Respiratory Syncytial Virus Gene Research Articles

Pathogenetic investigation of an outbreak of upper respiratory tract infection in a kindergarten in Baiyin City, Gansu Province

This study focuses on the cases(mainly characterized by respiratory symptoms such as cough, runny nose, fever, sore throat, and nasal congestion)of an outbreak of upper respiratory tract infections in a kindergarten in Jingyuan County, Baiyin City, Gansu Province, in May 2023. The epidemiological data were collected, and pharyngeal swab specimens were also obtained from the patients. The specimens of the research participants were subjected to respiratory multi-pathogen testing, and the positive specimens were further analyzed by sequencing the second hypervariable region (HRV2) of the G gene of respiratory syncytial virus (RSV) and constructing a phylogenetic tree. A total of 90 patients were collected, with an incidence rate of 22.84% (90/394), and the highest incidence was observed in the junior class group at 29.55%. Among the 17 pharyngeal swab specimens collected, 16 specimens were identified with the A subtype of respiratory syncytial virus. Sequencing analysis confirmed that it was the A subtype ON1 genotype. Based on the aforementioned testing results, it can be concluded that the current epidemic was primarily caused by infection with the A subtype of respiratory syncytial virus. Following the implementation of intervention measures, the epidemic has been effectively controlled.

Identification of distinct genotypes in circulating RSV A strains based on variants in the virus replication-associated genes.

Respiratory syncytial virus (RSV) is a common cause of respiratory infection that often leads to hospitalization of infected younger children and older adults. RSV is classified into two strains, A and B, each with several subgroups or genotypes. One issue with the definition of these subgroups is the lack of a unified method of identification or genotyping. We propose that genotyping strategies based on the genes coding for replication-associated proteins could provide critical information on the replication capacity of the distinct subgroups, while clearly distinguishing genotypes. Here, we analyzed the virus replication-associated genes N, P, M2, and L from de novo assembled RSV A sequences obtained from 31 newly sequenced samples from hospitalized patients in Philadelphia and 78 additional publicly available sequences from different geographic locations within the United States. In-depth analysis and annotation of variants in the replication-associated proteins identified the polymerase protein L as a robust target for genotyping RSV subgroups. Importantly, our analysis revealed non-synonymous variations in L that were consistently accompanied by conserved changes in its co-factor P or the M2-2 protein, suggesting associations and interactions between specific domains of these proteins. Similar associations were seen among sequences of the related human metapneumovirus. These results highlight L as an alternative to other RSV genotyping targets and demonstrate the value of in-depth analyses and annotations of RSV sequences as it can serve as a foundation for subsequent in vitro and clinical studies on the efficiency of the polymerase and fitness of different virus isolates.IMPORTANCEGiven the historical heterogeneity of respiratory syncytial virus (RSV) and the disease it causes, there is a need to understand the properties of the circulating RSV strains each season. This information would benefit from an informative and consensus method of genotyping the virus. Here, we carried out a variant analysis that shows a pattern of specific variations among the replication-associated genes of RSV A across different seasons. Interestingly, these variation patterns, which were also seen in human metapneumovirus sequences, point to previously defined interactions of domains within these genes, suggesting co-variation in the replication-associated genes. Our results also suggest a genotyping strategy that can prove to be particularly important in understanding the genotype-phenotype correlation in the era of RSV vaccination, where selective pressure on the virus to evolve is anticipated. More importantly, the categorization of pneumoviruses based on these patterns may be of prognostic value.

Molecular epidemiology and characteristics of respiratory syncytial virus among hospitalized children in Guangzhou, China

BackgroundHuman respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection and hospitalization, especially in children. Highly mutagenic nature and antigenic diversity enable the RSV to successfully survive in human population. We conducted a molecular epidemiological study during 2017–2021 to investigate the prevalence and genetic characteristics of RSV.MethodsA total of 6499 nasopharyngeal (NP) swabs were collected from hospitalized children at Department of Pediatrics, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong, China. All NP swab specimens were preliminary screened for common respiratory viruses and then tested for RSV using specific PCR assays. Partial G genes of RSV were amplified for phylogenetic analysis and genetic characterization.ResultsThe overall detection rate for common respiratory viruses was 16.12% (1048/6499). Among those, 405 specimens (6.20%, 405/6499) were found positive for RSV. The monthly distribution of RSV and other respiratory viruses was variable, and the highest incidence was recorded in Autumn and Winter. Based on the sequencing of hypervariable region of G gene, 93 RSV sequences were sub-grouped into RSV-A (56, 60.2%) and RSV-B (37, 39.8%). There was no coinfection of RSV-A and RSV-B in the tested samples. Phylogenetic analysis revealed that RSV-A and RSV-B strains belonged to ON1 and BA9 genotypes respectively, indicating predominance of these genotypes in Guangzhou. Several substitutions were observed which may likely change the antigenicity and pathogenicity of RSV. Multiple glycosylation sites were noticed, demonstrating high selection pressure on these genotypes.ConclusionThis study illustrated useful information about epidemiology, genetic characteristics, and circulating genotypes of RSV in Guangzhou China. Regular monitoring of the circulating strains of RSV in different parts of China could assist in the development of more effective vaccines and preventive measures.

Reference material development for detection of human respiratory syncytial virus using digital PCR.

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) × 104 copies/μL for RSV-A and (5.3 ± 1.2) × 104 copies/μL for RSV-B. The developed RMs can be used as standards to evaluate the performance of RSV detection assays.

Detection of Respiratory Syncytial Virus in Suspected Asthmatic Children by Conventional RT-PCR Assays in Kathmandu

Respiratory Syncytial Virus (RSV) is the most common causative agent for lower respiratory tract infections in children particularly in infants. At least 50% of children are infected with RSV by the age of 2 to 3 years of age. However, the diagnosis of the RSV infection and its association with risk factors for asthma in asthmatic children is not studied in Nepal. The objectives of this study were to determine the prevalence of RSV in asthmatic children. This work was a hospital based cross-sectional prospective study and conducted from September 2017 to February 2018. Ethical approval was obtained from the Institutional Review Committee (IRC) of Kanti Children’s Hospital, Maharajgunj. Suspected asthmatic children visiting the Special Asthma OPD of Kanti Children’s Hospital, Maharajgunj were selected with the help of asthma specialist using a set of inclusion criteria for asthma. A total of 32 nasal swab samples were obtained from suspected asthmatic children. Initially, samples were processed for RNA extraction. The extracted RNA was then used for cDNA synthesis followed by PCR using primers for the Nucleocapsid (N) gene of RSV. Out of total 32 samples, 9 (28%) samples were positive for the N gene of RSV. There was no significant association of different variables including age (P=0.187), sex (P=0.264), family history of asthma (P=0.115), passive smoking (P=0.88), birth weight (P=0.954), seasonality (P=0.298) and history of pneumonia (P=0.457) with the RSV infection in this study.

The protective immunity induced by intranasally inoculated serotype 63 chimpanzee adenovirus vector expressing human respiratory syncytial virus prefusion fusion glycoprotein in BALB/c mice.

Human respiratory syncytial virus (RSV) is a ubiquitous pediatric pathogen causing serious lower respiratory tract disease worldwide. No licensed vaccine is currently available. In this work, the coding gene for mDS-Dav1, the full-length and prefusion conformation RSV fusion glycoprotein (F), was designed by introducing the stabilized prefusion F (preF) mutations from DS-Cav1 into the encoding gene of wild-type RSV (wtRSV) F protein. The recombinant adenovirus encoding mDS-Cav1, rChAd63-mDS-Cav1, was constructed based on serotype 63 chimpanzee adenovirus vector and characterized in vitro. After immunizing mice via intranasal route, the rChAd63-mDS-Cav1 induced enhanced neutralizing antibody and F-specific CD8+ T cell responses as well as good immune protection against RSV challenge with the absence of enhanced RSV disease (ERD) in BALB/c mice. The results indicate that rChAd63-mDS-Cav1 is a promising mucosal vaccine candidate against RSV infection and warrants further development.

Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.

A multi-center study to determine genetic variations in the fusion gene of respiratory syncytial virus (RSV) from children

The fusion (F) protein of respiratory syncytial virus (RSV) is the major target of immunoprophylactic monoclonal antibodies (mAbs) and vaccines. Recently reported mutations in F gene antigenic sites can vary among RSV types A and B. To further understand mutations in RSV F proteins, we performed subtyping and F gene sequencing on 400 RSV-positive respiratory samples collected at four pediatric hospitals within the United States from children under 2 years old between 2018 and 2020. RSV B was predominant in 2018-2019 and RSV A in 2019-2020 (55.5% and 85.5% respectively). Compared to the reference sequence, all RSV B samples had at least one antigenic polymorphism with the most changes at sites AM14/V (100%) and Ø (93.3%) followed by II (5.8%), IV (3.9%), and p27 (2.9%). The most frequent mutations among RSV B for AM14/V site were in L172Q (100%), S173L (100%), and K191R (95.2%) while for Ø site they were in I206M (93.3%) and Q209R (93.3%). Conversely, polymorphisms were observed in only 15.3% of RSV A samples overall, specifically at antigenic sites p27 (5.9%), IV (3.0%), II (2.5%), AM14/V (2.0%), I (2.0%), and Ø (0.5%). Among RSV A cases, T122A at p27 (n=10) and S276N at II (n=3) were the most common substitution sites. S276N at site II was found in both RSV types. Although polymorphisms in F proteins of RSV B were more common than those in RSV A samples, changes in both subtypes were observed in key F antigenic sites which could potentially impact the efficacy of mAb therapies and vaccines.

Genome analysis of human respiratory syncytial virus in Fujian Province, Southeast China.

Respiratory syncytial virus (RSV) is one of the main causes of acute respiratory infections (ARI)leading to a heavy disease burden. Reports on RSV in China are limited, especially in Fujian Province, and RSV whole-genome sequences in Fujian Province are not reported. This study aimed to explore the genomic characteristics of RSV to provide evidence for the development of vaccines and medicines. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify and obtain the attachment (G) gene and whole-genome fragments from the positive samples. Genetic evolution as well as nucleotide and amino acid homology analyses of the virus sequences were conducted to identify any amino acid mutations. A total of 72 RSV-positive cases were collected, and 53G gene sequences were obtained using polymerase chain reaction (PCR) amplification. The ON1 and BA11 genotypes were found to be dominant using the Basic Local Alignment Search Tool (BLAST) on the NCBI website. The 40 genotype ON1 sequences had high nucleotide identity (95.3%-99.8%) and amino acid similarity (92.5%-100%), whereas the 13 BA11 genotype sequenceshad 97.3%-99.6% nucleotide identity and 94.8%-99.7% amino acid similarity. Compared to the ON1 prototype (JN257693) and BA11 prototype (AY333364), the obtained sequences had no nucleotide insertions or deletions, indicating high similarity among the samples. A total of 17 RSV whole genome sequences were obtained, 10 of which were genotype ON1 and seven were genotype BA11. Certain amino acid mutations were found in the antigen site and epitope of the fusion (F) protein but not in the G protein. Glycosylation analyses of specific RSV genes revealed high positive selection rates for the gene, and the N- and O-linked glycosylation sequences in the F gene were relatively conserved. From July 2018 to January 2020, ON1 and BA11 were the most prevalent RSV genotypes in Fujian Province. A high nucleotide identity and amino acid similarity were observed between the reference strain and the obtained strains, as well as among the sequences of the obtained isotypes. Certain amino acid mutations occur at the antigen site and the epitope of the F protein.

Novel and extendable genotyping system for human respiratory syncytial virus based on whole-genome sequence analysis.

BackgroundHuman 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.MethodsWe 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.ResultsIn this study, we first statistically examined the phylogenetic incongruence, rate variation for each RSV gene sequence and WGS. We then proposed a new RSV genotyping system based on a comparative analysis of WGS and the temporal distribution of strains. We also provide an RSV classification tool to perform RSV genotype assignment and a publicly accessible up‐to‐date instance of Nextstrain where the phylogenetic relationship of all genotypes can be explored.ConclusionsThis revised RSV genotyping system will provide important information for disease surveillance, epidemiology, and vaccine development.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes.

RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

Inhibitory Effect of Sargassum fusiforme and Its Components on Replication of Respiratory Syncytial Virus In Vitro and In Vivo.

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

Internal N6-methyladenosine modification controls the innate immune response to viral RNA

Abstract Internal N6-methyladenosine (m6A) modification is one of the most prevalent and abundant modifications of RNA. The m6A methylation is catalyzed by host methyltransferases (METTL3 and METTL14) and can be reversibly removed by host demethylases (FTO and ALKBH5). Recent studies have revealed that RNA of many DNA and RNA viruses contain m6A methylation and that viral m6A can play a pro-viral or anti-viral role. However, the biological functions of viral RNA m6A methylation remain poorly understood. In this study, we found that genome and antigenome of human respiratory syncytial virus (RSV), a non-segmented negative-sense RNA virus, are m6A methylated and that viral m6A methylation positively regulates RSV replication and gene expression. Interestingly, virion RNAs purified from RSV grown in METTL3-knockout cells induced significantly higher type I interferon responses than virion RNAs purified from RSV grown in wild type cells, demonstrating that viral RNA m6A methylation inhibits innate immunity. Similarly, inactivation of m6A methylation in RSV genome and/or antigenome by site-directed mutagenesis induced significantly higher innate immune responses than wild type RSV RNA. Mechanistically, these m6A-unmodified or m6A-deficient RSV RNAs trigger a higher expression of pattern recognition receptors (such as RIG-I and MDA5), enhance their binding affinity to the innate immune RNA sensors, and enhance the phosphorylation of downstream transcription factor IRF3, leading to an enhanced activation of type I interferon signaling pathway. Taken together, our study identified a novel biological function of viral m6A methylation, which is to serves as a molecular marker for host innate immunity to discriminate self from nonself RNAs.

Non-gradient and genotype-dependent patterns of RSV gene expression.

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand RNA virus (NSV) and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3' promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription extending across the genome, with the highest level of mRNA coming from the most promoter-proximal gene, the first nonstructural (NS1) gene, and mRNA levels from subsequent genes dropping until reaching a minimum at the most promoter-distal gene, the polymerase (L) gene. However, recent reports show non-gradient levels of mRNA, with higher than expected levels from the attachment (G) gene. It is unknown to what extent different transcript stabilities might shape measured mRNA levels. It is also unclear whether patterns of RSV gene expression vary, or show strain- or genotype-dependence. To address this, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and in cotton rats infected with RSV isolates belonging to four genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were non-gradient and genotype-specific, where mRNA levels from the G gene exceeded those from the more promoter-proximal nucleocapsid (N) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. Our results indicate that gene expression from a small but diverse set of RSV genotypes is non-gradient and genotype-dependent. We propose novel models of RSV transcription that can account for non-gradient transcription.

Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

Anti-Respiratory Syncytial Virus Activity of Plantago asiatica and Clerodendrum trichotomum Extracts In Vitro and In Vivo.

The herbs Plantago asiatica and Clerodendrum trichotomum have been commonly used for centuries in indigenous and folk medicine in tropical and subtropical regions of the world. In this study, we show that extracts from these herbs have antiviral effects against the respiratory syncytial virus (RSV) in vitro cell cultures and an in vivo mouse model. Treatment of HEp2 cells and A549 cells with a non-cytotoxic concentration of Plantago asiatica or Clerodendrum trichotomum extract significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and also blocked syncytia formation. Interestingly, oral inoculation with each herb extract significantly improved viral clearance in the lungs of BALB/c mice. Based on reported information and a high-performance liquid chromatography (HPLC) analysis, the phenolic glycoside acteoside was identified as an active chemical component of both herb extracts. An effective dose of acteoside exhibited similar antiviral effects as each herb extract against RSV in vitro and in vivo. Collectively, these results suggest that extracts of Plantago asiatica and Clerodendrum trichotomum could provide a potent natural source of an antiviral drug candidate against RSV infection.

Parainfluenza virus 5 (PIV5) amplifying virus-like particles expressing respiratory syncytial virus (RSV) antigens protect mice against RSV infection

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in children under one year of age. In addition to causing severe respiratory diseases in children, it is also a major cause of morbidity and mortality among the elderly and immunocompromised individuals. RSV is the most common cause of lower respiratory tract infections, yet there are currently no licensed vaccines. A parainfluenza virus 5 (PIV5)-based amplifying virus-like particle (AVLP), which enables the use of PIV5 RNA transcription/replication machinery to express gene of interest, has recently been developed. We evaluated the PIV5-based AVLP system as a vaccine platform for RSV by incorporating the fusion protein (F) gene and the transcription factor protein (M2-1) gene of RSV into the PIV5-AVLP backbone (AVLP-F and AVLP-M2-1, respectively). Mice immunized with a single dose of the AVLP-F or AVLP-M2-1 developed RSV-F or RSV-M2-1-specific immune responses, respectively. Both vaccine candidates elicited antigen-specific cell-mediated responses at levels comparable to or higher than an RSV infection. Most importantly, each vaccine was able to induce protection against RSV A2 challenge in the mouse model. These results indicate the potential of the PIV5-based AVLP system as a platform for vaccines against RSV infection.

Development of Luciferase Immunoprecipitation Systems (LIPS) Assay to Detect IgG Antibodies against Human Respiratory Syncytial Virus G-Glycoprotein.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants and the elderly. Although there is no licensed vaccine, RSV-F and -G glycoproteins are targets for vaccine development and therapeutics. We developed an assay that can detect anti-RSV-G IgG antibodies, either as a biomarker of natural exposure or immunization. RSV genes encoding native and mutated G (mG) proteins from subgroups A and B strains were cloned, expressed as luciferase-tagged proteins, and tested individually to detect anti-RSV-G specific IgG antibodies using a high-throughput luciferase immunoprecipitation system (LIPS-G). RSV monoclonal antibodies and polyclonal antisera specifically bound in the LIPS-GA and/or -GB assays; whereas anti-RSV-F and -N, and antisera against measles virus or human metapneumovirus did not bind. Anti-RSV-GA and -GB IgG responses detected in mice infected intranasally with RSV-A or -B strains were subtype specific. Subtype specific anti-RSV-GA or -GB IgG responses were also detected using paired serum samples from infants while human adolescent serum samples reacted in both LIPS-GA and -GB assays, reflecting a broader experience.

Analysis of genetic variability of respiratory syncytial virus groups A and B in Kuwait.

Respiratory syncytial virus (RSV) is the most frequently identified viral agent in infants, children, and elderly people with acute respiratory tract infections (ARTIs). This study is the only one of its kind in Kuwait, and its purpose was to investigate the genetic variability of the G protein gene in RSV strains prevalent in Kuwait. Respiratory samples were collected from patients with ARTIs in various hospitals in Kuwait and subjected to reverse transcription PCR (RT-PCR) amplifying a fragment of the G gene of RSV. A total of 305 samples were collected between January and mid-December 2016, and 77 (25.2%) were positive for RSV. Group A viruses were predominant over group B viruses; the RSV-A group was detected in 52 (67.5%) of the positive samples, while the RSV-B group was detected in 25 (32.5%) of the positive samples. Phylogenetic analysis showed that all RSV-A strains grouped into eight clusters of identical sequences of untyped strains. Twelve RSV-B strains, on the other hand, belonged to the RSV-B/BA10 genotype, while the rest were untyped. These data suggest that new and untyped strains of RSV-A group likely predominated in Kuwait and that the BA10 genotype of the RSV-B group became the dominant genotype in the 2016 season.