubility and the unexpectedly weaker Th1-type IgG2a response., Discussion: The results from this study support further NP-S177Q vaccine candidate development., Competing Interests: RD is an inventor on US patent filing No. 62/588,022. The remaining 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 © 2023 Bergeron, Murray, Juarez, Nangle, DuBois and Tripp.)

Introduction: In the Northeast US, respiratory viruses such as influenza and respiratory syncytial virus (RSV), which were largely suppressed by COVID-19-related social distancing, made an unprecedented resurgence during 2022, leading to a substantial rise in viral co-infections. However, the relative rates of co-infection with seasonal respiratory viruses over this period have not been assessed., Methods: Here we reviewed multiplex respiratory viral PCR data (BioFire FilmArray™ Respiratory Panel v2.1 [RPP]) from patients with respiratory symptoms presenting to our medical center in New York City to assess co-infection rates of respiratory viruses, which were baselined to total rates of infection for each virus. We examined trends in monthly RPP data from adults and children during November 2021 through December 2022 to capture the full seasonal dynamics of respiratory viruses across periods of low and high prevalence., Results: Of 50,022 RPPs performed for 34,610 patients, 44% were positive for at least one target, and 67% of these were from children. The overwhelming majority of co-infections (93%) were seen among children, for whom 21% of positive RPPs had two or more viruses detected, as compared to just 4% in adults. Relative to children for whom RPPs were ordered, children with co-infections were younger (3.0 vs 4.5 years) and more likely to be seen in the ED or outpatient settings than inpatient and ICU settings. In children, most viral co-infections were found at significantly reduced rates relative to that expected from the incidence of each virus, especially those involving SARS-CoV-2 and influenza. SARS-CoV-2 positive children had an 85%, 65% and 58% reduced rate of co-infection with influenza, RSV, and Rhino/enteroviruses, respectively, after compensating for the incidence of infection with each virus (p< 0.001)., Discussion: Our results demonstrate that most respiratory viruses peaked in different months and present in co-infections less than would be expected based on overall rates of infection, suggesting a viral exclusionary effect between most seasonal respiratory viruses, including SARS-CoV-2, influenza and RSV. We also demonstrate the significant burden of respiratory viral co-infections among children. Further work is necessary to understand what predisposes certain patients for viral co-infection despite this exclusionary effect., 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 © 2023 Weidmann, Green, Berry and Wu.)

A wide variety of viruses replicate in liquid-like viral factories. Non-segmented negative stranded RNA viruses share a nucleoprotein (N) and a phosphoprotein (P) that together emerge as the main drivers of liquid-liquid phase separation. The respiratory syncytial virus includes the transcription antiterminator M 2-1 , which binds RNA and maximizes RNA transcriptase processivity. We recapitulate the assembly mechanism of condensates of the three proteins and the role played by RNA. M 2-1 displays a strong propensity for condensation by itself and with RNA through the formation of electrostatically driven protein-RNA coacervates based on the amphiphilic behavior of M 2-1 and finely tuned by stoichiometry. M 2-1 incorporates into tripartite condensates with N and P, modulating their size through an interplay with P, where M 2-1 is both client and modulator. RNA is incorporated into the tripartite condensates adopting a heterogeneous distribution, reminiscent of the M 2-1 -RNA IBAG granules within the viral factories. Ionic strength dependence indicates that M 2-1 behaves differently in the protein phase as opposed to the protein-RNA phase, in line with the subcompartmentalization observed in viral factories. This work dissects the biochemical grounds for the formation and fate of the RSV condensates in vitro and provides clues to interrogate the mechanism under the highly complex infection context.

Background: Viruses are associated with pediatric community-acquired pneumonia (CAP) but are also common in the upper airways of healthy children. We have determined the contribution of respiratory viruses and bacteria by comparing children with CAP and hospital controls., Methods: Children less than 16 years old with radiologically confirmed CAP (n = 715) were enrolled over an 11-year period. Children admitted for elective surgery during the same period served as controls (n = 673). Nasopharyngeal aspirates were tested for 20 respiratory pathogens by semiquantitative polymerase chain reaction tests and cultivated for bacteria and viruses. We used logistic regression to calculate adjusted odds ratios [aOR; 95% confidence intervals (CIs)], and estimated population-attributable fractions (95% CI)., Results: At least 1 virus was detected in 85% of cases and 76% of controls, and greater than or equal to 1 bacterium was detected in 70% of cases and controls. The presence of respiratory syncytial virus (RSV) (aOR, 16.6; 95% CI: 9.81-28.2), human metapneumovirus (HMPV) (13.0; 6.17-27.5) and Mycoplasma pneumoniae (27.7; 8.37-91.6) were most strongly associated with CAP. For RSV and HMPV, there were significant trends between lower cycle-threshold values indicating higher viral genomic loads, and higher aORs for CAP. The population-attributable fraction estimates of RSV, HMPV, human parainfluenza virus, influenza virus and M. pneumoniae were 33.3% (32.2-34.5), 11.2% (10.5-11.9), 3.7% (1.0-6.3), 2.3% (1.0-3.6) and 4.2% (4.1-4.4), respectively., Conclusions: RSV, HMPV and M. pneumoniae were most strongly related to pediatric CAP and accounted for half of all cases. There were positive trends between increasing viral genomic loads of RSV and HMPV, and higher odds for CAP., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Human respiratory syncytial virus (RSV) is a severe threat to children and a main cause of acute lower respiratory tract infections. Nevertheless, the intra-host evolution and inter-regional diffusion of RSV are little known. In this study, we performed a systematic surveillance in hospitalized children in Hubei during 2020-2021, in which 106 RSV-positive samples were detected both clinically and by metagenomic next generation sequencing (mNGS). RSV-A and RSV-B groups co-circulated during surveillance with RSV-B being predominant. About 46 high-quality genomes were used for further analyses. A total of 163 intra-host nucleotide variation (iSNV) sites distributed in 34 samples were detected, and glycoprotein (G) gene was the most enriched gene for iSNVs, with non-synonymous substitutions more than synonymous substitutions. Evolutionary dynamic analysis showed that the evolutionary rates of G and NS2 genes were higher, and the population size of RSV groups changed over time. We also found evidences of inter-regional diffusion from Europe and Oceania to Hubei for RSV-A and RSV-B, respectively. This study highlighted the intra-host and inter-host evolution of RSV, and provided some evidences for understanding the evolution of RSV., (Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Purpose of Review: Respiratory syncytial virus (RSV) continues to be a major cause of severe lower respiratory tract infection in infants, young children, and older adults. In this review, changes in the epidemiology of RSV during the coronavirus disease 2019 (COVID-19) pandemic are highlighted together with the role which increased molecular surveillance efforts will have in future in assessing the efficacy of vaccines and therapeutics., Recent Findings: The introduction of nonpharmaceutical intervention (NPIs) strategies during the COVID-19 pandemic between 2020 and 2022 resulted in worldwide disruption to the epidemiology of RSV infections, especially with respect to the timing and peak case rate of annual epidemics. Increased use of whole genome sequencing along with efforts to better standardize the nomenclature of RSV strains and discrimination of RSV genotypes will support increased monitoring of relevant antigenic sites in the viral glycoproteins. Several RSV vaccine candidates based on subunit, viral vectors, nucleic acid, or live attenuated virus strategies have shown efficacy in Phase 2 or 3 clinical trials with vaccines using RSVpreF protein currently the closest to approval and use in high-risk populations. Finally, the recent approval and future use of the extended half-life human monoclonal antibody Nirsevimab will also help to alleviate the morbidity and mortality burden caused by annual epidemics of RSV infections., Summary: The ongoing expansion and wider coordination of RSV molecular surveillance efforts via whole genome sequencing will be crucial for future monitoring of the efficacy of a new generation of vaccines and therapeutics., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Although fewer children have been affected by the severe form of the coronavirus disease 2019 (COVID-19), community-acquired pneumonia (CAP) continues to be the leading global cause of child hospitalizations and deaths., Aim: This study investigated the incidence of respiratory syncytial virus (RSV) as well its subtypes (RSV A and B), adenovirus (ADV), rhinovirus (HRV), metapneumovirus (HMPV), coronavirus (NL63, OC43, 229E and HKU1), parainfluenza virus subtypes (PI1, PI2 and PI3), bocavirus and influenza A and B viruses (FluA and FluB) in children diagnosed with CAP during the COVID-19 pandemic., Methods: A total of 200 children with clinically confirmed CAP were initially recruited, of whom 107 had negative qPCR results for SARS-CoV-2 and were included in this study. Viral subtypes were identified using a real-time polymerase chain reaction in the nasopharyngeal swab samples., Results: Viruses were identified in 69.2% of the patients. RSV infections were the most frequently identified (65.4%), with type RSV B being the most prevalent (63.5%). In addition, HCoV 229E and HRV were detected in 6.5% and 3.7% of the patients, respectively. RSV type B was associated with severe acute respiratory infection (ARI) and a younger age (less than 24 months)., Conclusions: New strategies for preventing and treating viral respiratory infections, particularly RSV infections, are necessary.

Background: Safe and effective respiratory syncytial virus (RSV) vaccines remain elusive. This was a phase I/II trial (NCT02927873) of ChAd155-RSV, an investigational chimpanzee adenovirus-RSV vaccine expressing 3 proteins (fusion, nucleoprotein, and M2-1), administered to 12-23-month-old RSV-seropositive children followed up for 2 years after vaccination., Methods: Children were randomized to receive 2 doses of ChAd155-RSV or placebo (at a 1:1 ratio) (days 1 and 31). Doses escalated from 0.5 × 1010 (low dose [LD]) to 1.5 × 1010 (medium dose [MD]) to 5 × 1010 (high dose [HD]) viral particles after safety assessment. Study end points included anti-RSV-A neutralizing antibody (Nab) titers through year 1 and safety through year 2., Results: Eighty-two participants were vaccinated, including 11, 14, and 18 in the RSV-LD, RSV-MD, and RSV-HD groups, respectively, and 39 in the placebo groups. Solicited adverse events were similar across groups, except for fever (more frequent with RSV-HD). Most fevers were mild (≤38.5°C). No vaccine-related serious adverse events or RSV-related hospitalizations were reported. There was a dose-dependent increase in RSV-A Nab titers in all groups after dose 1, without further increase after dose 2. RSV-A Nab titers remained higher than prevaccination levels at year 1., Conclusions: Three ChAd155-RSV dosages were found to be well tolerated. A dose-dependent immune response was observed after dose 1, with no observed booster effect after dose 2. Further investigation of ChAd155-RSV in RSV-seronegative children is warranted., Clinical Trials Registration: NCT02927873., Competing Interests: Potential conflicts of interest. J. D. D. reports personal fees/grants from GSK, Sanofi Pasteur and MSD and nonfinancial support from Sanofi Pasteur and MSD. X. S. L. reports grants from Cevaxin. Outside the submitted work, C. E. reports support from GSK and ViiV for scientific congress attendance. F. M. T. reports personal fees and nonfinancial support from GSK, Medimmune, Pfizer, MSD, Sanofi Pasteur, Astra Zeneca, and Seqirus. J. M. L. reports grants from GSK and Janssen, personal fees from Pfizer, and nonfinancial support from Immunovaccine. J. B. D. reports consulting fees from Sanofi Pasteur. S. E. reports personal fees and grants from GSK, Sanofi, and Vifor and grants from Abbott and Pfizer. P. M., T. L. A. N., V. N., W. W., Y. Z., I. D., A. L., A. G. L., and N. V. are employees of the GSK group of companies or were employees during the conduct of the study. P. M., W. W., I. D., and A. L. hold shares in the GSK group of companies. The institutions of J. D. D., A. C., A. A. B., F. M. T., J. M. L., J. B. D., and S. E. received funds from GSK for conducting the present work. Outside the submitted work, the institution of F. M. T. received funds from GSK, Ablynx, Jansen, Regeneron, Pfizer, MSD, Novavax, Roche, Astra Zeneca and Seqirus, and the institution of J. B. D. received grants from Merck and Medimmune/Astra Zeneca. 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) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The development of a vaccine against human respiratory syncytial virus (HRSV) has been hampered by enhanced respiratory disease due to the Th2-biased immune response. In the present study, MA103 and aluminum phosphate (Adju-Phos) adjuvants were used to verify the immunogenicity of the recombinant fusion (RBF) protein (F protein expressed by Escherichia coli). Both adjuvants significantly increased the neutralizing antibody titer and number of interferon gamma (IFN-γ)-secreting CD4+ T cells in mice. Based on the immunoglobulin G1 (IgG1)/IgG2a and IFN-γ/interleukin 4-secreting CD4+ T cell ratio, however, MA103 significantly enhanced the Th1-biased immune response. The pathological damage to the lung in the RBF/MA103 group was less than what was seen in the RBF/Adju-Phos group. Additionally, the number of HRSV copies in the lungs of the RBF/MA103 group decreased by approximately 3 × log 10 . These results suggested that MA103 provides better protection against HRSV in mice.

This study assesses the circulation of human respiratory syncytial virus (HRSV) genotypes before, during, and toward the end of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in children and determines the influence of the pandemic on HRSV circulation patterns and evolution. Phylogenetic analysis of the hypervariable glycoprotein G gene was performed on 221/261 (84.7%) HRSV-positive samples and shows two separated clusters, one belonging to HRSV-A (129/221) and another to HRSV-B (92/221). All Slovenian HRSV-A strains contained the 72-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GA2.3.5. All Slovenian HRSV-B strains similarly contained a 60-nucleotide-long duplicated region in the attachment glycoprotein G gene and were classified as lineage GB5.0.5a. During the 3-year period (2018-2021) covered by the study, no significant differences were observed within strains detected before the SARS-CoV-2 pandemic, during it, and after the implementation of nonpharmaceutical preventive measures. Slovenian HRSV-A strains seem to be more diverse than HRSV-B strains. Therefore, further whole-genome investigations would be required for better monitoring of the long-term impact of SARS-CoV-2 endemic circulation and the formation of new HRSV lineages and epidemiological patterns.

Infections due to human respiratory syncytial virus (HRSV) and human bocavirus (HBoV) can mediate the release of several pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α, which are usually associated with disease severity in children. In this study, the change in the expression profile of cytokines and chemokines were determined during HRSV, HBoV, and HRSV coinfection with HBoV in 75 nasopharyngeal aspirates (NPAs) samples, positive real-time reverse transcriptase PCR Assay (rRT-PCR) for HRSV ( n = 36), HBoV ( n = 23) infection alone or HRSV coinfection with HBoV ( n = 16). The samples were collected from hospitalized children. qPCR-based detection revealed that the levels of IL-6, IL-8, IL-10, IL-13, IL-33, and G-CSF were significantly ( p < 0.05) greater in patients than in controls. IL-4, IL-17, GM-CSF, and CCL-5 were significantly elevated in children with HRSV coinfection with HBoV than in other groups ( p < 0.05). TNF-α, IL-6, IL-8, IL-10, IL-13, and IL-33 in children with HRSV were significantly increased in severe infections compared to mild infections. Whereas, IL-10, IL-13, and IL-33 were significantly increased in severe infection in compared a mild infection in children with HBoV. Further large-scale investigations involving isolates are needed to enhance our knowledge of the association between viral infections and cytokine expression patterns during the different stages of HRSV and HBoV infection.

Background: Respiratory syncytial virus (RSV) is associated with acute respiratory infection. We sought to identify RSV variants associated with prolonged infection., Methods: Among healthy term infants we identified those with prolonged RSV infection and conducted (1) a human genome-wide association study (GWAS) to test the dependence of infection risk on host genotype, (2) a viral GWAS for association with prolonged RSV infection using RSV whole-genome sequencing, (3) an analysis of all viral public sequences, (4) an assessment of immunological responses, and (5) a summary of all major functional data. Analyses were adjusted for viral/human population structure and host factors associated with infection risk., Results: We identified p.E123K/D and p.P218T/S/L in G protein that were associated with prolonged infection (Padj = .01). We found no evidence of host genetic risk for infection. The RSV variant positions approximate sequences that could bind a putative viral receptor, heparan sulfate., Conclusions: Using analysis of both viral and host genetics we identified a novel RSV variant associated with prolonged infection in otherwise healthy infants and no evidence supporting host genetic susceptibility to infection. As the capacity of RSV for chronicity and its viral reservoir are not defined, these findings are important for understanding the impact of RSV on chronic disease and endemicity., Competing Interests: Potential conflicts of interest. 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. Funding for this work has been supplied by the NIH and the SNSF. Funding to pay the Open Access publication charges for this article was provided by NIH. Tina Hartert; consulting fees: Sanofi Pasteur scientific advisory board RSV vaccines, participation on a data safety monitoring board: Pfizer. Larry J. Anderson; Grants or contracts (exlcuding funding for this project): CDC contract for herpes simplex antibody testing, subcontract with Sciogen on an NIH SBIR for RSV vaccines, contract with Pfizer to serologic testing studies of RSV, consulting fees: Janssen scientific advisory board RSV vaccines, ADVI scientific advisory board RSV vaccines, and Bavarian Nordic scientific advisory board, patents planned, issued or pending: RSV vaccines and Immune treatment, and RSV VLP vaccines., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Respiratory syncytial virus (RSV) is a leading cause of hospitalization and infant mortality worldwide. There are currently no approved vaccines against RSV, and immunoprophylaxis with the mAb palivizumab is limited to extremely vulnerable infants in resource-rich settings due to its high cost and the need for monthly injections throughout the RSV season. Nirsevimab (formerly MEDI8897) is a highly potent, long-acting, human, recombinant mAb that received approval for the prevention of RSV infection in newborns and infants during their first RSV season from the EMA and the UK's Medicines and Healthcare products Regulatory Agency in November 2022 based on positive results in Phase 2b and 3 clinical trials. Nirsevimab targets the highly conserved site Ø of the prefusion conformation of the RSV fusion (F) protein and contains a triple amino acid substitution in the Fc domain that extends its half-life, allowing for a single dose to cover a typical RSV season in regions with temperate climates. In this article I review key attributes of nirsevimab with an emphasis on pharmacology, pharmacokinetics, antiviral activity, and the potential for resistance and escape variants. I also summarize current progress in clinical trials and consider future research priorities., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Respiratory syncytial virus (RSV) is the leading cause of mortality and morbidity in children under the age of five. Despite this, there is still a lack of safe and effective vaccines and antiviral agents for clinical use. Andrographolide exerts antiviral functions against a variety of viruses, but whether (and how) it exerts antiviral effects on RSV remains unclear., Methods and Results: In vitro RSV infection models using A549 and 16HBE cell lines were established, and the effects of andrographolide on RSV were analyzed via RSV N gene load and proinflammatory cytokine levels. The RNA transcriptome was sequenced, and data were analyzed by R software. Andrographolide-related target genes were extracted via network pharmacology using online databases. Lentiviral transfection was applied to knockdown the heme oxygenase-1 gene (Hmox1, HO-1). Results showed that andrographolide suppressed RSV replication and attenuated subsequent inflammation. Network pharmacology and RNA sequencing analysis indicated that the hub gene HO-1 may play a pivotal role in the anti-RSV effects of andrographolide. Furthermore, andrographolide exerted antiviral effects against RSV partially by inducing HO-1 but did not activate the antiviral interferon response., Conclusion: Our findings demonstrated that andrographolide exerted anti-RSV activity by up-regulating HO-1 expression in human airway epithelial cells, providing novel insights into potential therapeutic targets and drug repurposing in RSV infection., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence., Methods: A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1-4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets., Findings: We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0-9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000-56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32-0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses., Interpretation: Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications., Funding: CDC Foundation., Competing Interests: Declaration of interests BH, DD, VC-H, AB, and BJW are employees of Verily Life Sciences. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

Background: During the coronavirus disease 2019 (COVID-19) pandemic, human parainfluenza type 3 (HPIV-3) and respiratory syncytial virus (RSV) circulation increased as nonpharmaceutical interventions were relaxed. Using data from 175 households (n = 690 members) followed between November 2020 and October 2021, we characterized HPIV-3 and RSV epidemiology in children aged 0-4 years and their households., Methods: Households with ≥1 child aged 0-4 years were enrolled; members collected weekly nasal swabs (NS) and additional NS with respiratory illnesses (RI). We tested NS from RI episodes in children aged 0-4 years for HPIV-3, RSV, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using reverse-transcriptase polymerase chain reaction (RT-PCR). Among children with HPIV-3 or RSV infection, we tested contemporaneous NS from household members. We compared incidence rates (IRs) of RI with each virus during epidemic periods and identified household primary cases (the earliest detected household infection), and associated community exposures., Results: 41 of 175 (23.4%) households had individuals with HPIV-3 (n = 45) or RSV (n = 46) infections. Among children aged 0-4 years, RI IRs /1000 person-weeks were 8.7 [6.0, 12.2] for HPIV-3, 7.6 [4.8, 11.4] for RSV, and 1.9 [1.0, 3.5] for SARS-CoV-2. Children aged 0-4 years accounted for 35 of 36 primary HPIV-3 or RSV cases. Children attending childcare or preschool had higher odds of primary infection (odds ratio, 10.81; 95% confidence interval, 3.14-37.23)., Conclusions: Among children aged 0-4 years, RI IRs for HPIV-3 and RSV infection were 4-fold higher than for SARS-CoV-2 during epidemic periods. HPIV-3 and RSV were almost exclusively introduced into households by young children., Competing Interests: Potential conflicts of interest. M. K. H. and M. D. K. report grant support from the CDC during this study and from Pfizer that is unrelated to this work. J. O. and K. W. report grant support from the CDC and the National Institutes of Health (NIH) during this study. M. L. is an employee of Merck & Co, Inc, with responsibilities related to device development. R. A. K. reports grant support from the CDC and support from the NIH, Sanofi Pasteur, and the Bill and Melinda Gates Foundation for research pertaining to respiratory syncytial virus vaccines. All remaining authors: No reported conflicts of interest. 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) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections and hospitalization in infants and young children. Here, we analyzed the genetic diversity of RSV using partial G gene sequences in 84 RSV-A and 78 RSV- B positive samples collected in Seoul, South Korea, for 10 consecutive years, from 2010 to 2019. Our phylogenetic analysis revealed that RSV-A strains were classified into either the ON1 (80.9%) or NA1 (19.0%) genotypes. On the other hand, RSV-B strains demonstrated diversified clusters within the BA genotype. Notably, some sequences designated as BA-SE, BA-SE1, and BA-DIS did not cluster with previously identified BA genotypes in the phylogenetic trees. Despite this, they did not meet the criteria for the assignment of a new genotype based on recent classification methods. Selection pressure analysis identified three positive selection sites (amino acid positions 273, 274, and 298) in RSV-A, and one possible positive selection site (amino acid position 296) in RSV-B, respectively. The mean evolutionary rates of Korean RSV-A from 1999 to 2019 and RSV-B strains from 1991 and 2019 were estimated at 3.51 × 10-3 nucleotides (nt) substitutions/site/year and 3.32 × 10-3 nt substitutions/site/year, respectively. The population dynamics in the Bayesian skyline plot revealed fluctuations corresponding to the emergence of dominant strains, including a switch of the dominant genotype from NA1 to ON1. Our study on time-scaled cumulative evolutionary analysis contributes to a better understanding of RSV epidemiology at the local level in South Korea., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kim 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.)

Background: Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness., Objectives: Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses., Study Design: Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32., Results: In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested., Conclusions: This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Respiratory syncytial virus (RSV) continues to pose serious threats to pediatric populations due to the lack of a vaccine and effective antiviral drugs. RSV fusion (F) glycoprotein mediates viral-host membrane fusion and is a key target for neutralizing antibodies. We generated 23 full-human monoclonal antibodies (hmAbs) against prefusion F protein (pre-F) from a healthy adult with natural RSV infection by single B cell cloning technique. A highly potent RSV-neutralizing hmAb, named as 25-20, is selected, which targets a new site Ø-specific epitope. Site-directed mutagenesis and structural modelling analysis demonstrated that 25-20 mainly targets a highly conserved hydrophobic region located at the a4 helix and a1 helix of pre-F, indicating a site of vulnerability for drug and vaccine design. It is worth noting that 25-20 uses an unreported inferred germline (iGL) that binds very poorly to pre-F, thus high levels of somatic mutations are needed to gain high binding affinity with pre-F. Our observation helps to understand the evolution of RSV antibody during natural infection. Furthermore, by in silico prediction and experimental verification, we optimized 25-20 with KD values as low as picomolar range. Therefore, the optimized 25-20 represents an excellent candidate for passive protection against RSV infection., (© 2023. Science China Press.)

We sequenced 54 respiratory syncytial virus (RSV) genomes collected during 2021-22 and 2022-23 outbreaks in Washington, USA, to determine the origin of increased RSV cases. Detected RSV strains have been spreading for >10 years, suggesting a role for diminished population immunity from low RSV exposure during the COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic and related public health intervention measures have been reported to have resulted in the reduction of infections caused by influenza viruses and other common respiratory viruses. However, the influence may be varied in areas that have different ecological, economic, and social conditions. This study investigated the changing epidemiology of 8 common respiratory pathogens, including Influenza A (IFVA), Influenza B (IFVB), Respiratory syncytial virus (HRSV), rhinovirus (RV), Human metapneumovirus Adenovirus, Human bocavirus, and Mycoplasma pneumoniae, among hospitalized children during spring and early summer in 2019-2021 in two hospitals in Hainan Island, China, in the COVID-19 pandemic era. The results revealed a significant reduction in the prevalence of IFVA and IFVB in 2020 and 2021 than in 2019, whereas the prevalence of HRSV increased, and it became the dominant viral pathogen in 2021. RV was one of the leading pathogens in the 3 year period, where no significant difference was observed. Phylogenetic analysis revealed close relationships among the circulating respiratory viruses. Large scale studies are needed to study the changing epidemiology of seasonal respiratory viruses to inform responses to future respiratory virus pandemics., (© 2023 Wiley Periodicals LLC.)

Background: Influenza and respiratory syncytial (RSV) viruses are expected to co-circulate with SARS-CoV-2 in the upcoming seasons and clinical differential diagnosis between them is difficult. Laboratory-based RT-PCR is a gold standard diagnostic method for influenza, RSV and SARS-CoV-2. The objective of this study was to estimate the diagnostic performance of a novel point-of-care RT-PCR assay STANDARD M10 Flu/RSV/SARS-CoV-2 (SD Biosensor) in a large number of clinical specimens with diversified (co)-infection patterns and viral loads., Methods: This was a retrospective study, in which all samples were tested in both STANDARD M10 Flu/RSV/SARS-CoV-2 index and Allplex SARS-CoV-2/Respiratory Panel 1 (Seegene) reference kits. Samples with discordant results were further processed in a third resolver test (Resp-4-Plex, Abbott)., Results: A total of 1,019 naso-/oropharyngeal samples (50.3% positive for at least one virus) were processed in both STANDARD M10 Flu/RSV/SARS-CoV-2 and Allplex assays and the overall between-assay agreement was as high as 94.6%. Positive percent agreement of the STANDARD M10 Flu/RSV/SARS-CoV-2 was 100%, 96.6%, 97.3% and 99.4% for influenza A, B, RSV and SARS-CoV-2, respectively. The corresponding negative percent agreement was 99.7%. 100%, 100% and 98.4%, respectively. The expected positive and negative predictive values for all viruses were constantly above 96% in a reasonable range of disease prevalence., Conclusions: STANDARD M10 Flu/RSV/SARS-CoV-2 is a reliable RT-PCR assay able to detect influenza A, influenza B, RSV and SARS-CoV-2 in one hour or less, fostering a rapid differential diagnosis of common respiratory viruses., Competing Interests: Declaration of Competing Interest A.D. was previously a full-time employee of Seqirus, a pharmaceutical company that manufactures and markets influenza vaccines, and received speaker fees from SD Biosensor. The other 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 © 2023. Published by Elsevier B.V.)

Background: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology., Methods: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process., Results: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR., Conclusion: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load., (© 2023 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Respiratory syncytial virus (RSV) has a significant health burden in children, older adults, and the immunocompromised. However, limited effort has been made to identify emergence of new RSV genotypes' frequency of infection and how the combination of nasopharyngeal microbiome and viral genotypes impact RSV disease outcomes. In an observational cohort designed to capture the first infant RSV infection, we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes, during which the 2012/2013 season was dominated by RSV-A, whereas 2013 and 2014 was dominated by RSV-B. We found non-G-72nt-duplicated RSV-A strains were more frequent in male infants ( P  = 0.02), whereas G-72nt-duplicated genotypes (which is ON1 lineage) were seen equally in both males and females. DESeq2 testing of the nasal microbiome showed Haemophilus was significantly more abundant in infants with RSV-A infection compared to infants with RSV-B infection (adjusted P  = 0.002). In addition, the broad microbial clustering of the abundant genera was significantly associated with infant sex ( P  = 0.03). Overall, we show sex differences in infection by RSV genotype and host nasopharyngeal microbiome, suggesting an interaction between host genetics, virus genotype, and associated nasopharyngeal microbiome. IMPORTANCE Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in young children and is responsible for high hospitalization rates and morbidity in infants and the elderly. To understand how the emergence of RSV viral genotypes and viral-respiratory microbiome interactions contribute to infection frequency and severity, we utilized an observational cohort designed to capture the first infant RSV infection we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes. We found non-G-72nt-duplicated RSV-A genotypes were more frequent in male infants, whereas G-72nt-duplicated RSV-A strains (ON1 lineage) were seen equally in both males and females. Microbiome analysis show Haemophilus was significantly more abundant in infants with RSV-A compared to infants with RSV-B infection and the microbial clustering of the abundant genera was associated with infant sex. Overall, we show sex differences in RSV genotype-nasopharyngeal microbiome, suggesting an interaction host genetics-virus-microbiome interaction.

Following the extensive non-pharmaceutical interventions (NPIs) and behavioral changes in the wake of the SARS-CoV-2 pandemic, an interseasonal rise in respiratory syncytial virus (RSV) cases was observed in Germany in 2021. The aim of this study was to characterize the local molecular epidemiology of RSV infections in comparison to the three pre-pandemic seasons. Additionally, clinical data were retrieved from patient charts to determine the clinical significance of RSV infections. RSV detections peaked in calendar week 40 of 2021, 18 weeks earlier than the usual peak observed in the three pre-pandemic seasons. Sequence analysis revealed a close phylogenetic relatedness regardless of the season of origin. A significantly higher amount of pediatric cases (88.9% of all cases, p < 0.001) was observed for season 2021/2022. For the pediatric cases, significant differences were observed for an increased number of siblings in the household ( p = 0.004), a lower rate of fever ( p = 0.007), and a reduced amount of co-infections ( p = 0.001). Although the mean age of the adult patients was significantly younger (47.1 vs. 64.7, p < 0.001), high rates of comorbidities, lower respiratory tract infections and intensive care unit admissions prevailed. The NPIs in the wake of the SARS-CoV-2 pandemic had a tremendous impact on the epidemiologic characteristics and seasonality of RSV and warrant further epidemiologic studies of this important pathogen.

Introduction: Human respiratory syncytial virus (HRSV) infection causes significant morbidity, and no effective treatments are currently available. Viral infections induce substantial metabolic changes in the infected cells to optimize viral production. Metabolites that reflect the interactions between host cells and viruses provided an opportunity to identify the pathways underlying severe infections., Objective: To better understand the metabolic changes caused by HRSV infection, we analyzed temporal metabolic profiling to provide novel targets for therapeutic strategies for inhaled HRSV infection., Methods: The epithelial cells and BALB/c mice were infected with HRSV. Protein and mRNA levels of inflammation factors were measured by using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Untargeted metabolomics, lipidomics and proteomics were performed using liquid chromatography coupled with mass spectrometry to profile the metabolic phenotypic alterations in HRSV infection., Results: In this study, we evaluated the inflammatory responses in vivo and in vitro and investigated the temporal metabolic rewiring of HRSV infection in epithelial cells. We combined metabolomics and proteomic analyses to demonstrate that the redox imbalance was further provoked by increasing glycolysis and anaplerotic reactions. These responses created an oxidant-rich microenvironment that elevated reactive oxygen species levels and exacerbated glutathione consumption., Conclusion: These observations indicate that adjusting for metabolic events during a viral infection could represent a valuable approach for reshaping the outcome of infections., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infections worldwide. While historically RSV research has been focused on children, data on RSV infection in adults are limited. The goal of this study was to establish the prevalence of RSV in community-dwelling Italian adults and analyze its genetic variability during the 2021/22 winter season., Methods: In this cross-sectional study, a random sample of naso-/oropharyngeal specimens from symptomatic adults seeking for SARS-CoV-2 molecular testing between December 2021 and March 2022 were tested for RSV and other respiratory pathogens by means of reverse-transcription polymerase chain reaction. RSV-positive samples were further molecularly characterized by sequence analysis., Results: Of 1,213 samples tested, 1.6% (95% CI: 0.9-2.4%) were positive for RSV and subgroups A (44.4%) and B (55.6%) were identified in similar proportions. The epidemic peak occurred in December 2021, when the RSV prevalence was as high as 4.6% (95% CI: 2.2-8.3%). The prevalence of RSV detection was similar (p = 0.64) to that of influenza virus (1.9%). All RSV A and B strains belonged to the ON1 and BA genotypes, respectively. Most (72.2%) RSV-positive samples were also positive for other pathogens being SARS-CoV-2, Streptococcus pneumoniae and rhinovirus the most frequent. RSV load was significantly higher among mono-detections than co-detections., Conclusion: During the 2021/22 winter season, characterized by the predominant circulation of SARS-CoV-2 and some non-pharmaceutical containment measures still in place, a substantial proportion of Italian adults tested positive for genetically diversified strains of both RSV subtypes. In view of the upcoming registration of vaccines, establishment of the National RSV surveillance system is urgently needed., (© 2023. The Author(s).)

Parainfluenza virus 5 (PIV5) is a negative-sense, single-stranded RNA virus that can infect humans and many species of animals. Infection in these reservoir hosts is generally asymptomatic and has few safety concerns. Emerging evidence has shown that PIV5 is a promising vector for developing vaccines against human infectious diseases caused by coronaviruses, influenza, respiratory syncytial virus, rabies, HIV, or bacteria. In this review, we summarize recent progress and highlight the advantages and strategies of PIV5 as a vaccine vector to improve future vaccine design and application for clinical trials., (© 2023 Wiley Periodicals LLC.)

Background: Acute lower respiratory tract infections (ALRIs) are one one of the leading causes of morbidity and mortality among people of all ages worldwide, particularly in low- and middle-income countries (LMICs). The purpose of this study was to determine epidemiological characteristics of respiratory viruses in acute respiratory infection (ARI) patients during the COVID-19 pandemic in Yaoundé, Cameroon., Methods: Patients were monitored for respiratory symptoms as part of the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viral infections. Patients of all ages with respiratory symptoms less than 5 days were considered. Sociodemographic and clinical data as well as nasopharyngeal samples was collected from patients. Nasopharyngeal samples were tested for SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) using real-time reverse-transcription polymerase chain reaction methods. Virus distribution and demographic data were analyzed with R version 2.15.1., Results: From July 2020 to October 2021, 1120 patients were included. The overall viral detection rate was 32.5%, including 9.5% for RSV, 12.6% for influenza virus and 12.8% for SARS-CoV-2. Co-infections were detected in 6.9% of positive cases. While RSV and influenza virus showed seasonal trends, SARS-CoV-2 was detected throughout the study period., Conclusion: We found that during COVID-19 pandemic, respiratory viruses play an important role in etiology of influenza-like illness in Cameroon, and this observation was true for patients of all ages., (© 2023 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Objectives: Despite the global impact of the Respiratory Syncytial Virus (RSV) infection in children, only one monoclonal antibody (Palivizumab) has been approved for clinical use. However, advances in the knowledge of RSV immunology may enable the development of safe and effective new vaccines and monoclonal antibodies in a few years. The purpose of this review is to summarize available data on approved and developing passive and active immunizations against RSV in childhood and pregnancy., Data Source: A non-systematic review of RSV immunoprophylaxis in childhood and pregnancy was carried out in PubMed, path.org and clinical trial registries, without language restrictions, up to September 2022., Data Synthesis: Three monoclonal antibodies and 17 active immunization candidates are under development in phase 1 to 3 clinical studies. Regarding the first group, Nirsevimab is a monoclonal antibody with a prolonged half-life whose approval for clinical use is expected in the next months. Among the vaccines under development, six techniques are being used: protein subunit, viral particles, live attenuated virus, recombinant viral vector, chimeric, and mRNA. The first two approaches are being tested primarily in pregnancy, while the others are being developed for the pediatric population., Conclusions: The approval of extended half-life monoclonal antibodies is the next expected advance in RSV prevention, although the costs may be a barrier to the implementation. Regarding active immunizations, maternal and infant vaccination are complementary strategies and there are many promising candidates in clinical studies using different platforms., Competing Interests: Conflicts of interest Author Renato T. Stein declares that he participated in the Advisory Boards of the following companies: Abbvie, SANOFI, Pfizer and AstraZeneca, but that his participation has not influenced the writing of this article., (Copyright © 2022. Published by Elsevier Editora Ltda.)

The study was planned to carry out the molecular characterization of the respiratory syncytial virus (RSV) circulating strains and to elucidate the gene expression of autophagy and mammalian target of rapamycin (mTOR) signaling pathways in children with acute lower respiratory tract infection (ALRTI). Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected to the detection of RSV. Of them, 31 RSV positive strains were subjected for sequencing. Semi-quantitative gene expression analysis for mTOR signaling and autophagy pathway genes was performed in respiratory tract epithelial cells using 25 RSV positive cases, and 10 age and sex matched healthy control subjects. Five representative genes were selected for each pathway and subjected to SYBR green real-time polymerase chain reaction. RSV was positive in 69 (47.6%) samples and the representative (n = 31) RSV strains belonged to RSV-A. Thirty-one strains of RSV-A on phylogenetic analysis clustered with the novel ON1 genotype having 72 bp nucleotide duplicationby targeting the ecto-domain portion of the G gene. Further, the stains belonged to lineage 1 (51.6%), followed by lineage 3 (29%) and lineage 2 (19.4%). Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1, and TSC1 genes of the mTOR pathway were significantly downregulated in RSV positive patients. Thus, RSV infection inducing autophagy pathway genes (NPC1 and ATG3) and suppressing mTOR signaling pathway genes (AKT1, mTOR, and TSC1) to possibly evade the host immune system through dysregulating these pathways for its way of survival within the host., (© 2023 Wiley Periodicals LLC.)

To better understand the circulation pattern and genetic characterization of human respiratory syncytial virus (HRSV) in China during 2008-2021, a total of 3967 HVR2 sequences were obtained from 20 provinces in China for phylogenetic analysis and sequence variation analysis. The results showed that the HRSV subtype presented the prevalence pattern of "ABBAABAABAAABB." Further genotyping identified seven genotypes for HRSVA and nine genotypes for HRSVB. Multiple genotypes of HRSV were cocirculating during 2008-2015, while ON1 and BA9 became the only predominant genotypes for HRSVA and HRSVB, respectively, since 2015. A genotype switch from NA1 to ON1 for HRSVA occurred in approximately 2014, while genotype BA9 of HRSVB had been the predominant genotype for at least 14 years. ON1 strains could be divided into four lineages with no temporal or geographical distribution tendency. In contrast, BA9 strains could be divided into three lineages with noticeable temporal clustering. Sequence variation analysis showed that two ON1 sequences in 2017 had 10 nucleotide deletion and compensatory extension at the C-terminal; 15 BA9 sequences during 2019-2021 had novel insertions between K225 and E226, along with 6 identical amino acid variant sites. This study further enriched the genetic data of HRSV circulating in China and provided an important basis for the development of HRSV vaccines and drugs as well as the formulation of prevention and control strategies., (© 2023 Wiley Periodicals LLC.)

Isolation of human respiratory syncytial virus (HRSV) from clinical samples and storage of isolates for long period remains a considerable problem. We describe in detail the optimized conditions of HRSV isolation and cultivation in three cell cultures HeLa, HEp-2, and Vero. HRSV was detected in 35.2% (166/471) specimens by real-time PCR from symptomatic infants and children up to 15 years from October 2017 to March 2018 in Russia. HRSV-positive samples were used for virus isolation in HeLa, HEp-2, and Vero cells in different manners (in monolayer or suspension). To optimize the conditions of HRSV cultivation, these cell cultures were treated or not with receptor-destroying enzyme (RDE). Ten isolates were successfully obtained by the way of infection of the suspension of cells with subsequent RDE treatment. Among them, several isolates induced the cytopathogenic effect (CPE) by the syncytium formation in both Hela and HEp-2 cell cultures. The genetic analysis revealed that the manners of isolation by using monolayer or suspension and subsequent RDE treatment did not influence the nucleotide and amino acid structures of obtained HRSVs. The CPE characteristics of obtained viruses were the same in HeLa, HEp-2, and Vero cell cultures, and were described as large syncytium up to 150 microns or more in size with the nuclei peripheral location and an optically bright zone in the center of the formation. We showed that infection of cell suspension with the subsequent RDE treatment increased the chance of HRSVs isolation from clinical samples., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Elena Alexandrovna Prokopyeva, Olga Grigoryevna Kurskaya, Mariya Vladimirovna Solomatina, Ivan Andreevich Sobolev, Tereza Araevna Saroyan, Nikita Artemyevich Dubovitskiy, Anastasiya Alexandrovna Derko, Peter Mikchailovich Kozhin, Tatyana Yurievna Alikina, Marsel Rasimovich Kabilov, Alexander Mikchailovich Shestopalov, Kirill Alexandrovich Sharshov.)

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Reverse transcription polymerase chain reaction (RT-PCR) on respiratory tract swabs has become the gold standard for sensitive and specific detection of influenza virus, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this retrospective analysis, we report on the successive implementation and routine use of multiplex RT-PCR testing for patients admitted to the Internal Medicine Emergency Department (ED) at a tertiary care center in Western Austria, one of the hotspots in the early coronavirus disease 2019 (COVID-19) pandemic in Europe. Our description focuses on the use of the Cepheid ® Xpert ® Xpress closed RT-PCR system in point-of-care testing (POCT). Our indications for RT-PCR testing changed during the observation period: From the cold season 2016/2017 until the cold season 2019/2020, we used RT-PCR to diagnose influenza or RSV infection in patients with fever and/or respiratory symptoms. Starting in March 2020, we used the RT-PCR for SARS-CoV-2 and a multiplex version for the combined detection of all these three respiratory viruses to also screen subjects who did not present with symptoms of infection but needed in-hospital medical treatment for other reasons. Expectedly, the switch to a more liberal RT-PCR test strategy resulted in a substantial increase in the number of tests. Nevertheless, we observed an immediate decline in influenza virus and RSV detections in early 2020 that coincided with public SARS-CoV-2 containment measures. In contrast, the extensive use of the combined RT-PCR test enabled us to monitor the re-emergence of influenza and RSV detections, including asymptomatic cases, at the end of 2022 when COVID-19 containment measures were no longer in place. Our analysis of PCR results for respiratory viruses from a real-life setting at an ED provides valuable information on the epidemiology of those infections over several years, their contribution to morbidity and need for hospital admission, the risk for nosocomial introduction of such infection into hospitals from asymptomatic carriers, and guidance as to how general precautions and prophylactic strategies affect the dynamics of those infections.

Background: Little information is available on the costs of respiratory syncytial virus (RSV) in Vietnam or other low- and middle-income countries. Our study estimated the costs of LRTIs associated with RSV infection among children in southern Vietnam., Methods: We conducted a prospective cohort study evaluating household and societal costs associated with LRTIs stratified by RSV status and severity among children under 2 years old who sought care at a major pediatric referral hospital in southern Vietnam. Enrollment periods were September 2019-December 2019, October 2020-June 2021 and October 2021-December 2021. RSV status was confirmed by a validated RT-PCR assay. RSV rapid detection antigen (RDA) test performance was also evaluated. Data on resource utilization, direct medical and non-medical costs, and indirect costs were collected from billing records and supplemented by patient-level questionnaires. All costs are reported in 2022 US dollars., Results: 536 children were enrolled in the study, with a median age of 7 months (interquartile range [IQR] 3-12). This included 210 (39.2%) children from the outpatient department, 318 children (59.3%) from the inpatient respiratory department (RD), and 8 children (1.5%) from the intensive care unit (ICU). Nearly 20% (105/536) were RSV positive: 3.9 percent (21/536) from the outpatient department, 15.7% (84/536) from the RD, and none from the ICU. The median total cost associated with LRTI per patient was US$52 (IQR 32-86) for outpatients and US$184 (IQR 109-287) for RD inpatients. For RSV-associated LRTIs, the median total cost per infection episode per patient was US$52 (IQR 32-85) for outpatients and US$165 (IQR 95-249) for RD inpatients. Total out-of-pocket costs of one non-ICU admission of RSV-associated LRTI ranged from 32%-70% of the monthly minimum wage per person (US$160) in Ho Chi Minh City. The sensitivity and the specificity of RSV RDA test were 88.2% (95% CI 63.6-98.5%) and 100% (95% CI 93.3-100%), respectively., Conclusion: These are the first data reporting the substantial economic burden of RSV-associated illness in young children in Vietnam. This study informs policymakers in planning health care resources and highlights the urgency of RSV disease prevention., (© 2023. The Author(s).)

Messenger RNA (mRNA) vaccines against COVID-19 are the first authorized biological preparations developed using this platform. During the pandemic, their administration has been proven to be a life-saving intervention. Here, we review the main advantages of using mRNA vaccines, identify further technological challenges to be met during the development of the mRNA platform, and provide an update on the clinical progress on leading mRNA vaccine candidates against different viruses that include influenza viruses, human immunodeficiency virus 1, respiratory syncytial virus, Nipah virus, Zika virus, human cytomegalovirus, and Epstein-Barr virus. The prospects and challenges of manufacturing mRNA vaccines in low-income countries are also discussed. The ongoing interest and research in mRNA technology are likely to overcome some existing challenges for this technology (e.g., related to storage conditions and immunogenicity of some components of lipid nanoparticles) and enhance the portfolio of vaccines against diseases for which classical formulations are already authorized. It may also open novel pathways of protection against infections and their consequences for which no safe and efficient immunization methods are currently available., (© 2023 Wiley Periodicals LLC.)

Respiratory syncytial virus (RSV) is a serious human respiratory pathogen, but no RSV vaccine has been licensed. Many vaccine candidates are focused on the viral F protein since the F protein is more conserved than the viral G protein across RSV strains and serotypes; thus, the F protein is thought more likely to induce a broader range of protection from infection. However, it is the G protein that binds the likely receptor, CX3CR1, in lung ciliated epithelial cells, raising the question of the importance of the G protein in vaccine candidates. Using virus-like particle (VLP) vaccine candidates, we have directly compared VLPs containing only the prefusion F protein (pre-F), only the G protein, or both glycoproteins. We report that VLPs containing both glycoproteins bind to anti-F-protein-specific monoclonal antibodies differently than do VLPs containing only the prefusion F protein. In RSV-naive cotton rats, VLPs assembled with only the pre-F protein stimulated extremely weak neutralizing antibody (NAb) titers, as did VLPs assembled with G protein. However, VLPs assembled with both glycoproteins stimulated quite robust neutralizing antibody titers, induced improved protection of the animals from RSV challenge compared to pre-F VLPs, and induced significantly higher levels of antibodies specific for F protein antigenic site 0, site III, and the AM14 binding site than did VLPs containing only the pre-F protein. These results indicate that assembly of pre-F protein with G protein in VLPs further stabilized the prefusion conformation or otherwise altered the conformation of the F protein, increasing the induction of protective antibodies. IMPORTANCE Respiratory syncytial virus (RSV) results in significant disease in infants, young children, and the elderly. Thus, development of an effective vaccine for these populations is a priority. Most ongoing efforts in RSV vaccine development have focused on the viral fusion (F) protein; however, the importance of the inclusion of G in vaccine candidates is unclear. Here, using virus-like particles (VLPs) assembled with only the F protein, only the G protein, or both glycoproteins, we show that VLPs assembled with both glycoproteins are a far superior vaccine in a cotton rat model compared with VLPs containing only F protein or only G protein. The results show that the presence of G protein in the VLPs influences the conformation of the F protein and the immune responses to F protein, resulting in significantly higher neutralizing antibody titers and better protection from RSV challenge. These results suggest that inclusion of G protein in a vaccine candidate may improve its effectiveness.

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N 0 P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.

During the COVID-19 pandemic in 2021, Japan experienced an outbreak of respiratory syncytial virus (RSV) infection. A total of 51 RSV cases were detected in infant specimens, including 38 rhinorrhea and 13 nasopharyngeal swabs, collected at the Tokyo Metropolitan Institute of Public Health. Of the 51 cases, 12 were RSV-A and 39 were RSV-B. The G protein gene sequence of RSV-A belonged to the ON1 genotype, whereas RSV-B belonged to the BA9 genotype; thus, different types of RSV were detected during the same period, suggesting that the unusual 2021 RSV season was not due to a single strain or genotype. Of all RSV-positive cases, the proportion of patients aged ≥2 years was 56.8% in 2021, higher than the 31.2% reported in the past 5 years. This indicates that infants aged <1 year who were originally susceptible to RSV infection were less likely to be infected with RSV because of the COVID-19 control measures. The 2021 epidemic peaked in the 28th week, 9 weeks earlier than the average from 2016 to 2020. Therefore, it seems necessary to accumulate and analyze further data, such as factors that led to the outbreak and the characteristics of the detected viruses in 2021.

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA., Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories., Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping., Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets., Competing Interests: No conflict of interest is declared by authors., (© 2023 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)