Respiratory Syncytial Virus Genotypes Research Articles

Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008-2017.

In this study, we investigated the molecular evolution and phylodynamics of respiratory syncytial virus (RSV) over 10 consecutive seasons (2008–2017) and the genetic variability of the RSV genotypes ON1 and BA in central Taiwan. The ectodomain region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the second hypervariable region of the G protein in RSV ON1 and BA were analyzed. A total of 132 RSV-A and 81 RSV-B isolates were obtained. Phylogenetic analysis revealed that the NA1, ON1, and BA9 genotypes were responsible for the RSV epidemics in central Taiwan in the study period. For RSV-A, the NA1 genotype predominated during the 2008–2011 seasons. The ON1 genotype was first detected in 2011 and replaced NA1 after 2012. For RSV-B, the BA9 and BA10 genotypes cocirculated from 2008 to 2010, but the BA9 genotype has predominated since 2012. Amino acid sequence alignments revealed the continuous evolution of the G gene in the ectodomain region. The predicted N-glycosylation sites were relatively conserved in the ON1 (site 237 and 318) and BA9 (site 296 and 310) genotype strains. Our results contribute to the understanding and prediction of the temporal evolution of RSV at the local level.

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.

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.

Determination of genetic characterization and circulation pattern of Respiratory Syncytial Virus (RSV) in children with a respiratory infection, Tehran, Iran, during 2018-2019

The RSV-associated disease accounts for a significant health burden particularly in infants and young children who need to be hospitalized. Since continuous surveillance of circulating RSV genotypes is crucial worldwide, this study aimed to investigate the genetic diversity of RSV circulating strains causing upper or lower acute respiratory infection. Our attention was geared towards studying the cases hospitalized or outpatient in children younger than 2 years of age in Iran during 2018/2019. In this study, nasopharyngeal swabs collected from 206 children who presented with respiratory infection symptoms, were admitted to the referral pediatric ward of Bahrami children's hospital in Tehran, Iran. RSV-positive samples were detected via Nested RT-PCR. The glycoprotein gene was sequenced, and virus genotypes were confirmed through phylogenetic analysis by the MEGA X program. A total of 74 (35.92%) samples tested positive for RSV. Among them, sequencing was done in 10 specimens from 2018 (RSV-A: RSV-B=4:6) and 19 specimens from 2019 (RSV-A: RSV-B=16:3). According to phylogenetic analysis, all RSV-A strains were assigned as ON1 genotype and RSV-B strains were assigned as BA9 genotype. A new N-glycosylation site in Iranian BA9 and positive selection in ON1 genotype was observed. Phylogenetic characterization of strains in the current study revealed co-circulation of ON1 and BA9 as the only prevalent genotypes of both RSV-A and –B groups.

Genotype and genetic characteristics of human respiratory syncytial virus circulating in Quanzhou, 2018-2019

Objective: To study the prevalence and genetic characteristics of human respiratory syncytial virus (HRSV) in Quanzhou city, from 2018 to 2019. Methods: A total of 141 throat swabs were collected from children patients of lower respiratory tract infection in Quanzhou children Hospital, Fujian Province from November 2018 to May 2019. RT-PCR was used to amplify the 3 'end of G gene HRSV. Sequencer 5.0 and MEGA5.05 softwares were used for sequence editing, phylogenetic tree construction and genotyping analysis. Results: Twenty-five samples were positive for HRSV. Seventeen samples succeeded to obtain the target gene, including 13 of HRSVA and 4 of HRSVB. Two genotypes were identified: ON1 genotype (13 samples, HRSVA) and BA9 genotype (4 samples, HRSVB). Five strains of ON1 genotype sequences were clustered with the ON1 sequences prevalent in Beijing, Changchun and Zhejiang from 2012 to 2015 (cluster1); one strain (FJ19-02) was clustered with the sequences of ON1 genotype circulating in many regions of China from 2012 to 2015 (cluster2); Seven strains were clustered independently (cluster FJ). FJ18-02, FJ19-14 and FJ19-15 of HRSVB were clustered with the BA9 genotype sequences prevalent in Changchun, Jilin Province in 2015, while FJ19-13 was closely related to the BA9 genotype sequences prevalent in Guangzhou and Zhejiang Province in 2013. Both the ON1 and BA9 genotypes showed variations of nucleotide and amino acid in 72 and 60 insertion segments. Amino acid mutation (H266L) only occurred among the sequence of cluster-FJ, and the mutations of H261Q and Q265L only appeared in strain FJ19-13. Conclusion: BA9 and ON1 genotypes were prevalent in Quanzhou city, from 2018 to 2019. Cluster-FJ was a newly discovered independent transmission chain, which may continue to circulate in local Quanzhou area.

Transmission of Respiratory Syncytial Virus genotypes in Cali, Colombia.

BackgroundColombia's climatological variety, added to pathogen diversity, creates local niches for infectious diseases. In Bogotá, respiratory syncytial virus causes 30%‐52% of the cases of respiratory infections. In coastal or inter‐Andean cities with higher temperature and longer dry seasons, frequency of this virus is 7%‐13%. By 2017, increased hospitalizations due to airway infections occurred in regions whose weather is differently influenced by “El Niño Southern Oscillation” than in Bogotá, although microbial diversity might have also been involved.MethodsFor Cali, an inter‐Andean city with warm tropical weather, records of respiratory syncytial virus from 2014 to 2018, in children two years old or younger, were analyzed, and genotypes transmitted during 2016‐2017 were identified based on partial sequences of glycoprotein G.ResultsMost cases of respiratory syncytial virus in Cali occur in the first semesters, with peaks expressed around March‐April, without a clear association with pluviosity. Unlike the biannual rotating pattern of Bogotá, co‐circulation of types A and B was detected. As years pass, transmission seasons are becoming longer and frequencies of the virus augment. The viral genotypes identified follow international trends with dominance of Ontario and Buenos Aires clades. Similar to other isolates in these clades, viruses from Cali exhibit glycosylation variability that may account for their fitness.ConclusionsThe pattern of respiratory syncytial virus transmission in Cali differs from that in Bogotá. Its epidemiology is shifting and will remain so with the advent of novel respiratory diseases. This may impact the introduction of vaccination schemes for these or other respiratory viruses.

Molecular epidemiology of Respiratory Syncytial Virus in children in sub‐Saharan Africa

This study investigated the molecular epidemiology of respiratory syncytial virus (RSV) among febrile children with acute respiratory tract infection in Ghana, Gabon, Tanzania and Burkina Faso between 2014-2017 as well as the evolution and diversification of RSV strains from other sub-Saharan countries. Pharyngeal swabs were collected at four study sites (Agogo, Ghana: n=490; Lambaréné, Gabon: n=182; Mbeya, Tanzania: n=293; Nouna, Burkina Faso: n=115) and analysed for RSV and other respiratory viruses using rtPCR. For RSV positive samples, sequence analysis of the second hypervariable region of the G-gene was performed. A dataset of RSV strains from sub-Saharan Africa (2011-2017) currently available in GenBank was compiled. Phylogenetic analysis was conducted to identify the diversity of circulating RSV genotypes. In total, 46 samples tested RSV positive (Ghana n=31 (6.3%), Gabon n=4 (2.2%), Tanzania n=9 (3.1%), Burkina Faso n=2 (1.7%)). The most common RSV co-infection was with rhinovirus. All RSV-A strains clustered with genotype ON1 strains with a 72-nucleotide duplication and all RSV-B strains belonged to genotype BAIX. Phylogenetic analysis of amino acid sequences from sub-Saharan Africa revealed the diversification into 11 different ON1 and 22 different BAIX lineages and differentiation of ON1 and BAIX strains into potential new sub-genotypes, provisionally named ON1-NGR, BAIX-KEN1, BAIX-KEN2 and BAIX-KEN3. The study contributes to an improved understanding of the molecular epidemiology of RSV infection in sub-Saharan Africa. It provides the first phylogenetic data for RSV from Tanzania, Gabon and Burkina Faso and combines it with RSV strains from all other sub-Saharan countries currently available in GenBank.

Molecular epidemiology of respiratory syncytial virus in children in sub-Saharan Africa.

This study investigated the molecular epidemiology of respiratory syncytial virus (RSV) among febrile children with acute respiratory tract infection in Ghana, Gabon, Tanzania and Burkina Faso between 2014 and 2017 as well as the evolution and diversification of RSV strains from other sub-Saharan countries. Pharyngeal swabs were collected at four study sites (Agogo, Ghana: n=490; Lambaréné, Gabon: n=182; Mbeya, Tanzania: n=293; Nouna, Burkina Faso: n=115) and analysed for RSV and other respiratory viruses using rtPCR. For RSV-positive samples, sequence analysis of the second hypervariable region of the G gene was performed. A dataset of RSV strains from sub-Saharan Africa (2011-2017) currently available in GenBank was compiled. Phylogenetic analysis was conducted to identify the diversity of circulating RSV genotypes. In total, 46 samples were tested RSV positive (Ghana n=31 (6.3%), Gabon n=4 (2.2%), Tanzania n=9 (3.1%) and Burkina Faso n=2 (1.7%)). The most common RSV co-infection was with rhinovirus. All RSV A strains clustered with genotype ON1 strains with a 72-nucleotide duplication and all RSV B strains belonged to genotype BAIX. Phylogenetic analysis of amino acid sequences from sub-Saharan Africa revealed the diversification into 11 different ON1 and 22 different BAIX lineages and differentiation of ON1 and BAIX strains into potential new sub-genotypes, provisionally named ON1-NGR, BAIX-KEN1, BAIX-KEN2 and BAIX-KEN3. The study contributes to an improved understanding of the molecular epidemiology of RSV infection in sub-Saharan Africa. It provides the first phylogenetic data for RSV from Tanzania, Gabon and Burkina Faso and combines it with RSV strains from all other sub-Saharan countries currently available in GenBank.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Evolution of respiratory syncytial virus genotype BA in Kilifi, Kenya, 15\xa0years on

Respiratory syncytial virus (RSV) is recognised as a leading cause of severe acute respiratory disease and deaths among infants and vulnerable adults. Clinical RSV isolates can be divided into several known genotypes. RSV genotype BA, characterised by a 60-nucleotide duplication in the G glycoprotein gene, emerged in 1999 and quickly disseminated globally replacing other RSV group B genotypes. Continual molecular epidemiology is critical to understand the evolutionary processes maintaining the success of the BA viruses. We analysed 735 G gene sequences from samples collected from paediatric patients in Kilifi, Kenya, between 2003 and 2017. The virus population comprised of several genetically distinct variants (n = 56) co-circulating within and between epidemics. In addition, there was consistent seasonal fluctuations in relative genetic diversity. Amino acid changes increasingly accumulated over the surveillance period including two residues (N178S and Q180R) that mapped to monoclonal antibody 2D10 epitopes, as well as addition of putative N-glycosylation sequons. Further, switching and toggling of amino acids within and between epidemics was observed. On a global phylogeny, the BA viruses from different countries form geographically isolated clusters suggesting substantial localized variants. This study offers insights into longitudinal population dynamics of a globally endemic RSV genotype within a discrete location.

Genetic Characterization of G Protein in Respiratory Syncytial Virus ON-1 Genotype in Tehran

Aim: We investigated the genetic characterization of the respiratory syncytial virus (RSV) ON-1 genotypes and their different lineages based on the G gene among children <2 years of age presenting with acute respiratory tract infections in Tehran, Iran. Materials & methods: A phylogenetic tree from the Iranian samples and ON-1 strains of various parts of the world were constructed. The amino acid composition of the RSV G protein of the ON-1 genotype was mapped. Results: Human RSV ON-1 genotypes from the Iranian samples clustered in three lineages. The most common amino acid substitutions were as follows: X218Q, I240S, L289P, Y304H and L310P. Conclusion: Continuing molecular epidemiological surveys in other regions of Iran will provide deeper insight into the nature of this replacement of the dominant RSV genotype from GA2 to ON-1 in Iran.

Predominance of ON1 and BA9 genotypes of respiratory syncytial virus (RSV) in Bulgaria, 2016-2018.

The objectives of this study were to investigate the prevalence of respiratory syncytial virus (RSV) infections in Bulgaria, to characterize the genetic diversity of the RSV strains, and to perform amino acid sequence analysis of the RSV G protein. Clinical, epidemiological data and nasopharyngeal swabs were prospectively collected from children aged less than 5 years presenting with acute respiratory infections from October 2016 to September 2018. Real-time polymerase chain reaction for 12 respiratory viruses, and sequencing, phylogenetic, and amino acid analyses of the RSV G gene/protein were performed. Of the 875 children examined, 645 (73.7%) were positive for at least one viral respiratory pathogen. RSV was the most commonly detected virus (26.2%), followed by rhinoviruses (15%), influenza A (H3N2) (9.7%), adenoviruses (9%), bocaviruses (7.2%), human metapneumovirus (6.1%), parainfluenza viruses 1/2/3 (5.8%), influenza type B (5.5%), and A(H1N1)pdm09 (3.4%). The detection rate for RSV varied across two winter seasons (36.7% vs 20.3%). RSV-B cases outnumbered those of the RSV-A throughout the study period. RSV was the most common virus detected in patients with bronchiolitis (45.1%) and pneumonia (24%). Phylogenetic analysis indicated that all the sequenced RSV-A strains belonged to the ON1 genotype and the RSV-B strains were classified as BA9 genotype. Amino acid substitutions at 15 and 22 positions of the HVR-2 were identified compared with the ON1 and BA prototype strains,respectively. This study revealed the leading role of RSV as a causative agent of serious respiratory illnesses in early childhood, year-on-year fluctuations in RSV incidence, the dominance of RSV-B, and relatively low genetic diversity in the circulating RSV strains.

Molecular epidemiology of human respiratory syncytial virus (HRSV) in Iranian military trainees with acute respiratory symptoms in 2017.

Background and Objectives:Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in many populations, including military recruits receiving basic training. Therefore, this study was set out to determine the molecular epidemiology, genotype and phylogenetic features of RSVs in patients with respiratory infection as a case study.Materials and Methods:In this study, military barracks of Tehran, Iran, between January to March 2017 exposed to respiratory diseases were used for sampling. Throat swabs were taken, a reverse transcriptase-polymerase chain reaction (RTPCR) assay was performed to identify RSV and then the genotyping and phylogenetic analyses of RSVs in patients with a respiratory infection.Results:Among 400 Iranian military trainees with respiratory symptoms, RSV infection was identified in 2.75% (11/400) using RT-PCR. Sequencing showed the incidence of type A (2.5%, n=10) to be much higher than type B (0.25%, n=1); Sore throat was the most common symptom among RSV patients. Phylogenetic analysis revealed that the majority of strains from the studied samples were more consistent with those from the Philippines and the US strains.Conclusion:This study is the first to document RSV as a major cause of acute respiratory illness among military trainees in Iran. The prevalence of RSV is substantial in the cold season and the prevalence of genotype A is dominant in the country, leading to take essential steps in preparing a preventive vaccine against this viral infection.

Molecular characterization of respiratory syncytial virus circulating in Tunisia between 2015 and 2018.

Introduction. Respiratory syncytial virus (RSV) is the most frequently identified viral agent in children with lower respiratory tract infection (LRTI). No data are available to date regarding RSV genotypes circulating in Tunisia.Aim. The aim of the present study was to investigate the genetic variability of the glycoprotein G gene in Tunisian RSV strains.Methodology. Nasopharyngeal aspirates were collected from infants hospitalized for LRTI in five Tunisian hospitals. All specimens were screened for RSV by a direct immunofluorescence assay (DIFA). To molecularly characterize Tunisian RSV strains, a phylogenetic analysis was conducted. Randomly selected positive samples were subjected to reverse transcription PCR amplifying the second hyper-variable region (HVR2) of the G gene.Results. Among a total of 1417 samples collected between 2015 and 2018, 394 (27.8 %) were positive for RSV by DIFA. Analysis of 61 randomly selected RSV strains revealed that group A RSV (78.7 %) predominated during the period of study as compared to group B RSV (21.3 %). The phylogenetic analysis showed that two genotypes of RSV-A were co-circulating: the ON1 genotype with a 72-nt duplication in HVR2 of the G gene was predominant (98.0 % of RSV-A strains), while one RSV-A strain clustered with the NA1 genotype (2.0 %). Concerning Tunisian group B RSV strains, all sequences contained a 60-nt insertion in HVR2 and a clustered BA10 genotype.Conclusion. These data suggest that RSV-A genotype ON1 and RSV-B genotype BA10, both with duplications in the G gene, were widely circulating in the Central coastal region of Tunisia between 2015 and 2018.

Towards a unified classification for human respiratory syncytial virus genotypes.

Since the first human respiratory syncytial virus (HRSV) genotype classification in 1998, inconsistent conclusions have been drawn regarding the criteria that define HRSV genotypes and their nomenclature, challenging data comparisons between research groups. In this study, we aim to unify the field of HRSV genotype classification by reviewing the different methods that have been used in the past to define HRSV genotypes and by proposing a new classification procedure, based on well-established phylogenetic methods. All available complete HRSV genomes (>12,000 bp) were downloaded from GenBank and divided into the two subgroups: HRSV-A and HRSV-B. From whole-genome alignments, the regions that correspond to the open reading frame of the glycoprotein G and the second hypervariable region (HVR2) of the ectodomain were extracted. In the resulting partial alignments, the phylogenetic signal within each fragment was assessed. Maximum likelihood phylogenetic trees were reconstructed using the complete genome alignments. Patristic distances were calculated between all pairs of tips in the phylogenetic tree and summarized as a density plot in order to determine a cutoff value at the lowest point following the major distance peak. 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 HRSV genotypes. We define a genotype using the following criteria: a bootstrap support of 70 per cent for the respective clade and a maximum patristic distance between all members of the clade of ≤0.018 substitutions per site for HRSV-A or ≤0.026 substitutions per site for HRSV-B. By applying this definition, we distinguish twenty-three genotypes within subtype HRSV-A and six genotypes within subtype HRSV-B. Applying the genotype criteria on subsampled data sets confirmed the robustness of the method.

Systematic Review of the Respiratory Syncytial Virus (RSV) Prevalence, Genotype Distribution, and Seasonality in Children from the Middle East and North Africa (MENA) Region.

Respiratory syncytial virus (RSV) is one of the most common viruses to infect children worldwide and is the leading cause of lower respiratory tract illness (LRI) in infants. This study aimed to conduct a systematic review by collecting and reviewing all the published knowledge about the epidemiology of RSV in the Middle East and North Africa (MENA) region. Therefore, we systematically searched four databases; Embase, Medline, Scopus, and Cochrane databases from 2001 to 2019 to collect all the information related to the RSV prevalence, genotype distribution, and seasonality in children in MENA region. Our search strategy identified 598 studies, of which 83 met our inclusion criteria, which cover the past 19 years (2000–2019). Odds ratio (OR) and confidence interval (CI) were calculated to measure the association between RSV prevalence, gender, and age distribution. An overall prevalence of 24.4% (n = 17,106/69,981) of respiratory infections was recorded for RSV. The highest RSV prevalence was reported in Jordan (64%, during 2006–2007) and Israel (56%, 2005–2006). RSV A subgroup was more prevalent (62.9%; OR = 2.9, 95%CI = 2.64–3.13) than RSV B. RSV was most prevalent in children who were less than 12 months old (68.6%; OR = 4.7, 95%CI = 2.6–8.6) and was higher in males (59.6%; OR = 2.17, 95%CI = 1.2–3.8) than in female infants. Finally, the highest prevalence was recorded during winter seasons in all countries, except for Pakistan. RSV prevalence in the MENA region is comparable with the global one (24.4% vs. 22%). This first comprehensive report about RSV prevalence in the MENA region and our data should be important to guide vaccine introduction decisions and future evaluation.

Molecular epidemiology of respiratory syncytial virus for 28 consecutive seasons (1990-2018) and genetic variability of the duplication region in the G gene of genotypes ON1 and BA in South Korea

We investigated the molecular epidemiology of respiratory syncytial virus (RSV) isolated from children during 28 consecutive seasons (1990-2018) and the genetic variability of the duplication region of RSV genotypes ON1 and BA in South Korea. RSV was identified using culture-based methods in Hep-2 cells and was grouped as RSV-A or RSV-B by an immunofluorescence assay. The second hypervariable region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the duplication region of RSV ON1 and BA were analyzed. A total of 670 RSV-A and 233 RSV-B isolates were obtained. For RSV-A, the NA1 genotype predominated during the 2004/2005-2011/2012 seasons. The ON1 genotype was first detected in 2011 and has since replaced all other genotypes. For RSV-B, the GB3 genotype predominated during the 1999/2000-2005/2006 seasons, but the BA genotype also replaced all other genotypes of RSV-B after the first season in which it was isolated (2005/2006). In ON1 and BA genotype RSV strains, novel sequence types of the duplication region of the G gene were identified in 50-95% and 33-80% of the isolates, respectively, in each season. The ON1 and BA9 genotypes are responsible for the current epidemics of RSV infection in South Korea. The sequences in the duplication region of the G gene have evolved continuously and might be sufficient for the identification of specific strains of the RSV-A ON1 and RSV-B BA genotypes.

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.

Circulating Respiratory Syncytial Virus Genotypes and Genetic Variability of the G Gene during 2017 and 2018/2019 Seasonal Epidemics Isolated from Children with Lower Respiratory Tract Infections in Daejeon, Korea

BackgroundRespiratory syncytial virus (RSV) is a major pathogen causing respiratory tract infections in infants and young children. The aim of this study was to confirm the genetic evolution of RSV causing respiratory infections in children at Daejeon in Korea, through G gene analysis of RSV-A and RSV-B strains that were prevalent from 2017 to 2019.MethodsPediatric patients admitted for lower respiratory tract infections at The Catholic University of Korea Daejeon St. Mary's Hospital in the 2017 and 2018/2019 RSV seasonal epidemics, who had RSV detected via multiplex polymerase chain reaction (PCR) were included. The nucleic acid containing RSV-RNA isolated from each of the patients' nasal discharge during standard multiplex PCR testing was stored. The G gene was sequenced and phylogenetic analysis was performed using MEGA X program and the genotype was confirmed.ResultsA total of 155 specimens including 49 specimens from 2017 and 106 specimens from 2018-2019 were tested. The genotype was confirmed in 18 specimens (RSV-A:RSV-B = 4:14) from 2017 and 8 specimens (RSV-A:RSV-B = 7:1) from 2018/2019. In the phylogenetic analysis, all RSV-A type showed ON1 genotype and RSV-B showed BA9 genotype.ConclusionRSV-B belonging to BA9 in 2017, and RSV-A belonging to ON1 genotype in 2018/2019 was the most prevalent circulating genotypes during the two RSV seasons in Daejeon, Korea.

Respiratory syncytial virus A genotype classification based on systematic intergenotypic and intragenotypic sequence analysis

Respiratory syncytial virus (RSV), a leading cause of lower respiratory tract infections, is classified in two major groups (A and B) with multiple genotypes within them. Continuous changes in spatiotemporal distribution of RSV genotypes have been recorded since the identification of this virus. However, there are no established criteria for genotype definition, which affects the understanding of viral evolution, immunity, and development of vaccines. We conducted a phylogenetic analysis of 4,353 RSV-A G gene ectodomain sequences, and used 1,103 complete genome sequences to analyze the totallity of RSV-A genes. Intra- and intergenotype p-distance analysis and identification of molecular markers associated to specific genotypes were performed. Our results indicate that previously reported genotypes can be classified into nine distinct genotypes: GA1-GA7, SAA1, and NA1. We propose the analysis of the G gene ectodomain with a wide set of reference sequences of all genotypes for an accurate genotype identification.