Genomic Characterization of Respiratory Syncytial Virus during 2022-23 Outbreak, Washington, USA.

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.

Impact of COVID-19 on the changing pattern of human orthopneumovirus (respiratory syncytial virus) infection in Iran.

BACKGROUND: Human orthopneumovirus (HOPV) or respiratory syncytial virus (RSV) is one of the important causes of acute respiratory infections (ARIs) during the cold months of the year worldwide. Many countries have reported an absence of ARIs due to HOPV during the winter of 2020-2021 associated with preventive measures to reduce the spread of SARS-CoV2. However, with the reduction of COVID-19 public health restrictions and the absence of immunity in the community due to the lack of exposure in the previous season, many countries had a delayed HOPV outbreak. Here we reported the impact of COVID-19 on the changing pattern of HOPV infection in Iran. METHODS: Throat and nasopharyngeal swab samples were collected from patients (children and adults) with ARIs and sent to the Iran National Influenza Center. After RNA extraction, Real time RT-PCR was performed for HOPV detection. RESULTS: In 260 samples collected from patients with ARIs in three different groups, which included children in March 2021, pilgrims in July 2022, and outpatients during November and December 2022, no HOPV was detected in any group. CONCLUSIONS: The lack of HOPV activity in Iran during the winter of 2020-2021 and then the resurgence in spring 2022 and again the absence of activity in summer and autumn 2022 was extraordinary in the HOPV epidemiology, and probably due to the implementation of public health non-pharmaceutical interventions to reduce the spread of SARS-CoV2. Although it is not possible to keep such restrictions, similar methods can be taken to control outbreaks caused by respiratory viruses.

Proposal for Human Respiratory Syncytial Virus Nomenclature below the Species Level.

Human respiratory syncytial virus (HRSV) 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 HRSV to circulate annually. Globally, research has increased the number of HRSV genomic sequences available. To ensure accurate molecular epidemiology analyses, we propose a uniform nomenclature for HRSV-positive samples and isolates, and HRSV sequences, namely: HRSV/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.

Molecular epidemiology of respiratory syncytial virus among children and adults in India 2016 to 2018.

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections among children less than 5 years of age and the elderly. This study intended to determine the circulating genotypes of RSV among severe acute respiratory illness (SARI) cases during the period 2016-2018 in India, among hospitalized acute febrile illness cases of age ranging from 1 to 65 years. Throat/nasopharyngeal swab samples were subjected for testing RSV and subgroups by real-time reverse transcriptase polymerase chain reaction (RT-PCR), further sequencing and phylogenetic analysis were performed for the second hypervariable region of the G gene. RSV-A and B subtypes co-circulated during the years 2016, 2017, and 2018, with RSV-A as the dominant subtype in 2016, and RSV-B as the dominant subgroup in 2017 and 2018. Phylogenetic analysis revealed that the circulating genotypes of RSV were GA2 (16/16), of RSV-A, and GB5 (23/23) of RSV-B in the South, North, and Northeast region of India during the period between 2016 and 2018. Here we report the first study comprising the distribution of RSV-A and B genotypes in the different geographic regions of India among children and adults during the year 2016 to 2018. We also report GA2.3.7 lineage of GA2 genotype for the first time in India to the best of our knowledge.

Antibody development for preventing the human respiratory syncytial virus pathology.

Human respiratory syncytial virus (hRSV) is the most important etiological agent causing hospitalizations associated with respiratory diseases in children under 5 years of age as well as the elderly, newborns and premature children are the most affected populations. This viral infection can be associated with various symptoms, such as fever, coughing, wheezing, and even pneumonia and bronchiolitis. Due to its severe symptoms, the need for mechanical ventilation is not uncommon in clinical practice. Additionally, alterations in the central nervous system -such as seizures, encephalopathy and encephalitis- have been associated with cases of hRSV-infections. Furthermore, the absence of effective vaccines or therapies against hRSV leads to elevated expenditures by the public health system and increased mortality rates for the high-risk population. Along these lines, vaccines and therapies can elicit different responses to this virus. While hRSV vaccine candidates seek to promote an active immune response associated with the achievement of immunological memory, other therapies -such as the administration of antibodies- provide a protective environment, although they do not trigger the activation of the immune system and therefore do not promote an immunological memory. An interesting approach to vaccination is the use of virus-neutralizing antibodies, which inhibit the entry of the pathogen into the host cells, therefore impairing the capacity of the virus to replicate. Currently, the most common molecule targeted for antibody design against hRSV is the F protein of this virus. However, other molecular components of the virus -such as the G or the N hRSV proteins- have also been explored as potential targets for the control of this disease. Currently, palivizumab is the only monoclonal antibody approved for human use. However, studies in humans have shown a protective effect only after the administration of at least 3 to 5 doses, due to the stability of this vaccine. Furthermore, other studies suggest that palivizumab only has an effectiveness close to 50% in high-risk infants. In this work, we will review different strategies addressed for the use of antibodies in a prophylactic or therapeutic context and their ability to prevent the symptoms caused by hRSV infection of the airways, as well as in other tissues such as the CNS.

Molecular Profiling of Inflammatory Mediators in Human Respiratory Syncytial Virus and Human Bocavirus Infection.

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.

Development and Validation of Rapid In-House Diagnostic ELISA Kits for Detection of Human Orthopneumovirus in Clinical Samples.

Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) > indirect antigen-capture ELISA (90.6%) > direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections.

Full Genome Characterization of Respiratory Syncytial Virus Causing a Fatal Infection in an Immunocompromised Patient in Tunisia.

Human orthopneumovirus (HRSV) is a virus belonging to the Pneumovirus genus that causes lower respiratory tract infections (LRTI) in infants worldwide. In Tunisia, thousands of infants hospitalized for LRTI are found to be positive for HRSV but no whole genome sequences of HRSV strains circulating in this country are available thus far. In this study, five nasal swab samples collected at different time points from a three-month-old female baby with severe immunodeficiency that was hospitalized for acute bronchiolitis were investigated by next generation sequencing. The Tunisian sequences from this study originated from samples collected in 2021, belong to the ON1 genotype of HRSV-A, and are clustered with European sequences from 2019 and not from 2020 or 2021. This is most likely related to local region-specific transmission of different HRSV-A variants due to the COVID-19 related travel restrictions. Overall, this is the first report describing the whole genome sequence of HRSV from Tunisia. However, more sequence data is needed to better understand the genetic diversity and transmission dynamic of HRSV.

ON-1 and BA-IX Are the Dominant Sub-Genotypes of Human Orthopneumovirus A&B in Riyadh, Saudi Arabia.

Human orthopneumovirus (HOPV) is the major viral pathogen responsible for lower respiratory tract infections (LRTIs) in infants and young children in Riyadh, Saudi Arabia. Yet, predominant HOPV subtypes circulating in this region and their molecular and epidemiological characteristics are not fully ascertained. A total of 300 clinical samples involving nasopharyngeal aspirates (NPAs), throat swabs, and sputum were collected during winter seasons of 2019/2020 and 2021/2022 for HOPV subtyping and genotyping. Of the 300 samples, HOPV was identified in 55 samples (18.3%) with a distinct predominance of type A viruses (81.8%) compared to type B viruses (18.2%). Importantly, the ON1 strain of HOPV-A and BA-IX strain of HOPV-B groups were found to be responsible for all the infections. Sequence analysis revealed a duplication region within 2nd HVR of G protein gene of ON1 and BA-IX strains. This nucleotide duplication exerted a profound effect on protein length and affinity towards cell receptors. Further, these modifications may aid the HOPV in immune evasion and recurrent infections. Data from this study showed that ON-1 genotype of HOPV-A and BA-IX genotype of HOPV-B were dominant in Riyadh, Saudi Arabia. Further, a duplication of sequence within 2nd HVR of G protein gene was found.

Practical Validation of United States Centers for Disease Control and Prevention Assays for the Detection of Human Respiratory Syncytial Virus in Pediatric Inpatients in Japan.

The World Health Organization initiated a global surveillance system for respiratory syncytial virus (RSV) in 2015, and the pilot surveillance is ongoing. The real-time RT-PCR RSV assays (Pan-RSV and duplex assays) developed by the United States Centers for Disease Control and Prevention are applied as the standard assays. To introduce these as standard assays in Japan, their practicality was evaluated using 2261 specimens obtained from pediatric inpatients in Japan, which were collected from 2018 to 2021. Although the Pan-RSV and duplex assays had similar analytical sensitivities, they yielded 630 (27.9%) and 786 (34.8%) RSV-positive specimens, respectively (p < 0.001). Although sequencing analysis showed mismatches in the reverse primer used in the Pan-RSV assay, these mismatches did not affect its analytical sensitivity. The analysis of read numbers of RSV isolates from air−liquid interface culture of human bronchial/tracheal epithelial cells showed that the duplex assay had a greater number of reads than did the Pan-RSV assay. Therefore, the duplex assay has superior detection performance compared with the Pan-RSV assay, but the two assays have similar analytical sensitivities.

An Ultra-Rapid Real-Time RT-PCR Method Using PCR1100 for Detecting Human Orthopneumovirus.

Human orthopneumovirus, also known as the respiratory syncytial virus (RSV), is a leading cause of respiratory tract infections in children worldwide. The World Health Organization has taken steps toward establishing a global surveillance system for RSV, based on the global influenza surveillance and response system initiated in 2015. The US Centers for Disease Control and Prevention (CDC) has developed a genetic detection method based on real-time reverse transcription polymerase chain reaction (RT-PCR), which is used in global RSV surveillance. In Japan, immunoassay-based rapid antigen detection kits are widely used for the detection of RSV. In this study, an ultra-rapid real-time RT-PCR method for the rapid detection of RSV was developed using the PCR1100 device based on the US CDC assay in order to detect RSV in comparable time to rapid test kits. The ultra-rapid real-time RT-PCR could detect RSV viral RNA in less than 20 min while maintaining sensitivity and specificity comparable to conventional real-time RT-PCR using large installed instruments. Furthermore, combining ultra-rapid real-time RT-PCR with the M1 Sample Prep kit reduced the total working time for the detection of RSV from clinical specimen to less than 25 min, suggesting this method could be used for point-of-care RSV testing.

Toward unified molecular surveillance of RSV: A proposal for genotype definition.

BACKGROUND: 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. METHODS: 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. RESULTS: We compared the patterns of sequence clustering of complete genomes; the three surface glycoproteins genes (SH, G, and F, single and concatenated); the ectodomain and the 2nd hypervariable region of G gene. Although complete genome analysis achieved the best resolution, the F, G, and G-ectodomain phylogenies showed similar topologies with statistical support comparable to complete genome. Based on the widespread geographic representation and large number of available G-ectodomain sequences, this region was chosen as the minimum region suitable for RSV genotyping. A genotype was defined as a monophyletic cluster of sequences with high statistical support (≥80% bootstrap and ≥0.8 posterior probability), with an intragenotype p-distance ≤0.03 for both subgroups and an intergenotype p-distance ≥0.09 for RSV-A and ≥0.05 for RSV-B. In this work, the number of genotypes was reduced from 13 to three for RSV-A (GA1-GA3) and from 20 to seven for RSV-B (GB1-GB7). Within these, two additional levels of classification were defined: subgenotypes and lineages. Signature amino acid substitutions to complement this classification were also identified. CONCLUSIONS: We propose an objective protocol for RSV genotyping suitable for adoption as an international standard to support the global expansion of RSV molecular surveillance.

Contribution of Resident Memory CD8+ T Cells to Protective Immunity Against Respiratory Syncytial Virus and Their Impact on Vaccine Design.

Worldwide, human respiratory syncytial virus (RSV) is the most common etiological agent for acute lower respiratory tract infections (ALRI). RSV-ALRI is the major cause of hospital admissions in young children, and it can cause in-hospital deaths in children younger than six months old. Therefore, RSV remains one of the pathogens deemed most important for the generation of a vaccine. On the other hand, the effectiveness of a vaccine depends on the development of immunological memory against the pathogenic agent of interest. This memory is achieved by long-lived memory T cells, based on the establishment of an effective immune response to viral infections when subsequent exposures to the pathogen take place. Memory T cells can be classified into three subsets according to their expression of lymphoid homing receptors: central memory cells (TCM), effector memory cells (TEM) and resident memory T cells (TRM). The latter subset consists of cells that are permanently found in non-lymphoid tissues and are capable of recognizing antigens and mounting an effective immune response at those sites. TRM cells activate both innate and adaptive immune responses, thus establishing a robust and rapid response characterized by the production of large amounts of effector molecules. TRM cells can also recognize antigenically unrelated pathogens and trigger an innate-like alarm with the recruitment of other immune cells. It is noteworthy that this rapid and effective immune response induced by TRM cells make these cells an interesting aim in the design of vaccination strategies in order to establish TRM cell populations to prevent respiratory infectious diseases. Here, we discuss the biogenesis of TRM cells, their contribution to the resolution of respiratory viral infections and the induction of TRM cells, which should be considered for the rational design of new vaccines against RSV.