ON1 Genotype Research Articles

Differences in viral load among human respiratory syncytial virus genotypes in hospitalized children with severe acute respiratory infections in the Philippines.

BackgroundHuman respiratory syncytial virus (HRSV) is a leading viral etiologic agent of pediatric lower respiratory infections, including bronchiolitis and pneumonia. Two antigenic subgroups, HRSV-A and B, each contain several genotypes. While viral load may vary among HRSV genotypes and affect the clinical course of disease, data are scarce regarding the actual differences among genotypes. Therefore, this study estimated and compared viral load among NA1 and ON1 genotypes of HRSV-A and BA9 of HRSV-B. ON1 is a newly emerged genotype with a 72-nucleotide duplication in the G gene as observed previously with BA genotypes in HRSV-B.FindingsChildren <5 years of age with an initial diagnosis of severe or very severe pneumonia at a hospital in the Philippines from September 2012 to December 2013 were enrolled. HRSV genotypes were determined and the viral load measured from nasopharyngeal swabs (NPS). The viral load of HRSV genotype NA1 were significantly higher than those of ON1 and BA9. Regression analysis showed that both genotype NA1 and younger age were significantly associated with high HRSV viral load.ConclusionsThe viral load of NA1 was higher than that of ON1 and BA9 in NPS samples. HRSV genotypes may be associated with HRSV viral load. The reasons and clinical impacts of these differences in viral load among HRSV genotypes require further evaluation.

Alternate circulation and genetic variation of human respiratory syncytial virus genotypes in Chengdu, West China, 2009-2014.

Human respiratory syncytial virus (HRSV) is a major pathogen that causes worldwide seasonal epidemic disease in infants due to its genetic variations. However, published information on the molecular epidemiology of HRSV was never reported particularly in Chengdu of West China. During five consecutive seasons (from 2009 to 2014), 433 (23.7%) of 1827 samples from hospitalized patients were identified as HRSV positive. Epidemiological characteristics of HRSV revealed that subtype A viruses (62.7%) prevailed in the first three epidemic seasons and faded in the next two seasons, while subtype B viruses (37.3%) kept circulating in five epidemic periods. According to the phylogenetic analysis of glycoprotein (G) gene, five HRSV genotypes NA1, ON1, BA9, BA-C, and CB1 were found in Chengdu. The predominant circulating genotype changed from NA1 in the period of 2010-2012 to BA9 of 2013-2014. The newly emerging ON1 was first reported in West China in October 2013. The early genotypes BA-C and CB1 were replaced by the prevailing BA9 after the third epidemic peak. Genetic mutations in glycosylation sites of G protein were found in HRSV variants, suggesting the virus is able to escape the immune recognition and attack. This study elucidated the local HRSV epidemic was associated with the alternate circulation of multiple genotypes and with the change of glycosylation sites of G protein. J. Med. Virol. 89:32-40, 2017. © 2016 Wiley Periodicals, Inc.

A molecular epidemiological study of human respiratory syncytial virus in Croatia, 2011–2014

Human respiratory syncytial virus (HRSV) causes common respiratory tract infections in infants, young children and the elderly. The diversity of HRSV strains circulating in Croatia was investigated throughout a period of four consecutive years from March 2011–March 2014. The analysis was based on sequences from the second hypervariable region of the G gene. A predominance of HRSV group A was observed in the first three years of the study, while group B became slightly predominant during the first few months of 2014. Overall, 76% of viruses belonged to group A including the genotypes NA1, ON1 and GA5. NA1 was by far the most common genotype within group A in 2011–2013; however, only ON1 and a few GA5 viruses were detected in the first three months of 2014. The majority of group B strains were of genotype BA9 (97%), and a few BA10 genotypes were detected. BA9 had the highest substitution rate of all the detected genotypes, followed by ON1.Multiple analyses showed that HRSV group A strains were more diverse than group B strains. Gly at residue 232 (previously described to be specific for ON1) was also detected in three NA1 strains, which were phylogenetically placed on separate branches within the NA1 genotype. For all genotypes, the diversity was higher at the amino acid level than at the nucleotide level, although positive selection of mutations was shown for only a few sites using four different methods of codon-based analysis of selective pressure. More codons were predicted to be negatively selected.The complexity of the HRSV pools present during each epidemic peak was determined and compared to previous epidemiological data. In addition to presenting genetic versatility of HRSV in this geographic region, the collected sequences provide data for further geographical and temporal comparative analyses of HRSV and its evolutionary pathways.

Epidemiology and Molecular Characterization of Human Respiratory Syncytial Virus in Senegal after Four Consecutive Years of Surveillance, 2012-2015.

BackgroundThe burden of respiratory syncytial virus (RSV) infection remains poorly defined in Africa. To address this, we carried out a descriptive and retrospective pilot study, with a focus on the epidemiology of RSV in Senegal after 4 years of surveillance.Methodology and ResultsFrom January 2012 to October 2015 swabs were collected from consenting ILI outpatients. Viral detection was performed using RV16 kit enabling direct subtyping of RSV-A and B. For the molecular characterization of HRSV, the second hypervariable region of the Glycoprotein (G) gene was targeted for sequencing. We enrolled 5338 patients with 2803 children younger than five years of age (52.5%). 610 (11.4%) were positive for RSV infection: 276 (45.2%) were group A infections, 334 (54.8%) were group B infections and 21 (3.4%) were A/B co-infections. RSV detection rate is significantly higher (P < 0.0001) in children below 5 years. We noted that the annual distribution of RSV varied substantially by season and for the predominant subtype. Globally, results show a clear circulation pattern in the second half of each year; between June and September and possibly extended into November. The majority of RSV-A strains from Senegal clustered with strains that were previously assigned NA1 and novel ON1 genotype sequences. RSV-B sequences from Senegal clustered with the BA9 genotype. At the amino acid level, RSV-A strains from Senegal show proximity with the genotype ON1 characterized by a 72 nt insertion in G, resulting in 24 extra amino acids of which 23 are duplications of aa 261–283.ConclusionGlobally our results show a clear circulation pattern of RSV in the second half of each year, between June and September and possibly extending into November, with children under 5 being more susceptible. Molecular studies identified the novel strains ON1 and BA9 as the major genotypes circulating in Senegal between 2012 and 2015.

Association of RSV-A ON1 genotype with Increased Pediatric Acute Lower Respiratory Tract Infection in Vietnam

Since the initial discovery of RSV-A ON1 in Canada in 2010, ON1 has been reported worldwide, yet information regarding its clinical impact and severity has been controversial. To investigate the clinical relevance of RSV-A ON1,acute respiratory infection (ARI) cases enrolled to our population-based prospective pediatric ARI surveillance at Khanh Hoa General Hospital, Central Vietnam from January 2010 through December 2012 were studied. Clinical-epidemiological information and nasopharyngeal samples were collected. Multiplex PCR assays were performed for screening 13 respiratory viruses. RSV-positive samples were further tested for subgroups (A/B) and genotypes information by sequencing the G-glycoprotein 2nd hypervariable region. Statistical analysis was performed to evaluate the clinical-epidemiological characteristics of RSV-A ON1. A total of 1854 ARI cases were enrolled and 426 (23.0%) of them were RSV-positive. During the study period, RSV-A and B had been co-circulating. NA1 was the predominant RSV-A genotype until the appearance of ON1 in 2012. RSV-related ARI hospitalization incidence significantly increased after the emergence of ON1. Moreover, multivariate analysis revealed that risk of lower respiratory tract infection was 2.26 (95% CI: 1.37–3.72) times, and radiologically-confirmed pneumonia was 1.98 (95% CI: 1.01–3.87) times greater in ON1 compared to NA1 cases. Our result suggested that ON1 ARI cases were clinically more severe than NA1.

Clinical characterisation and phylogeny of respiratory syncytial virus infection in hospitalised children at Red Cross War Memorial Children's Hospital, Cape Town.

BackgroundRespiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children in both the community and hospital setting.MethodsThe clinical presentation, patient and phylogenetic characteristicsof laboratory-confirmed cases of RSV, as well as risk factors for nosocomial infectionat Red Cross War Memorial Children’s Hospital in Cape Town were analysed. A multiplex PCR assay that detects 7 respiratory viruses was used to identify RSV nucleic acid on respiratory specimens.ResultsA total of 226 children were studied, ages ranging between 1 week and 92.5 months (median: 2.8 months, IQR: 1.3–6.3 months) and 51.8 % were males. The median duration of symptoms prior to diagnosis was 2 days (IQR: 1–4 days). Nosocomial infections wereidentified in 22 (9.7 %) children. There were pre-existing medical conditions in 113 (50.0 %) excluding HIV, most commonly prematurity (n = 58, 50.0 %) and congenital heart disease (n = 34, 29.3 %). The commonest presenting symptoms were cough (196, 86.7 %), difficulty in breathing (115, 50.9 %) and fever (91, 41.6 %).A case fatality rate of 0.9 % was recorded. RSV group A predominated (n = 181, 80.1 %) while group B accounted for only 45 (19.9 %) of the infections. The prevalent genotypes were NA1 (n = 127,70.1 %), ON1 (n = 45,24.9 %) and NA2 (n = 9,5.0 %) for group A while the only circulating RSV B genotype was BA4. There was no significant difference in the genotype distribution between the nosocomial and community-acquired RSV infections. Age ≥ 6 months was independently associated with nosocomial infection.ConclusionsA large percentage of children with RSV infection had pre-existing conditions. Approximately one tenth of the infections were nosocomial with age 6 months or older being a risk factor. Though both RSV groups co-circulated during the season, group A was predominant and included the novel ON1 genotype. Continued surveillance is necessary to identify prevalent and newly emerging genotypes ahead of vaccine development and efficacy studies.

Long-Term Shedding of Influenza Virus, Parainfluenza Virus, Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.

Characteristics of Respiratory Syncytial Virus isolated from Acute Respiratory Infectious Disease in Busan

Human respiratory syncytial virus (HRSV) is known as the leading cause of respiratory tract illness in infancy and elderly children worldwide. We investigate the prevalence pattern and genetic characteristics in the second variable region G protein gene of HRSV during 5 consecutive seasons from 2010 to 2015. A total of 4,793 specimens (throat swabs) were collected from patients with acute respiratory tract. HRSV were evaluated and classified as HRSV A (n=111) or HRSV B (n=64) by real-time RT-PCR or RT-PCR. In general HRSV were detected in winter season. Coughing, fever, rhinorrhea and sputum were confirmed main symptoms in patients with HRSV. There were no significant differences in clinical characteristics or severity according to the HRSV subgroup infections. Out of 175 HRSV positive samples, 94 samples were successfully sequenced using G gene. Phylogenetic analysis revealed that 62 HRSV-A strains clustered into genotypes ON1 (n=54, 87.1%), NA1 (n=7), NA2 (n=1) and 32 HRSV-B strains clustered into three genotypes: BA4 (n=28, 87.5%), BA5 (n=2), BA6 (n=2). These results provide a better understanding of HRSV prevalence pattern and genetic characteristics.

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013.

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.

Genetic diversity and evolutionary insights of respiratory syncytial virus A ON1 genotype: global and local transmission dynamics.

Human respiratory syncytial virus (RSV) A ON1 genotype, first detected in 2010 in Ontario, Canada, has been documented in 21 countries to date. This study investigated persistence and transmission dynamics of ON1 by grouping 406 randomly selected RSV-positive specimens submitted to Public Health Ontario from August 2011 to August 2012; RSV-A-positive specimens were genotyped. We identified 370 RSV-A (181 NA1, 135 NA2, 51 ON1 3 GA5) and 36 RSV-B positive specimens. We aligned time-stamped second hypervariable region (330 bp) of G-gene sequence data (global, n = 483; and Ontario, n = 60) to evaluate transmission dynamics. Global data suggests that the most recent common ancestor of ON1 emerged during the 2008–2009 season. Mean evolutionary rate of the global ON1 was 4.10 × 10−3 substitutions/site/year (95% BCI 3.1–5.0 × 10−3), not significantly different to that of Ontario ON1. The estimated mean reproductive number (R0 = ∼ 1.01) from global and Ontario sequences showed no significant difference and implies stability among global RSV-A ON1. This study suggests that local epidemics exhibit similar underlying evolutionary and epidemiological dynamics to that of the persistent global RSV-A ON1 population. These findings underscore the importance of continual molecular surveillance of RSV in order to gain a better understanding of epidemics.

Epidemiologic characteristics and the relationship with disease severity of respiratory syncytial virus genotypes from children with lower respiratory tract infection in the southern Zhejiang province

To investigate the epidemiological characteristics of respiratory syncytial virus (RSV) subtypes and genotypes in southern Zhejiang province, and to determine whether RSV genotypes are correlated with the disease severity of lower respiratory tract infection (LRTI). Nasopharyngeal secretions (NPS) from children under 5 years of age who were hospitalized with LRTI during 5 consecutive seasons from July 1, 2009 to June 30, 2014 were collected. RSV antigen was determined using direct immunofluorescence (DIF). Two hundred strains of RSV were randomly selected from each epidemic season. RNA was extracted and identified as subtype A or B by using reverse transcription-polymerase chain reaction (RT-PCR), and randomly selected strains of the full length attachment (G) genes of both subtype A and subtype B were amplified by PCR and sequencing. Clinical data were collected, and the disease severity between different genotypes were compared simultaneously. Of the total 1 000 randomly selected RSV positive samples, 462 (46.2%) and 538 (53.8%) samples were identified as subtype A and B, respectively. It was found that subtype B predominated in the 2009-2010 and 2012-2014 epidemic seasons and subtype A in 2010-2012 epidemic seasons. A total of 112 strains of complete sequences of G genes were obtained, including four subtype A genotypes NA1, NA4, GA2 and ON1, and six subtype B genotypes BA8-10, BA-C, CB1, and GB2. Phylogenetic analysis revealed that 39/52 (75.0%) subtype A strains were classified as NA1 genotype, followed by ON1 genotype (10/52,19.2%) and 44/60 (73.3%) subtype B strains were classified as BA9 genotype, followed by BA8 genotype (6/60,10.0%). BA9 was the predominant genotype among subtype B except 2010-2011 epidemic season, while NA1 was the predominant genotype among subtype A except 2013-2014 epidemic season. Only ON1 and BA9 genotypes were checked out during 2013-2014 epidemic season. There was statistically significant difference in the average severity score of illness in 39 cases infected with NA1 genotype (4.154) and 44 cases of BA9 genotype (3.341) (U=642.500, P<0.05). Furthermore, in the rate of oxygen uptake, the percentage of those infected with NA1 genotype (33.3%) was higher than those infected with BA9 genotype (13.6%) (χ2=4.544, P<0.05). However, there were no significant difference in the age, clinical symptoms, the percentage of intensive care unit admission, length of hospitalization and the outcome of the disease between NA1 and BA9 infection. The shift of predominant RSV subtype from 2009 to 2014 were B-A-A-B-B in the southern areas of Zhejiang province. Multiple genotypes co-circulated during five RSV epidemic seasons. NA1 and BA9 genotypes were the predominant genotypes of subtype A and B, respectively. Compared with infection with BA9 genotype, NA1 genotype infection was associated with more severe disease and proportion of patients needed oxygen therapy was higher.

Characteristics and Their Clinical Relevance of Respiratory Syncytial Virus Types and Genotypes Circulating in Northern Italy in Five Consecutive Winter Seasons.

In order to investigate the genetic diversity and patterns of the co-circulating genotypes of respiratory syncytial virus (RSV) and their possible relationships with the severity of RSV infection, we studied all of the RSV-positive nasopharyngeal samples collected from children during five consecutive winters (2009–2010, 2010–2011, 2011–2012, 2012–2013 and 2013–2014). The RSVs were detected using the respiratory virus panel fast assay and single-tube RT-PCR, their nucleotides were sequenced, and they were tested for positive selection. Of the 165 positive samples, 131 (79.4%) carried RSV-A and 34 (20.6%) RSV-B; both groups co-circulated in all of the study periods, with RSV-A predominating in all the seasons except for winter 2010–2011, which had a predominance of RSV-B. Phylogenetic analysis of the RSV-A sequences identified genotypes NA1 and ON1, the second replacing the first during the last two years of the study period. The RSV-B belonged to genotypes BA9 and BA10. BA9 was detected in all the years of the study whereas BA only desultorily. Comparison of the subjects infected by RSV-A and RSV-B types did not reveal any significant differences, but the children infected by genotype A/NA1 more frequently had lower respiratory tract infections (p<0.0001) and required hospitalisation (p = 0.007) more often than those infected by genotype A/ON1. These findings show that RSV has complex patterns of circulation characterised by the periodical replacement of the predominant genotypes, and indicate that the circulation and pathogenic role of the different RSV strains should be investigated as each may have a different impact on the host. A knowledge of the correlations between types, genotypes and disease severity may also be important in order to be able to include the more virulent strains in future vaccines.

Rapid replacement of prevailing genotype of human respiratory syncytial virus by genotype ON1 in Beijing, 2012–2014

Human respiratory syncytial virus (HRSV) is the most common viral pathogen causing lower respiratory infections in infants and young children worldwide. HRSV ON1 genotype in subgroup A with a characteristic of a 72 nucleotide duplication in the second highly variable region of attachment glycoprotein gene, has been reported in some countries since it was first detected in clinical samples collected in Canada in 2010. In this study, 557 HRSV antigen-positive nasopharyngeal aspirates were randomly selected during 2012/2013 to 2013/2014 HRSV seasons in Beijing for subgroup typing and for ON1 genotype screening by using a PCR based method developed for easily identifying genotype ON1 out of strains of subtype A. It was found that subgroup B was dominant in the 2012/2013 season and sudden shift of subgroup dominance from B to A and rapid replacement of previously prevailing NA1 genotype by ON1 genotype occurred in the 2013/2014 season. Reversible amino acid replacement in the G protein gene was found in a new branch of ON1 genotype. The evolutionary rate of the 351 global ON1 sequences was estimated to 7.34×10−3 nucleotide substitutions per site per year (95% highest probability density intervals, HPD, 5.71×10−3 to 9.04×10−3), with the time of most recent common ancestor dating back to June 2009.

Molecular epidemiology and molecular characterization of respiratory syncytial viruses at a tertiary care university hospital in Catalonia (Spain) during the 2013–2014 season

BackgroundHuman respiratory syncytial virus (HRSV) is the main cause of lower respiratory tract infections among infants and young children. ObjectivesThe molecular epidemiology and characterization of HRSV strains detected at a Spanish tertiary hospital during the 2013–2014 season is reported. Study designPhylogenetic analysis and molecular characterization of HRSV laboratory-confirmed respiratory samples were performed, based on coding sequences of G and F proteins. ResultsHRSV infection was laboratory-confirmed in respiratory samples from 320 patients of which 223 (70%) were less than 2 years of age and none undergoing Palivizumab treatment. HRSV was detected at varying levels throughout the season with a maximum rate in the week 52/2013, right before the beginning of the influenza epidemic. Whilst both HRSV groups were found co-circulating, HRSV-B group clearly predominated. The phylogenetic analyses from 139 HVR-2 sequences revealed that most characterized strains belonged to ON1 and BA9 genotypes. Three different phylogenetic subgroups could be distinguished within these genotypes. In addition, three strains (out of the 52 randomly selected) were carrying amino acid substitutions in the epitope A of the F protein, one of them previously related to Palivizumab resistance. ConclusionsThe results of the present study highlight the importance of a continuous HRSV surveillance to monitor not only the introduction of new genotypes on circulation but also the emergence of viral variants with new genetic characteristics that can affect the antigenicity features and the susceptibility to the only current prophylaxis treatment and also for the future development of HRSV vaccines.

Early Evolution of Human Respiratory Syncytial Virus ON1 Strains: Analysis of the Diversity in the C-Terminal Hypervariable Region of Glycoprotein Gene within the First 3.5 Years since Their Detection

Objective: Characterization of the phylogeny and diversity of human respiratory syncytial virus (HRSV) genotype ON1 that occurred during its early evolution (within the first 3.5 years since the detection of the first ON1 strains). ON1 strains have a 72-nucleotide-long in-frame duplication within the second hypervariable domain of the glycoprotein gene (HVR2). Methods: All available HVR2 sequences of strains belonging to the ON1 genotype published prior to June 20, 2014 were collected. Multiple sequence alignments, phylogeny, phylogeography, sequence clustering and putative protein analyses were performed. Results: The worldwide spread and diversification of ON1 strains are presented. Only in a minority of ON1 strains do the two replicas remain identical, and various ON1 strains possess common differences between the first and the second copy (segments A and B). Mutations of the progenitor sequence were more frequent in segment B, a higher overall diversity on the protein level and more putative glycosylation sites exist in segment B, and, unlike in segment A, positive selection acts on that protein region. Conclusions: The fast spread of the novel HRSV genotype ON1 has been accompanied by its rapid concurrent diversification. Differences in variability of the two replicas within HVR2 were detected, with C-terminal replica being more variable.

Novel respiratory syncytial virus (RSV) genotype ON1 predominates in Germany during winter season 2012-13.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization especially in young children with respiratory tract infections (RTI). Patterns of circulating RSV genotypes can provide a better understanding of the molecular epidemiology of RSV infection. We retrospectively analyzed the genetic diversity of RSV infection in hospitalized children with acute RTI admitted to University Hospital Heidelberg/Germany between October 2012 and April 2013. Nasopharyngeal aspirates (NPA) were routinely obtained in 240 children younger than 2 years of age who presented with clinical symptoms of upper or lower RTI. We analyzed NPAs via PCR and sequence analysis of the second variable region of the RSV G gene coding for the attachment glycoprotein. We obtained medical records reviewing routine clinical data. RSV was detected in 134/240 children. In RSV-positive patients the most common diagnosis was bronchitis/bronchiolitis (75.4%). The mean duration of hospitalization was longer in RSV-positive compared to RSV-negative patients (3.5 vs. 5.1 days; p<0.01). RSV-A was detected in 82.1%, RSV-B in 17.9% of all samples. Phylogenetic analysis of 112 isolates revealed that the majority of RSV-A strains (65%) belonged to the novel ON1 genotype containing a 72-nucleotide duplication. However, genotype ON1 was not associated with a more severe course of illness when taking basic clinical/laboratory parameters into account. Molecular characterization of RSV confirms the co-circulation of multiple genotypes of subtype RSV-A and RSV-B. The duplication in the G gene of genotype ON1 might have an effect on the rapid spread of this emerging RSV strain.

Molecular evolution of human respiratory syncytial virus attachment glycoprotein (G) gene of new genotype ON1 and ancestor NA1

We conducted a comprehensive genetic analysis of the C-terminal 3rd hypervariable region of the attachment glycoprotein (G) gene in human respiratory syncytial virus subgroup A (HRSV-A) genotype ON1 (93 strains) and ancestor NA1 (125 strains). Genotype ON1 contains a unique mutation of a 72 nucleotide tandem repeat insertion (corresponding to 24 amino acids) in the hypervariable region. The Bayesian Markov chain Monte Carlo (MCMC) method was used to conduct phylogenetic analysis and a time scale for evolution. We also calculated pairwise distances (p-distances) and estimated the selective pressure. Phylogenetic analysis showed that the analyzed ON1 and NA1 strains formed 4 lineages. A strain belonging to lineage 4 of ON1 showed wide genetic divergence (p-distance, 0.072), which suggests that it might be a candidate new genotype, namely ON2. The emergence of genotype NA1 was estimated to have occurred in 2000 (95% of highest probability density, HPD; 1997–2002) and that of genotype ON1 in 2005 (95% HPD; 2000–2010) based on the time-scaled phylogenetic tree. The evolutionary rate of genotype ON1 was higher than that of ancestral genotype NA1 (6.03×10−3 vs. 4.61×10−3 substitutions/site/year, p<0.05). Some positive and many negative selection sites were found in both ON1 and NA1 strains. The results suggested that the new genotype ON1 is rapidly evolving with antigenic changes, leading to epidemics of HRSV infection in various countries.

Infection with novel respiratory syncytial virus genotype Ontario (ON1) in adult hematopoietic cell transplant recipients, Texas, 2011-2013.

Respiratory syncytial virus (RSV) can cause severe respiratory disease in adult hematopoietic cell transplant (HCT) recipients. RSV subgroups A and B have evolved into multiple genotypes. We report on a recently described RSV genotype (ON1) in a cohort of adult HCT recipients in Texas. Twenty adult HCT recipients were enrolled as a part of an efficacy trial of ribavirin therapy. RSV identification and genotyping was performed using molecular techniques. RSV-specific neutralizing antibody (NAb) responses were measured. ON1 genotype was detected in 3 of 6 patients in the 2011-2012 season and in 8 of 14 patients in 2012-2013 season. Other genotypes detected were NA1 and BA. NAb levels were low at enrollment. Eight of 9 patients who cleared the RSV infection within 2 weeks mounted a ≥4-fold NAb response, compared with 2 of 8 who shed the virus for >2 weeks. The clinical course of those infected with ON1 was comparable to the course for individuals infected with other genotypes. This is the first report of RSV ON1 genotype in the United States, and ON1 genotype was dominant genotype in adult HCT recipients. Interestingly, faster viral clearance was associated with a ≥4-fold NAb response, likely indicating a reconstituted immune system.

The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under 5years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.

Rapid spread of the novel respiratory syncytial virus A ON1 genotype, central Italy, 2011 to 2013.

Respiratory infections positive for human respiratory syncytial virus (RSV) subtype A were characterised in children admitted to hospitals in Rome and Ancona (Italy) over the last three epidemic seasons. Different strains of the novel RSV-A genotype ON1, first identified in Ontario (Canada) in December 2010, were detected for the first time in Italy in the following 2011/12 epidemic season. They bear an insertion of 24 amino acids in the G glycoprotein as well as amino acid changes likely to change antigenicity. By early 2013, ON1 strains had spread so efficiently that they had nearly replaced other RSV-A strains. Notably, the RSV peak in the 2012/13 epidemic season occurred earlier and, compared with the previous two seasons, influenza-like illnesses diagnoses were more frequent in younger children; bronchiolitis cases had a less severe clinical course. Nonetheless, the ON1-associated intensive care unit admission rate was similar, if not greater, than that attributable to other RSV-A strains. Improving RSV surveillance would allow timely understanding of the epidemiological and clinicopathological features of the novel RSV-A genotype.