Respiratory Syncytial Virus Subgroup Research Articles

Epidemiology and Clinical Presentations of Respiratory Syncytial Virus Subgroups A and B Detected with Multiplex Real-Time PCR.

Respiratory syncytial virus (RSV) is one of the most important pathogenic infections of children and requires in-depth research worldwide, and especially in developing countries. We used a novel multiplex real-time PCR to test 5483 patients (≤ 14 years old) hospitalized with respiratory illness in Guangzhou, China, over a 3-year period. Of these patients, 729 were positive for RSV-A (51.2%, 373/729) or RSV-B (48.8%, 356/729), but none was infected with both viruses. Two seasonal peaks in total RSV were detected at the changes from winter to spring and from summer to autumn. RSV-B was dominant in 2013 and RSV-A in 2015, whereas RSV-A and RSV-B cocirculated in 2014. The clinical presentations of 645 RSV-positive patients were analyzed. Bronchiolitis, dyspnea, coryza, vomiting, poor appetite, and diarrhea occurred more frequently in RSV-A-positive than RSV-B-positive patients, whereas chill, headache, myalgia, debility, and rash etc. were more frequent in RSV-B-positive than RSV-A-positive patients, suggesting specific clinical characteristics for RSV-A and RSV-B. Coinfectons with other pathogens were common and diverse. Bronchiolitis, fever (≥ 38°C), and poor appetite were more frequent in patients with single RSV infections than in coinfected patients, suggesting the key pathogenic activity of RSV. Analysis of the relationships between the comparative viral load and clinical presentations showed significant differences in bronchiolitis, fever (≥ 38°C), and rash etc. among patients with different viral loads. This study provides a novel rapid method for detecting RSV subgroups, and provides new insights into the epidemiology and clinical implications of RSV.

Molecular evolution of respiratory syncytial virus subgroup A genotype NA1 and ON1 attachment glycoprotein (G) gene in central Vietnam

We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010–2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains. We also estimated the N-glycosylation sites within G gene hypervariable region. Amino acid substitutions under positive and negative selection pressure were examined using Conservative Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Internal Fixed Effects Likelihood (IFEL) and Mixed Effects Model for Episodic Diversifying Selection (MEME) models. The majority of central Vietnam ON1 strains detected in 2012 were classified into lineage 1 with few positively selected substitutions. As for the Vietnamese NA1 strains, four lineages were circulating during the study period with a few positive selection sites. Shifting patterns of the predominantly circulating NA1 lineage were observed in each year during the investigation period. Median p-distance of central Vietnam NA1 strains was wider (p-distance=0.028) than that of ON1 (p-distance=0.012). The molecular evolutionary rate of central Vietnam ON1 strains was estimated to be 2.55×10−2 (substitutions/site/year) and was faster than NA1 (7.12×10−3 (substitutions/site/year)). Interestingly, the evolutionary rates of both genotypes ON1 and NA1 strains from central Vietnam were faster than the global strains respectively. Furthermore, the shifts of N-glycosylation pattern within the G gene 3rd hypervariable region of Vietnamese NA1 strains were observed in each year. BSP analysis indicated the rapid growth of RSV-A effective population size in early 2012. These results suggested that the molecular evolution of RSV-A G gene detected in central Vietnam was fast with unique evolutionary dynamics.

Respiratory Syncytial Virus and Other Viral Infections among Children under Two Years Old in Southern Vietnam 2009-2010: Clinical Characteristics and Disease Severity.

BackgroundDespite a high burden of respiratory syncytial virus (RSV) infections among children, data on demographic and clinical characteristics of RSV are scarce in low and middle income countries. This study aims to describe the viral etiologies, the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lower respiratory tract infections (LRTI), focusing on RSV (prevalence, seasonality, subgroups, viral load) and its association with disease severity.MethodsA prospective study among children under two years of age, hospitalized with LRTI was conducted in two referral pediatric hospitals in Ho Chi Minh City, Vietnam, from May 2009 to December 2010. Socio-demographic, clinical data and nasopharyngeal swabs were collected on enrolment and discharge. Multiplex real-time RT-PCR (13 viruses) and quantitative RSV RT-PCR were used to identify viral pathogens, RSV load and subgroups.ResultsAmong 632 cases, 48% were RSV positive. RSV infections occurred at younger age than three other leading viral infections i.e rhinovirus (RV), metapneumovirus (MPV), parainfluenza virus (PIV-3) and were significantly more frequent in the first 6 months of life. Clinical severity score of RSV infection was significantly higher than PIV-3 but not for RV or MPV. In multivariate analysis, RV infection was significantly associated with severity while RSV infection was not. Among RSV infections, neither viral load nor viral co-infections were significantly associated with severity. Young age and having fever at admission were significantly associated with both RSV and LRTI severity. A shift in RSV subgroup predominance was observed during two consecutive rainy seasons but was not associated with severity.ConclusionWe report etiologies, the epidemiological and clinical characteristics of LRTI among hospitalized children under two years of age and risk factors of RSV and LRTI severity.

Molecular evolution of the fusion protein gene in human respiratory syncytial virus subgroup A

We studied the molecular evolution of the fusion protein (F) gene in the human respiratory syncytial virus subgroup A (HRSV-A). We performed time-scaled phylogenetic analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. We also conducted genetic distance (p-distance), positive/negative selection, and Bayesian skyline plot analyses. Furthermore, we mapped the amino acid substitutions of the protein. The MCMC-constructed tree indicated that the HRSV F gene diverged from the bovine RSV (BRSV) gene approximately 550years ago and had a relatively low substitution rate (7.59×10−4 substitutions/site/year). Moreover, a common ancestor of HRSV-A and -B diverged approximately 280years ago, which has since formed four distinct clusters. The present HRSV-A strains were assigned six genotypes based on F gene sequences and attachment glycoprotein gene sequences. The present strains exhibited high F gene sequence similarity values and low genetic divergence. No positive selection sites were identified; however, 50 negative selection sites were identified. F protein amino acid substitutions at 17 sites were distributed in the F protein. The effective population size of the gene has remained relatively constant, but the population size of the prevalent genotype (GA2) has increased in the last 10years. These results suggest that the HRSV-AF gene has evolved independently and formed some genotypes.

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats.

Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10 total PFU and 2.7 log10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. RSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

Human respiratory syncytial virus (RSV) is a major health problem and the main cause of hospitalization due to bronchiolitis. RSV is divided into two antigenic subgroups, RSV-A and -B that co-circulate worldwide. Rapid and sensitive detection is desirable for proper patient handling while assessment of viral load may help to evaluate disease severity and progression. Finally RSV subtyping is needed to determine the prevalence and pathogenicity of each RSV subgroup, as well as their sensitivity to treatment. In this study, we took into account the most recent circulating RSV variants and designed two quantitative TaqMan one-step RT-PCR assays to detect and quantify both RSV subgroups separately. Standard dilutions of transcripts of positive and negative polarities were included in the assay validation to assess potential differences in sensitivity on negative-sense genomes and positive-sense RNAs. In addition, RSV detection in respiratory specimens of different types and sampled in different populations was compared to commercially available RSV diagnostic tools. Altogether, the RSV-A and -B assays revealed sensitive and quantitative over a wide range of viral loads, with a slight improved sensitivity of the RSV-B assay on positive sense transcripts, and allowed accurate RSV subtyping. We thus provide a useful tool for both RSV diagnostics and research.

Sixteen years of evolution of human respiratory syncytial virus subgroup A in Buenos Aires, Argentina: GA2 the prevalent genotype through the years

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections (LRTI) in children worldwide. In recent years, several preclinical trials with vaccine candidates have been reported. It is in this sense that molecular epidemiological studies become important. Understanding viral dispersion patterns before and after the implementation of a vaccine can provide insight into the effectiveness of the control strategies. In this work we analyzed the molecular epidemiology of HRSV-A over a period of sixteen years (1999–2014) in Buenos Aires. By bioinformatic tools we analyzed 169 sequences of the G glycoprotein gene from hospitalized pediatric patients with LRTI. We found that GA2 was the most prevalent genotype (73.35%). GA5 genotype co-circulated in our region until 2009 when it was no longer detected, except in 2011. The recently globally emerging ON1 lineage with a 72-nt duplication increased its frequency to become the only lineage detected in Buenos Aires in 2014. By discrete phylogeographic analysis of global ON1 strains we could determine that Panama could be the location of the MRCA dated June 20, 2010; and this lineage could be introduced in Argentina from Spain in April 2011. This analysis also showed temporary and geographical clustering of ON1 strains observed as phylogenetic clades with strains exclusively associated from a single country, nevertheless among our 44 ON1 strains from three outbreaks (2012–2014) we could also detect posterior reintroductions and circulation from United States, Cuba, South Korea, and Spain. The continuous phylogeographic analysis of one sublineage of Argentine ON1 strains allowed us to establish that there could be a local clustering of some strains even in neighborhoods. This work shows the potential of this type of bioinformatic tools in the context of a future vaccine surveillance network to trace the spread of new genetic lineages in human populations.

Sensitivities of Respiratory Syncytial Virus Antigen-detection Kits Commercially Available in Japan

Respiratory syncytial virus (RSV) antigen-detection kits are used in clinical settings in Japan. Here, we compared the sensitivities of 12 different kits that are commercially available in Japan. Notably, these kits were developed based on the principle of immunochromatography. The comparison was performed using six different test specimens：standard laboratory strains of RSV subgroups A and B, and clinical specimens of nasal aspirates from patients infected with RSV subgroups A or B. The specimens were serially diluted with medium at half log concentration, and the dilutions were applied to the kit tests. The minimum detection limit was determined for each kit and for every specimen by investigating the maximum dilution rate for acquiring positive result. The absolute minimum detection limits were calculated based on the viral RNA copy number of the specimens and the maximum dilution rate ranged from 107 to 109 copies/mL, depending on the specimens and kits. Thus, the sensitivity was revealed to differ among the viral specimens examined using each kit and among the kits used to examine each viral specimen, though the difference was not significant and revealed a maximum of only 10 times. The results were compared with the geometric mean values of the results for all 12 kits which were separately calculated for all 6 specimens. Consequently, some kits with lower or higher sensitivities to all the tested specimens were identified, suggesting that some general differences in sensitivity exist among commercially available RSV antigen-detection kits, although the differences are not significant. Of course, selecting kit with a higher sensitivity would be preferable, but no large differences in sensitivity among the available kits examined in the current study. Therefore, other factors such as cost-effectiveness, convenience, time required for testing, etc., can be taken into account when selecting a kit for use in clinical settings.

Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children ,2 years of age. Little is known about RSV intra-host genetic diversity over the course of infection or about the immune pressures that drive RSV molecular evolution. We performed whole-genome deep-sequencing on 53 RSV-positive samples (37 RSV subgroup A and 16 RSV subgroup B) collected from the upper airways of hospitalized children in southern Vietnam over two consecutive seasons. RSV A NA1 and RSV B BA9 were the predominant genotypes found in our samples, consistent with other reports on global RSV circulation during the same period. For both RSV A and B, the M gene was the most conserved, confirming its potential as a target for novel therapeutics. The G gene was the most variable and was the only gene under detectable positive selection. Further, positively selected sites inG were found in close proximity to and in some cases overlapped with predicted glycosylation motifs, suggesting that selection on amino acid glycosylation may drive viral genetic diversity. We further identified hotspots and coldspots of intra-host genetic diversity in the RSV genome, some of which may highlight previously unknown regions of functional importance.

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8×10−3substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance>0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.

Relating plaque morphology to respiratory syncytial virus subgroup, viral load, and disease severity in children.

Background:Viral culture plaque morphology in human cell lines are markers for growth capability and cytopathic effect, and have been used to assess viral fitness and select preattenuation candidates for live viral vaccines. We classified respiratory syncytial virus (RSV) plaque morphology and analyzed the relationship between plaque morphology as compared to subgroup, viral load and clinical severity of infection in infants and children.Methods:We obtained respiratory secretions from 149 RSV-infected children. Plaque morphology and viral load was assessed within the first culture passage in HEp-2 cells. Viral load was measured by polymerase chain reaction (PCR), as was RSV subgroup. Disease severity was determined by hospitalization, length of stay, intensive care requirement, and respiratory failure.Results:Plaque morphology varied between individual subjects; however, similar results were observed among viruses collected from upper and lower respiratory tracts of the same subject. Significant differences in plaque morphology were observed between RSV subgroups. No correlations were found among plaque morphology and viral load. Plaque morphology did not correlate with disease severity.Conclusion:Plaque morphology measures parameters that are viral-specific and independent of the human host. Morphologies vary between patients and are related to RSV subgroup. In HEp-2 cells, RSV plaque morphology appears unrelated to disease severity in RSV-infected children.Supplementary informationThe online version of this article (doi:10.1038/pr.2015.122) contains supplementary material, which is available to authorized users.

A recombinant varicella vaccine harboring a respiratory syncytial virus gene induces humoral immunity

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is highly efficient and causes few adverse events; therefore, it is used worldwide. We previously constructed recombinant vOka (rvOka) harboring the mumps virus gene. Immunizing guinea pigs with rvOka induced the production of neutralizing antibodies against the mumps virus and VZV.Here, we constructed recombinant vOka viruses containing either the respiratory syncytial virus (RSV) subgroup A fusion glycoprotein (RSV A–F) gene or RSV subgroup B fusion glycoprotein (RSV B–F) gene (rvOka-RSV A–F or rvOka-RSV B–F). Indirect immunofluorescence and Western blot analyses confirmed the expression of each recombinant RSV protein in virus-infected cells. Immunizing guinea pigs with rvOka-RSV A–F or rvOka-RSV B–F led to the induction of antibodies against RSV proteins. These results suggest that the current varicella vaccine genome can be used to generate custom-made vaccine vectors to develop the next generation of live vaccines.

Functional Analysis of the 60-Nucleotide Duplication in the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein.

There are two subgroups of respiratory syncytial virus (RSV), A and B, and within each subgroup, isolates are further divided into clades. Several years ago, multiple subgroup B isolates which contained a duplication of 60 nucleotides in the glycoprotein (G) gene were described. These isolates were given a new clade designation of BA based on the site of isolation, Buenos Aires, Argentina. BA RSV strains have since become the predominant circulating clade of RSV B viruses. We hypothesized that the duplicated region in G serves to enhance the function of G in the virus life cycle. We generated recombinant viruses that express a consensus BA G gene or a consensus BA G gene lacking the duplication (GΔdup). We determined that the duplicated region functions during virus attachment to cells. Additionally, we showed that in vitro, the virus containing the duplication has a fitness advantage compared to the virus without the duplication. Our data demonstrate that the duplicated region in the BA strain G protein augments virus attachment and fitness. Respiratory syncytial virus (RSV) is an important pathogen for infants for which there is no vaccine. Different strains of RSV circulate from year to year, and the predominating strains change over time. Subgroup B RSV strains with a duplication in the attachment glycoprotein (G) emerged and then became the dominant B genotype. We found that a recombinant virus harboring the duplication bound more efficiently to cells and was more fit than a recombinant strain lacking the duplication. Our work advances a mechanism for an important natural RSV mutation.

Usefulness of a Newly Developed Immunochromatographic Assay Kit for the Detection of Respiratory Syncytial Virus

We evaluated the usefulness of IMMUNOCATCH-RSV (Eiken chemical Co., Ltd.) (IC-A), a newly developed immunochromatographic assay kit for detection of respiratory syncytial virus (RSV). For the clinical study, 210 nasal swabs and 134 nasopharyngeal aspirates were collected from pediatric patients with acute respiratory tract infections in 2013. Three immunochromatographic assay kits (IC-A, IC-B and IC-C), and the RT-PCR method were used for the detection of RSV. The detection times for IC-A, IC-B and IC-C were 8, 15 and 10 minutes, respectively. The positive rates for IC-A using nasal swabs and nasopharyngeal aspirates were 33.8% and 35.8%, respectively. For the nasal swab specimens, the total concordance rates of RT-PCR with IC-A, IC-B and IC-C were 96.2% (202/210), 89.5% (188/210), and 90.5% (143/158), respectively. As for the nasopharyngeal aspirates, the total concordance rates of RT-PCR with IC-A, IC-B and IC-C were 96.3% (129/134), 94.0% (125/133), and 97.7% (130/133), respectively. The minimum detection concentration of IC-A was 3.0 x 10(2) TCID50/mL for the RSV subgroup A strain, and 7.5 x 10 TCID50/mL for the RSV subgroup B strain. In conclusion, the current data indicate that IC-A is a useful kit for more rapid and accurate detection of RSV infection.

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.

Human Respiratory Syncytial Virus Infection and its Subgroups Among the Hospitalized Young Children With Acute Respiratory Infection

Background: Respiratory syncytial virus (RSV) can cause acute respiratory infection (ARI) in infants and young children. Objectives: This study was conducted to determine the incidence of RSV infection and its subgroups among children with ARI. Materials and Methods: A total of 100 throat samples were collected from hospitalized children with ARI in different hospitals across the Khuzestan province from June 2009 to April 2010. The samples were tested for RSV by the nested PCR. The product of positive RSV was sequenced to determine the RSV subgroup, followed by phylogenic tree. Results: Of total 100 patients, 29 (29%) including 16 (16%) male and 13 (13%) female were found positive for RSV infection. All the RSV positive patients were subgroup A dominant. High prevalence of RSV (8%) was found among the children under one year in contrast to 2% RSV incidence among the age group 6 years. Conclusions: This study revealed that RSV subgroup A is dominant among the young children especially in children less than one year of age.

Genetic Variation in Attachment Glycoprotein Genes of Human Respiratory Syncytial Virus Subgroups A and B in Children in Recent Five Consecutive Years

Human respiratory syncytial virus (HRSV) outranks other viral agents as the cause of respiratory tract diseases in children worldwide. Molecular epidemiological study of the virus provides useful information for the development of globally effective vaccine. We investigated the circulating pattern and genetic variation in the attachment glycoprotein genes of HRSV in Beijing during 5 consecutive seasons from 2007 to 2012. Out of 19,942 tested specimens, 3,160 (15.8%) were HRSV antigen-positive. The incidence of HRSV infection in males was significantly higher than in females. Of the total 723 (23.1%) randomly selected HRSV antigen-positive samples, 462 (63.9%) and 239 (33.1%) samples were identified as subgroup A and B, respectively. Subgroups A and B co-circulated in the 5 consecutive HRSV seasons, which showed a shifting mixed pattern of subgroup dominance. Complete G gene sequences were obtained from 190 HRSV-A and 72 HRSV-B by PCR for phylogenetic analysis. Although 4 new genotypes, NA3 and NA4 for HRSV-A and BA-C and CB1 for HRSV-B, were identified here, they were not predominant; NA1 and BA9 were the prevailing HRSV-A and -B genotypes, respectively. We provide the first report of a 9 consecutive nucleotide insertion in 3 CB1 genotype strains. One Beijing strain of ON1 genotype with a 72 nucleotide insertion was found among samples collected in February 2012. The reversion of codon states in glycosylation sites to previous ones were found from HRSV strains in this study, suggesting an immune-escape strategy of this important virus.

Development of a Sensitive Loop-Mediated Isothermal Amplification Assay That Provides Specimen-to-Result Diagnosis of Respiratory Syncytial Virus Infection in 30 Minutes

Rapid isothermal amplification methods have recently been introduced, and some of these methods offer significant advantages over PCR. The objective of this study was to develop a rapid and sensitive multiplex loop-mediated isothermal amplification (M-LAMP) assay for the detection of respiratory syncytial virus subgroups A and B (RSV A and B). We designed six primers each for the matrix gene of RSV A and the polymerase gene of RSV B and developed an M-LAMP assay by using a commercially available master mix and a real-time fluorometer (Genie II; Optigene, United Kingdom) that displays real-time amplification, time to positivity, and amplicon annealing temperature (Tm). The M-LAMP was evaluated against PCR by testing 275 nasopharyngeal (NP) specimens. The final optimized M-LAMP assay had a mean amplification time of 14.2 min (compared with 90 to 120 min for PCR) and had an analytical sensitivity of 1 genome equivalent (ge) for both RSV A and B. Using PCR as a comparator, M-LAMP had a sensitivity of 100% (81/81) and specificity of 100% (194/194). We also evaluated a 3- to 10-min specimen processing method involving vortexing with glass beads and heating to 98°C in M-swab medium (Copan Italia, Brescia, Italy) and found that this rapid processing method allowed detection of 37/41 (90.2%) of positives when we used extracted nucleic acid. In summary, the M-LAMP assay had excellent sensitivity and specificity for detecting RSV A and B in NP specimens and, when coupled with a rapid specimen preparation method, could provide a specimen-to-result diagnosis time of 30 min.

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008–2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63×10−3 and 4.56×10−3 substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.