Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity.

The human respiratory syncytial virus (hRSV) and the human metapneumovirus (hMPV) are important human respiratory pathogens from the Pneumoviridae family. Both are responsible for severe respiratory tract infections in infants, young children, elderly individuals, adults with chronic medical conditions, and immunocompromised patients. Despite their large impact on human health, vaccines for hRSV were only recently introduced, and only limited treatment options exist. Here we show that Ginkgolic acid (GA), a natural compound from the extract of Ginkgo biloba, with known antiviral properties for several viruses, efficiently inhibits these viruses' infectivity and spread in cultures in a dose-dependent manner. We demonstrate that the drug specifically affects the entry step during the early stages on the viruses' life cycle with no effect on post-entry and late stage events, including viral gene transcription, genome replication, assembly and particles release. We provide evidence that GA acts as an efficient antiviral for members of the Pneumoviridae family and has the potential to be used to treat acute infections.

Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

Genomic characterization of human respiratory syncytial virus circulating in Islamabad, Pakistan, during an outbreak in 2022-2023.

In this study, conducted at the National Institute of Health, Islamabad, during an outbreak of human respiratory syncytial virus (hRSV) from December 2022 to January 2023, the first whole-genome sequences of hRSV isolates from Islamabad, Pakistan, were determined. Out of 10 positive samples, five were sequenced, revealing the presence of two genotypes: RSV-A (GA2.3.5, ON1 strain) and RSV-B (GB5.0.5.a, BA-10 strain). A rare non-synonymous substitution (E232G) in G the protein and N276S in the F protein were found in RSV-A. In RSV-B, the unique mutations K191R, Q209R, and I206M were found in the F protein. These mutations could potentially influence vaccine efficacy and viral pathogenicity. This research underscores the importance of genomic surveillance for understanding RSV diversity and guiding public health responses in Pakistan.

The genomic evolutionary dynamics and global circulation patterns of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in young children and the second leading cause of infant death worldwide. While global circulation has been extensively studied for respiratory viruses such as seasonal influenza, and more recently also in great detail for SARS-CoV-2, a lack of global multi-annual sampling of complete RSV genomes limits our understanding of RSV molecular epidemiology. Here, we capitalise on the genomic surveillance by the INFORM-RSV study and apply phylodynamic approaches to uncover how selection and neutral epidemiological processes shape RSV diversity. Using complete viral genome sequences, we show similar patterns of site-specific diversifying selection among RSVA and RSVB and recover the imprint of non-neutral epidemic processes on their genealogies. Using a phylogeographic approach, we provide evidence for air travel governing the global patterns of RSVA and RSVB spread, which results in a considerable degree of phylogenetic mixing across countries. Our findings highlight the potential of systematic global RSV genomic surveillance for transforming our understanding of global RSV spread.

Pre-COVID-19-pandemic RSV epidemiology and clinical burden in pediatric primary care in Italy: a comparative analysis across two regions for the 2019/2020 season.

BACKGROUND: Respiratory syncytial virus (RSV) infection in children under 5 years have a significant clinical burden, also in primary care settings. This study investigates the epidemiology and burden of RSV in Italian children during the 2019/20 pre-pandemic winter season. METHODS: A prospective cohort study was conducted in two Italian regions. Children with Acute Respiratory Infection (ARI) visiting pediatricians were eligible. Nasopharyngeal swabs were collected and analyzed via multiplex PCR for RSV detection. A follow-up questionnaire after 14 days assessed disease burden, encompassing healthcare utilization and illness duration. Statistical analyses, including regression models, explored associations between variables such as RSV subtype and regional variations. RESULTS: Of 293 children with ARI, 41% (119) tested positive for RSV. Median illness duration for RSV-positive cases was 7 days; 6% required hospitalization (median stay: 7 days). Medication was prescribed to 95% (110/116) of RSV cases, with 31% (34/116) receiving antibiotics. RSV subtype B and regional factors predicted increased healthcare utilization. Children with shortness of breath experienced a 36% longer illness duration. CONCLUSIONS: This study highlights a significant clinical burden and healthcare utilization associated with RSV in pre-pandemic Italian primary care settings. Identified predictors, including RSV subtype and symptomatology, indicate the need for targeted interventions and resource allocation strategies. RSV epidemiology can guide public health strategies for the implementation of preventive measures.

Deviations in RSV epidemiological patterns and population structures in the United States following the COVID-19 pandemic.

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

Targeted metagenomics reveals association between severity and pathogen co-detection in infants with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants <12 months in Spain, the UK, and the Netherlands during 2017-20. We show, using targeted metagenomic sequencing of >100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03-9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity.

Resurgence of human respiratory syncytial virus during COVID-19 pandemic in Southern Brazil.

Respiratory Syncytial Virus (RSV) is an important cause of respiratory infection in humans. Severe cases are common in children ≤2 years old, immunocompromised individuals, and the elderly. In 2020, RSV infection reduced in Rio Grande do Sul (RS), southern Brazil; however, in 2021 resurgence of RSV was observed. This study analyzed epidemiological and genetic features of RSV infection cases reported in 2021 in RS. Nasopharyngeal samples collected from individuals with respiratory infection negative for SARS-CoV-2, Influenza A and B viruses were assessed for the presence of RSV by real time RT-qPCR. RSV-A and RSV-B genomic sequencing and phylogenetic reconstructions were performed for genotyping and clade characterization. Among 21,035 respiratory samples analyzed, 2,947 were positive for RSV, 947 of which were hospitalized patients. Positive cases were detected year-round, with the highest number in June-July (winter). Children <1 year comprised 56.28% (n = 533) of the hospitalized patients infected with RSV, whereas 14.46% (n = 137) were individuals >60 years. Of a total of 361 deaths, 14.68% (n = 53) were RSV positive, mostly patients >60 years old (73.58%, n = 39). Chronic kidney disease, cardiopathy, Down syndrome and neurological diseases were associated with RSV infection. RSV-A was identified in 58.5% (n = 117/200) of the patients, and RSV-B in 41.5% (n = 83/200). Of 95 RSV genomes recovered from SARI cases, 66 were RSV-A GA.2.3.5 genotype, while 29 were RSV-B GB.5.0.5a genotype. This study provides epidemiological and molecular data on RSV cases in RS during the COVID-19 pandemic and highlights that investigation of different respiratory viruses is essential for decision-making and disease prevention and control measures.

Severe Pneumonia Caused by Respiratory Syncytial Virus and Adenovirus in Children from 2 to 24 Months at Children's Hospital 1 in Ho Chi Minh City, Vietnam.

In Vietnam, due to the lack of facilities to detect respiratory viruses from patients' specimens, there are only a few studies on the detection of viral pathogens causing pneumonia in children, especially respiratory syncytial virus (RSV) and adenovirus (Adv). Here, we performed a cross-sectional descriptive prospective study on 138 children patients from 2 to 24 months old diagnosed with severe pneumonia hospitalized at the Respiratory Department of Children's Hospital 1 from November 2021 to August 2022. The number of patients selected in this study was based on the formula n = ([Z(1 - α/2)]2 × P [1 - P])/d2, with α = 0.05, p = 0.5, and d = 9%, and the sampling technique was convenient sampling until the sample size was met. A rapid test was used to detect RSV and Adv from the nasopharyngeal swabs and was conducted immediately after the patient's hospitalization. Laboratory tests were performed, medical history interviews were conducted, and nasotracheal aspirates were collected for multiplex real-time PCR (MPL-rPCR) to detect viral and bacterial pathogens. The results of the rapid test and the MPL-rPCR in the detection of both pathogens were the same at 31.9% (44/138) for RSV and 8.7% (7/138) for Adv, respectively. Using MPL-rPCR, the detection rate was 21% (29/138) for bacterial pathogens, 68.8% (95/138) for bacterial-viral co-infections, and 6.5% (9/138) for viral pathogens. The results showed few distinctive traits between RSV-associated and Adv-associated groups, and the Adv group children were more prone to bacterial infection than those in the RSV group. In addition, the Adv group experienced a longer duration of treatment and a higher frequency of re-hospitalizations compared to the RSV group. A total of 100% of Adv infections were co-infected with bacteria, while 81.82% of RSV co-infected with bacterial pathogens (p = 0.000009). This study might be one of the few conducted in Vietnam aimed at identifying viral pathogens causing severe pneumonia in children.

Human respiratory syncytial virus subgroups A and B outbreak in a kindergarten in Zhejiang Province, China, 2023.

Background: In May-June 2023, an unprecedented outbreak of human respiratory syncytial virus (HRSV) infections occurred in a kindergarten, Zhejiang Province, China. National, provincial, and local public health officials investigated the cause of the outbreak and instituted actions to control its spread. Methods: We interviewed patients with the respiratory symptoms by questionnaire. Respiratory samples were screened for six respiratory pathogens by real-time quantitative polymerase chain reaction (RT-PCR). The confirmed cases were further sequenced of G gene to confirm the HRSV genotype. A phylogenetic tree was reconstructed by maximum likelihood method. Results: Of the 103 children in the kindergarten, 45 were classified as suspected cases, and 25 cases were confirmed by RT-PCR. All confirmed cases were identified from half of classes. 36% (9/25) were admitted to hospital, none died. The attack rate was 53.19%. The median ages of suspected and confirmed cases were 32.7 months and 35.8 months, respectively. Nine of 27 confirmed cases lived in one community. Only two-family clusters among 88 household contacts were HRSV positive. A total of 18 of the G gene were obtained from the confirmed cases. Phylogenetic analyses revealed that 16 of the sequences belonged to the HRSV B/BA9 genotype, and the other 2 sequences belonged to the HRSV A/ON1 genotype. The school were closed on June 9 and the outbreak ended on June 15. Conclusion: These findings suggest the need for an increased awareness of HRSV coinfections outbreak in the kindergarten, when HRSV resurges in the community after COVID-19 pandemic.

Respiratory syncytial virus incidence and typing in the last six seasons in the north of Spain (Asturias). Genetic characterization during the SARS-CoV-2 pandemic.

Respiratory syncytial virus is associated with lower respiratory tract infections. As several types and genotypes can circulate at the same time, genomic characterization is important for timely epidemiological control and treatment measures. In the last 6 seasons (2017-2023), 191 236 nasopharyngeal swabs were processed for respiratory viruses to determine the etiology of acute respiratory infections, describe the incidence and distribution of RSV types and enrich the data of epidemiological molecular studies on RSV in Spain. The incidence of RSV reached 7% in the pre-pandemic season. RSV was most frequent in children under 5 years of age (12.6%), but was also significant in those over 70 years of age (5.63%). The measures taken to control SARS-CoV-2 infection were useful for RSV control and the incidence decreased to 1.8%, but caused a change in the types. Pre-pandemic, the majority circulating types were RSV-B/RSV-B/RSV-A and in the pandemic it was RSV-B/RSV-B. In the last season, RSV-B and RSV-A were detected in the same proportion. Genetic characterization showed three new clades. This has been taken into account to understand the epidemiology as well as the development of therapeutic and preventive strategies.

Non-SARS-CoV-2 respiratory viral detection and whole genome sequencing from COVID-19 rapid antigen test devices: a laboratory evaluation study.

BACKGROUND: There has been high uptake of rapid antigen test device use for point-of-care COVID-19 diagnosis. Individuals who are symptomatic but test negative on COVID-19 rapid antigen test devices might have a different respiratory viral infection. We aimed to detect and sequence non-SARS-CoV-2 respiratory viruses from rapid antigen test devices, which could assist in the characterisation and surveillance of circulating respiratory viruses in the community. METHODS: We applied archival clinical nose and throat swabs collected between Jan 1, 2015, and Dec 31, 2022, that previously tested positive for a common respiratory virus (adenovirus, influenza, metapneumovirus, parainfluenza, rhinovirus, respiratory syncytial virus [RSV], or seasonal coronavirus; 132 swabs and 140 viral targets) on PCR to two commercially available COVID-19 rapid antigen test devices, the Panbio COVID-19 Ag Rapid Test Device and Roche SARS-CoV-2 Antigen Self-Test. In addition, we collected 31 COVID-19 rapid antigen test devices used to test patients who were symptomatic at The Royal Melbourne Hospital emergency department in Melbourne, Australia. We extracted total nucleic acid from the device paper test strips and assessed viral recovery using multiplex real-time PCR (rtPCR) and capture-based whole genome sequencing. Sequence and genome data were analysed through custom computational pipelines, including subtyping. FINDINGS: Of the 140 respiratory viral targets from archival samples, 89 (64%) and 88 (63%) were positive on rtPCR for the relevant taxa following extraction from Panbio or Roche rapid antigen test devices, respectively. Recovery was variable across taxa: we detected influenza A in nine of 18 samples from Panbio and seven of 18 from Roche devices; parainfluenza in 11 of 20 samples from Panbio and 12 of 20 from Roche devices; human metapneumovirus in 11 of 16 from Panbio and 14 of 16 from Roche devices; seasonal coronavirus in eight of 19 from Panbio and two of 19 from Roche devices; rhinovirus in 24 of 28 from Panbio and 27 of 28 from Roche devices; influenza B in four of 15 in both devices; and RSV in 16 of 18 in both devices. Of the 31 COVID-19 devices collected from The Royal Melbourne Hospital emergency department, 11 tested positive for a respiratory virus on rtPCR, including one device positive for influenza A virus, one positive for RSV, four positive for rhinovirus, and five positive for SARS-CoV-2. Sequences of target respiratory viruses from archival samples were detected in 55 (98·2%) of 56 samples from Panbio and 48 (85·7%) of 56 from Roche rapid antigen test devices. 98 (87·5%) of 112 viral genomes were completely assembled from these data, enabling subtyping for RSV and influenza viruses. All 11 samples collected from the emergency department had viral sequences detected, with near-complete genomes assembled for influenza A and RSV. INTERPRETATION: Non-SARS-CoV-2 respiratory viruses can be detected and sequenced from COVID-19 rapid antigen devices. Recovery of near full-length viral sequences from these devices provides a valuable opportunity to expand genomic surveillance programmes for public health monitoring of circulating respiratory viruses. FUNDING: Australian Government Medical Research Future Fund and Australian National Health and Medical Research Council.

Bacterial small RNAs may mediate immune response differences seen in respiratory syncytial virus versus rhinovirus bronchiolitis.

Bronchiolitis, a viral lower respiratory infection, is the leading cause of infant hospitalization, which is associated with an increased risk for developing asthma later in life. Bronchiolitis can be caused by several respiratory viruses, such as respiratory syncytial virus (RSV), rhinovirus (RV), and others. It can also be caused by a solo infection (e.g., RSV- or RV-only bronchiolitis) or co-infection with two or more viruses. Studies have shown viral etiology-related differences between RSV- and RV-only bronchiolitis in the immune response, human microRNA (miRNA) profiles, and dominance of certain airway microbiome constituents. Here, we identified bacterial small RNAs (sRNAs), the prokaryotic equivalent to eukaryotic miRNAs, that differ between infants of the 35th Multicenter Airway Research Collaboration (MARC-35) cohort with RSV- versus RV-only bronchiolitis. We first derived reference sRNA datasets from cultures of four bacteria known to be associated with bronchiolitis (i.e., Haemophilus influenzae, Moraxella catarrhalis, Moraxella nonliquefaciens, and Streptococcus pneumoniae). Using these reference sRNA datasets, we found several sRNAs associated with RSV- and RV-only bronchiolitis in our human nasal RNA-Seq MARC-35 data. We also determined potential human transcript targets of the bacterial sRNAs and compared expression of the sRNAs between RSV- and RV-only cases. sRNAs are known to downregulate their mRNA target, we found that, compared to those associated with RV-only bronchiolitis, sRNAs associated with RSV-only bronchiolitis may relatively activate the IL-6 and IL-8 pathways and relatively inhibit the IL-17A pathway. These data support that bacteria may be contributing to inflammation differences seen in RSV- and RV-only bronchiolitis, and for the first time indicate that the potential mechanism in doing so may be through bacterial sRNAs.

Robust and sensitive amplicon-based whole-genome sequencing assay of respiratory syncytial virus subtype A and B.

Prevention of respiratory syncytial virus (RSV) infection is now a global health priority, with a long-acting monoclonal antibody and two RSV vaccines recently licenced for clinical use. Most licenced and candidate interventions target the RSV fusion (RSV-F) protein. New interventions may be associated with the spread of mutations, reducing susceptibility to antibody neutralization in RSV-F. There is a need for ongoing longitudinal global surveillance of circulating RSV strains. To achieve this large-scale genomic surveillance, a reliable, high-throughput RSV sequencing assay is required. Here we report an improved high-throughput RSV whole-genome sequencing (WGS) assay performed directly on clinical samples without additional enrichment, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. Using upper respiratory tract (URT) RSV-positive clinical samples obtained from a sentinel network of primary care providers and from hospital patients (29.7% and 70.2%, respectively; n = 1,037), collected over the period 2019 to 2023, this assay had a threshold of approximately 4 × 103 to 8 × 103 copies/mL (RSV-B and RSV-A sub-types, respectively) as the lowest amount of virus needed in the sample to achieve >96% of whole-genome coverage at a high-quality level. Using a Ct value of 31 as an empirical cut-off, the overall assay success rate of obtaining >90% genome coverage at a read depth minimum of 20 was 96.83% for clinical specimens successfully sequenced from a total of 1,071. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national programs for the surveillance of RSV genomic variation. IMPORTANCE: In this paper, we report an improved high-throughput respiratory syncytial virus (RSV) whole-genome sequencing (WGS) assay performed directly on clinical samples, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national and global programs for the surveillance of RSV genomic variation. The quality of sequence produced is essential for preparedness for new interventions in monitoring antigenic escape, where a single point mutation might lead to a reduction in antibody binding effectiveness and neutralizing activity, or indeed in the monitoring of retaining susceptibility to neutralization by existing and new interventions.

A cost-consequence analysis of the Xpert Xpress CoV-2/Flu/RSV plus test strategy for the diagnosis of influenza-like illnesses.

AIMS: Influenza-like illnesses (ILI) affect millions each year in the United States (US). Determining definitively the cause of symptoms is important for patient management. Xpert Xpress CoV-2/Flu/RSV plus (Xpert Xpress) is a rapid, point-of-care (POC), multiplex real-time polymerase chain reaction (RT-PCR) test intended for the simultaneous qualitative detection and differentiation of SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV). The objective of our analysis was to develop a cost-consequence model (CCM) demonstrating the clinico-economic impacts of implementing PCR testing with Xpert Xpress compared to current testing strategies. METHODS: A decision tree model, with a 1-year time horizon, was used to compare testing with Xpert Xpress alone to antigen POC testing and send-out PCR strategies in the US outpatient setting from a payer perspective. A hypothetical cohort of 1,000,000 members was modeled, a portion of whom develop symptomatic ILIs and present to an outpatient care facility. Our main outcome measure is cost per correct treatment course. RESULTS: The total cost per correct treatment course was $1,131 for the Xpert Xpress strategy compared with a range of $3,560 to $5,449 in comparators. POC antigen testing strategies cost more, on average, than PCR strategies. LIMITATIONS: Simplifying model assumptions were used to allow for modeling ease. In clinical practice, treatment options, costs, and diagnostic test sensitivity and specificity may differ from what is included in the model. Additionally, the most recent incidence and prevalence data was used within the model, which is not reflective of historical averages due to the SARS-CoV-2 pandemic. CONCLUSION: The Xpert Xpress CoV-2/Flu/RSV plus test allows for rapid and accurate diagnostic results, leading to reductions in testing costs and downstream healthcare resource utilization compared to other testing strategies. Compared to POC antigen testing strategies, PCR strategies were more efficient due to improved diagnostic accuracy and reduced use of confirmatory testing.

Factors Associated With Prolonged Respiratory Virus Detection From Polymerase Chain Reaction of Nasal Specimens Collected Longitudinally in Healthy Children in a US Birth Cohort.

BACKGROUND: Respiratory viral shedding is incompletely characterized by existing studies due to the lack of longitudinal nasal sampling and limited inclusion of healthy/asymptomatic children. We describe characteristics associated with prolonged virus detection by polymerase chain reaction (PCR) in a community-based birth cohort. METHODS: Children were followed from birth to 2 years of age in the PREVAIL cohort. Weekly nasal swabs were collected and tested using the Luminex Respiratory Pathogen Panel. Weekly text surveys were administered to ascertain the presence of acute respiratory illnesses defined as fever and/or cough. Maternal reports and medical chart abstractions identified healthcare utilization. Prolonged virus detection was defined as a persistently positive test lasting ≥4 weeks. Factors associated with prolonged virus detection were assessed using mixed effects multivariable logistic regression. RESULTS: From a sub-cohort of 101 children with ≥70% weekly swabs collected, a total of 1489 viral infections were detected. Prolonged virus detection was found in 23.4% of viral infections overall, 39% of bocavirus infections, 33% of rhinovirus/enterovirus infections, 14% of respiratory syncytial virus (RSV) A infections, and 7% of RSV B infections. No prolonged detection was found for influenza virus A or B, coronavirus 229E or HKU1, and parainfluenza virus 2 or 4 infections. First-lifetime infection with each virus, and co-detection of another respiratory virus were significantly associated with prolonged detection, while symptom status, child sex, and child age were not. CONCLUSIONS: Prolonged virus detection was observed in 1 in 4 viral infections in this cohort of healthy children and varied by pathogen, occurring most often for bocavirus and rhinovirus/enterovirus. Evaluating the immunological basis of how viral co-detections and recurrent viral infections impact duration of virus detection by PCR is needed to better understand the dynamics of prolonged viral shedding.

Epigenetic Control of Innate Immunity: Consequences of Acute Respiratory Virus Infection.

Infections caused by acute respiratory viruses induce a systemic innate immune response, which can be measured by the increased levels of expression of inflammatory genes in immune cells. There is growing evidence that these acute viral infections, alongside transient transcriptomic responses, induce epigenetic remodeling as part of the immune response, such as DNA methylation and histone modifications, which might persist after the infection is cleared. In this article, we first review the primary mechanisms of epigenetic remodeling in the context of innate immunity and inflammation, which are crucial for the regulation of the immune response to viral infections. Next, we delve into the existing knowledge concerning the impact of respiratory virus infections on the epigenome, focusing on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Influenza A Virus (IAV), and Respiratory Syncytial Virus (RSV). Finally, we offer perspectives on the potential consequences of virus-induced epigenetic remodeling and open questions in the field that are currently under investigation.

A potential bivalent mRNA vaccine candidate protects against both RSV and SARS-CoV-2 infections.

As the world continues to confront severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV) is also causing severe respiratory illness in millions of infants, elderly individuals, and immunocompromised people globally. Exacerbating the situation is the fact that co-infection with multiple viruses is occurring, something which has greatly increased the clinical severity of the infections. Thus, our team developed a bivalent vaccine that delivered mRNAs encoding SARS-CoV-2 Omicron spike (S) and RSV fusion (F) proteins simultaneously, SF-LNP, which induced S and F protein-specific binding antibodies and cellular immune responses in BALB/c mice. Moreover, SF-LNP immunization effectively protected BALB/c mice from RSV infection and hamsters from SARS-CoV-2 Omicron infection. Notably, our study pointed out the antigenic competition problem of bivalent vaccines and provided a solution. Overall, our results demonstrated the potential of preventing two infectious diseases with a single vaccine and provided a paradigm for the subsequent design of multivalent vaccines.

A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.