Establishing a Network of RSV Laboratories in Europe PROMISE aims at strengthening and harmonising RSV testing and surveillance in Europe and to support the publication of the Fortnightly Electronic Bulletin. The project builds on the experience that was gained in RESCEU in collaboration with the European Reference Laboratory Network for Human Influenza (ERLI-Net) of ECDC. The outputs of this task will promote and support harmonisation of RSV detection and characterisation methods. The harmonised data will be incorporated into the surveillance platform, and used to support the Fortnightly Electronic Bulletin. Support will also be provided for External Quality Assessments (EQA) of RSV diagnostics and characterisation techniques. Our workplan spreads from Nov 22 to April 24: Image Relevant Literature Molecular Diagnostics Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh-7 and 293T, permitted the propagation of recombinant green fluorescent protein–expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh-7 and 293T cells. We then confirmed that recombinant green fluorescent protein–expressing RSV was rescued in both Huh-7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh-7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II. Access the article 28 July 2023 The impact of laboratory characteristics on molecular detection of RSV in a European multicentre quality control study The performance of nucleic acid amplification techniques for detecting respiratory syncytial virus (RSV) was evaluated in 25 laboratories across Europe by an external quality assessment study. In addition, factors related to the diagnostic performance of laboratories were explored. The results of this quality control study show that the performance of laboratories for RSV diagnosis in Europe is good, with an overall correct score of 88%. The type of assay (nested or real-time PCR vs. commercial tests) was identified as a significant factor in predicting a correct result. Access the article 17 November 2022 Sequencing and Genotyping Eurosurveillance | Genomic characterisation of respiratory syncytial virus: a novel system for whole genome sequencing and full-length G and F gene sequences To advance our understanding of respiratory syncytial virus (RSV) impact through genomic surveillance, we describe two PCR-based sequencing systems, (i) RSVAB-WGS for generic whole-genome sequencing and (ii) RSVAB-GF, which targets major viral antigens, G and F, and is used as a complement for challenging cases with low viral load. These methods monitor RSV genetic diversity to inform molecular epidemiology, vaccine effectiveness and treatment strategies, contributing also to the standardisation of surveillance in a new era of vaccines. Access the Article 13 December 2023 Next-generation sequencing of human respiratory syncytial virus subgroups A and B genomes Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with Ct values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with Ct values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness. Access the Article 28 July 2023 A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses 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 reversetranscription 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. Document PROMISE A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses (1.23 MB / 0-S1386653223000458-MAIN) 30 March 2023 Global molecular diversity of RSV – the “INFORM RSV” study Respiratory syncytial virus (RSV) is a global cause of severe respiratory morbidity and mortality in infants. While preventive and therapeutic interventions are being developed, including antivirals, vaccines and monoclonal antibodies, little is known about the global molecular epidemiology of RSV. INFORM is a prospective, multicenter, global clinical study performed by ReSViNET to investigate the worldwide molecular diversity of RSV isolates collected from children less than 5 years of age. Document Promise Global molecular diversity of RSV – the “INFORM RSV” study (733.35 KB / PDF) 30 March 2023 Novel and extendable genotyping system for human RSV based on whole-genome sequence analysis Human respiratory syncytial virus (RSV) is one of the leading causes of respiratory infections, especially in infants and young children. Previous RSV sequencing studies have primarily focused on partial sequencing of G gene (200-300 nucleotides) for genotype characterization or diagnostics. However, the genotype assignment with G gene has not recapitulated the phylogenetic signal of other genes, and there is no consensus on RSV genotype definition. We conducted maximum likelihood phylogenetic analysis with 10 RSV individual genes and whole-genome sequence (WGS) that are published in GenBank. This revised RSV genotyping system will provide important information for disease surveillance, epidemiology, and vaccine development. Access this Article 17 November 2022 Sequencing and analysis of globally obtained human RSV A and B genomes Human respiratory syncytial virus (RSV) is the leading cause of respiratory tract infections in children globally, with nearly all children experiencing at least one infection by the age of two. Partial sequencing of the attachment glycoprotein gene is conducted routinely for genotyping, but relatively few whole genome sequences are available for RSV. The goal of our study was to sequence the genomes of RSV strains collected from multiple countries to further understand the global diversity of RSV at a whole-genome level. We collected RSV samples and isolates from Mexico, Argentina, Belgium, Italy, Germany, Australia, South Africa, and the USA from the years 1998-2010. Both Sanger and next-generation sequencing with the Illumina and 454 platforms were used to sequence the whole genomes of RSV A and B. Our analyses showed that RSV circulates on a global scale with the same predominant clades of viruses being found in countries around the world. However, the distribution of clades can change rapidly as new strains emerge. We did not observe a strong spatial structure in our trees, with the same three main clades of RSV co-circulating globally, suggesting that the evolution of RSV is not strongly regionalized. Access this Article 17 November 2022 Surveillance Respiratory syncytial virus infection and novel interventions The large global burden of respiratory syncytial virus (RSV) respiratory tract infections in young children and older adults has gained increased recognition in recent years. Recent discoveries regarding the neutralization-specific viral epitopes of the pre-fusion RSV glycoprotein have led to a shift from empirical to structure-based design of RSV therapeutics, and controlled human infection model studies have provided early-stage proof of concept for novel RSV monoclonal antibodies, vaccines and antiviral drugs. The world’s first vaccines and first monoclonal antibody to prevent RSV among older adults and all infants, respectively, have recently been approved. Large-scale introduction of RSV prophylactics emphasizes the need for active surveillance to understand the global impact of these interventions over time and to timely identify viral mutants that are able to escape novel prophylactics. In this Review, we provide an overview of RSV interventions in clinical development, highlighting global disease burden, seasonality, pathogenesis, and host and viral factors related to RSV immunity. Access the article 28 July 2023 New perspectives on respiratory syncytial virus surveillance at the national level: lessons from the COVID-19 pandemic Learning from the COVID-19 pandemic and considering the effects of this pandemic, we provide recommendations that can guide towards sustainable RSV surveillance with the potential to be integrated into the broader perspective of respiratory surveillance. Document PROMISE New perspectives on respiratory syncytial virus surveillance at the national level: lessons from the COVID-19 pandemic (751.63 KB / -LESSONS-FROM-THE-COVID-19-PANDEMIC) 6 April 2023 Toward unified molecular surveillance of RSV: A proposal for genotype definition Human respiratory syncytial virus (RSV) is classified into antigenic subgroups A and B. Thirteen genotypes have been defined for RSV-A and 20 for RSV-B, without any consensus on genotype definition. We evaluated clustering of RSV sequences published in GenBank until February 2018 to define genotypes by using maximum likelihood and Bayesian phylogenetic analyses and average p-distances. We propose an objective protocol for RSV genotyping suitable for adoption as an international standard to support the global expansion of RSV molecular surveillance. Access the article 11 November 2022 RSV, parainfluenza and influenza virus infection in young children with acute lower respiratory infection in rural Gambia We conducted population-based surveillance in rural Gambia using standardized criteria to identify and investigate children with acute lower respiratory infection (ALRI). Each month from February through December 2015, specimens from 50 children aged 2-23 months were randomly selected to test for respiratory syncytial (RSV), parainfluenza (PIV) and influenza viruses. One or more viruses were detected in 303/519 children (58.4%). RSV-A was detected in 237 and RSV-B in seven. The expected incidence of ALRI associated with RSV, PIV or influenza was 140 cases (95% CI, 131-149) per 1000 person-years; RSV incidence was 112 cases (95% CI, 102-122) per 1000 person-years. Multiple strains of RSV and influenza circulated during the year. RSV circulated throughout most of the year and was associated with eight times the number of ALRI cases compared to PIV or IV. Gambian RSV viruses were closely related to viruses detected in other continents. An effective RSV vaccination strategy could have a major impact on the burden of ALRI in this setting. Access the article 11 November 2022 Approaches to use the WHO RSV surveillance platform to estimate disease burden The World Health Organization (WHO) recently completed the first phase of a RSV surveillance pilot study in fourteen countries (two to three in each WHO region) building on the Global Influenza Surveillance and Response System (GISRS). This active surveillance strategy had several objectives including understanding RSV-related health burden in a variety of settings. A range of approaches can be used to estimate disease burden; most approaches could not be applied by participating countries in the WHO surveillance pilot. This article provides the recommendations made by WHO for strengthening and expanding the scope of the RSV surveillance in the next phase to enable burden estimation. Access the article 11 November 2022 Recommendations for RSV surveillance at national level Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infections (ALRI) and hospitalizations among young children and is globally responsible for many deaths in young children, especially in infants below 6 months of age. Furthermore, RSV is a common cause of severe respiratory disease and hospitalization among the elderly. The development of new candidate vaccines and monoclonal antibodies highlights the need for reliable surveillance of RSV. In the European Union (EU), no up-to-date general recommendations on RSV surveillance are currently available. Based on outcomes of a workshop with 29 European experts in the field of RSV virology, epidemiology and public health, we provide recommendations to develop a feasible and sustainable national surveillance strategy for RSV that will enable harmonization and data comparison at the European level. Access the article 11 November 2022 Current practices for RSV surveillance across the EU/EEA member states, 2017 Access the article 11 November 2022 Unified Nomenclature and Classsification GitHub Repositories. RSV Genotyping Consensus Consortium These 2 respositories contain the definitions of lineages for RSV-A and RSV-B following the proposal put forward in The unified proposal for classification of human respiratory syncytial virus below the subgroup level by Goya et al and the RSV Genotyping Consensus Consortium. Access the article 19 February 2024 The unified proposal for classification of human respiratory syncytial virus below the subgroup level A globally implemented unified classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. Here, we formulate the global consensus of HRSV classification based on the challenges and limitations of our previous proposals and the future of genomic surveillance. Access the article 19 February 2024 Novel and extendable genotyping system for human respiratory syncytial virus based on whole-genome sequence analysis Human respiratory syncytial virus (RSV) is one of the leading causes of respiratory infections, especially in infants and young children. Previous RSV sequencing studies have primarily focused on partial sequencing of G gene (200–300 nucleotides) for genotype characterization or diagnostics. However, the genotype assignment with G gene has not recapitulated the phylogenetic signal of other genes, and there is no consensus on RSV genotype definition. Methods: We conducted maximum likelihood phylogenetic analysis with 10 RSV individual genes and whole-genome sequence (WGS) that are published in GenBank. RSV genotypes were determined by using phylogenetic analysis and pair-wise node distances. Document PROMISE Novel and extendable genotyping system for human respiratory syncytial virus based on whole-genome sequence analysis (3.25 MB / PDF) 12 December 2022 Towards a unified classification for human RSV genotypes Since the first human respiratory syncytial virus (RSV) genotype classification in 1998, inconsistent conclusions have been drawn regarding the criteria that define RSV genotypes and their nomenclature, challenging data comparisons between research groups. In this study, we aim to unify the field of RSV genotype classification by reviewing the different methods that have been used in the past to define RSV genotypes and by proposing a new classification procedure, based on well-established phylogenetic methods. Our data show that neither the HVR2 fragment nor the G gene contains sufficient phylogenetic signal to perform reliable phylogenetic reconstruction. Therefore, whole-genome alignments were used to determine RSV genotypes. Access the article 17 November 2022 Proposal for Human RSV nomenclature below the species level Human respiratory syncytial virus (RSV) is the leading viral cause of serious pediatric respiratory disease, and lifelong reinfections are common. Its 2 major subgroups, A and B, exhibit some antigenic variability, enabling RSV to circulate annually. Globally, research has increased the number of RSV genomic sequences available. To ensure accurate molecular epidemiology analyses, we propose a uniform nomenclature for RSV-positive samples and isolates, and RSV sequences, namely: RSV/subgroup identifier/geographic identifier/unique sequence identifier/year of sampling. We also propose a template for submitting associated metadata. Universal nomenclature would help researchers retrieve and analyze sequence data to better understand the evolution of this virus. Access the article 17 November 2022 Diagnostic Protocols RT-PCR Document PROMISE EMC - PCR - RSV A/B (341.42 KB / PDF) 29 March 2023 Document PROMISE UCD - PCR - RSV A/B (330.07 KB / PDF) 06 February 2023 Document PROMISE RIVM – PCR – RSV A/B, Influenza B/Vic and B/Yam (363.77 KB / PDF) 06 February 2023 Document PROMISE MUW - PCR - RSV A/B (309.86 KB / PDF) 06 February 2023 This article was published on 2024-09-24
