Respiratory Syncytial Virus Subtypes Research Articles

GS-5806 Inhibits a Broad Range of Respiratory Syncytial Virus Clinical Isolates by Blocking the Virus-Cell Fusion Process.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. GS-5806, discovered via chemical optimization of a hit from a high-throughput antiviral-screening campaign, selectively inhibits a diverse set of 75 RSV subtype A and B clinical isolates (mean 50% effective concentration [EC50] = 0.43 nM). The compound maintained potency in primary human airway epithelial cells and exhibited low cytotoxicity in human cell lines and primary cell cultures (selectivity > 23,000-fold). Time-of-addition and temperature shift studies demonstrated that GS-5806 does not block RSV attachment to cells but interferes with virus entry. Follow-up experiments showed potent inhibition of RSV F-mediated cell-to-cell fusion. RSV A and B variants resistant to GS-5806, due to mutations in F protein (RSV A, L138F or F140L/N517I, and RSV B, F488L or F488S), were isolated and showed cross-resistance to other RSV fusion inhibitors, such as VP-14637, but remained fully sensitive to palivizumab and ribavirin. In summary, GS-5806 is a potent and selective RSV fusion inhibitor with antiviral activity against a diverse set of RSV clinical isolates. The compound is currently under clinical investigation for the treatment of RSV infection in pediatric, immunocompromised, and elderly patients.

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.

The impact of viral dynamics on the clinical severity of infants with respiratory syncytial virus bronchiolitis.

The impact of dynamic respiratory syncytial virus (RSV) load on the clinical severity of hospitalized infants with bronchiolitis has not been clarified. Nasopharyngeal aspirates were obtained from 60 infants who were diagnosed with bronchiolitis within 96 hr of wheezing onset upon admission and on days 3, 5, and 7 in the hospital, and 17 respiratory viruses were detected. The RSV load was quantified by real‐time qPCR for RSV subtypes A and B at different time points. Scoring criteria were used to evaluate the degree of severity. A total of 40 infants were determined to be RSV‐positive, nine were identified as RSV subtype A (RSVA), and 31 were RSV subtype B (RSVB). The peak RSV load was observed upon admission, and the RSV load decreased significantly over time; in addition, this decrease began to have significant differences on day 5. There was a positive correlation between the RSV load and the clinical score (r2 = 0.121 and P < 0.001). According to the clinical scores, the infants in the severe group tended to have higher RSV loads than those in the moderate and mild groups. Multivariate logistic regression models revealed that the viral load on day 3 was independently associated with the degree of severity. This study elucidated that a higher mean RSV load was associated with a more severe disease and a longer duration of hospitalization and symptoms. This study also clarified RSV replication in infants and provides a theoretical basis for specifying an anti‐RSV therapy strategy. J. Med. Virol. 87:1276–1284, 2015. © 2015 Wiley Periodicals, Inc.

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.

Molecular characterization of human respiratory syncytial virus subtype B: a novel genotype of subtype B circulating in China.

Human respiratory syncytial virus (HRSV) is major pathogen of lower respiratory tract infections in infants and young children worldwide. There have been many studies regarding HRSV subgroup A (HRSV-A) G protein genetic variability but little information about HRSV subtype B (HRSV-B) G protein genetic diversity and molecular evolution in China. Thus, a survey of the molecular epidemiology and evolution of the G protein in China is of high importance. In this study, the circulation and genetic diversity of HRSV in Chongqing, Southwestern China, from June 2009 to May 2013, were investigated. A total of 3,167 nasopharyngeal aspirates were obtained in this study, and it was found that HRSV-B predominated in the 2009-2010 and 2012-2013 epidemic seasons. This study identified the genetic variability of the glycoprotein G gene among 102 HRSV-B strains isolated by cell culture from Chongqing nasopharyngeal aspirates, and 68 Chinese HRSV-B sequences were deposited in GenBank. Genotyping and phylogenetic analysis revealed that the HRSV-B strains were clustered into three genotypes: BA (n = 111, 65.29%), GB3 (n = 5, 2.94%), and a new GB genotype (n = 54, 31.77%) named GB5. The GB5 strains varied from other genotypes in the central conserved region and N-glycosylation sites. The estimated evolutionary rate of Chinese HRSV-B was 2.01 × 10(-3) nucleotide substitutions/site/year, which is similar to the reports from Belgium and the Netherlands with 1.95 × 10(-3) and 2.78 × 10(-3) nucleotide substitutions/site/year, respectively. This study provides data on the circulating pattern and molecular characterization of HRSV-B genotypes in China during four consecutive years and may contribute to HRSV vaccine development.

Development of Safe and Effective RSV Vaccine by Modified CD4 Epitope in G Protein Core Fragment (Gcf)

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183–195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51–66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.

Respiratory viruses within homeless shelters in Marseille, France

BackgroundHomeless shelters are identified as places where humans are at high risk of acquiring respiratory disease. We previously reported the prevalence of the main respiratory diseases affecting a population of homeless in Marseille, France. Here, we investigated the prevalence of 10 respiratory viruses in a similar homeless population during 2 successive winter seasons.FindingsFollowing a clinical examination, we collected nasal specimens from which the RT-PCR detection of 10 respiratory viruses was performed through snapshot investigations. Among the 265 patients included, 150 (56.6%) reported at least one respiratory symptom of which 13 (8.7%) had positive swabs for at least one respiratory virus, and 115 patients reported any respiratory symptom of which 10 (8.7%) had positive swabs for respiratory virus. Overall, 23 patients had positive swabs for at least one respiratory virus. Human rhinovirus (HRV) was the predominant virus (13 isolates) followed by enteroviruses (3), human metapneumovirus (2), human coronavirus OC43 (2), 229E virus (2) and human respiratory syncytial virus subtype B (1). Among the patients infected with HRV, 10 were collected during the same snapshot.ConclusionsAlthough one half of the patients reported respiratory symptoms, the prevalence of respiratory viruses was within the range of that previously described in adult asymptomatic patients outside the homeless community. Most HRV-positive swabs were collected during the same snapshot suggesting a local outbreak. No influenza viruses were found despite the fact that one half of the patients were investigated during the peak of the seasonal influenza epidemic in Marseille.

Complete Genome Sequences of Two Human Respiratory Syncytial Virus Genotype A Strains from India, RSV-A/NIV1114046/11 and RSV-A/NIV1114073/11

Two complete genomes of human respiratory syncytial virus subtype A (HRSV-A), with and without a 72-nucleotide duplication in the C-terminal glycoprotein G gene, were sequenced and analyzed. Characterization of these genomes will improve understanding of the diversity, emergence, virulence, pathogenicity, and transmissibility of a novel RSV-A genotype with a 72-nucleotide G gene duplication.

Epidemiological characteristics of influenza virus and respiratory syncytial virus among children in Wuhan area from 2008 to 2012

To investigate the infection status and epidemiological characteristics of influenza virus and respiratory syncytial virus (RSV) in influenza-like illness (ILI) of children ( ≤ 14 years) in Wuhan area from 2008 to 2012. A total of 2854 cases of ILI patients ( ≤ 14 years) in a hospital of Wuhan were recruited in the study from July 2008 to June 2012. The sample of pharyngeal swab was collected from each patient, to extract the virus nucleic acids. Real-time fluorescent quantitation reverse transcription PCR (RT-PCR) method was applied to detect the subtypes of influenza virus and RSV, and then analyzed the time and age characteristics. Out of the 2854 cases, 758 (26.6%) were positive for influenza virus,including 547 (19.2%) influenza A virus positive samples and 211 (7.4%) influenza B virus positive samples. Usually, there were two peaks present in the annual curve of influenza virus, namely summer peak and winter/spring peak. The positive rate of influenza virus in 6-14 years old children (48.0%, 275/573) was significantly higher than that in 3-5 years old children (26.6%, 213/801) and that under 3 years old children (18.3%, 270/1480). The difference showed statistical significance (χ(2) = 187.432, P < 0.01). A total of 219 (7.7%) cases were positive for RSV,including 108 RSV-A positive samples and 112 RSV-B positive samples (1 co-infection). The epidemic of RSV showed an obvious seasonal pattern with peaks in autumn,winter and spring,which accounted for 96.8% (212/219) of all the cases; however, the annual incidence of RSV fluctuated greatly. The predominant subtype shifted every 2 years. RSV-B predominated during September 2008 and May 2009, December 2009 and March 2010, accounting for 76.6% (36/47) and 96.9% (62/64) respectively. RSV-A predominated during November 2010 and March 2011, September 2011 and April 2012, accounting for 92.5% (37/40) and 100.0% (48/48) respectively. With the increase of the age, the positive rate of RSV-A and RSV-B decreased gradually (RSV-A: χ(2) = 36.223, P < 0.01; RSV-B: χ(2) = 36.281, P < 0.01). The positive rates of RSV-A in children < 1,1,2,3,4,5-9 and 10-14 years old were 7.0% (26/373), 5.9% (39/662), 4.0% (18/445), 3.2% (13/406), 1.3% (3/236), 1.4% (7/517) and 0.9% (2/215) respectively; while, the positive rates of RSV-B in each age group were 6.4% (24/373), 6.0% (40/662), 4.5% (20/445), 4.4% (18/406), 1.3% (3/236), 1.0% (5/517) and 0.9% (2/215) respectively. The children aged 0-3 years old were more susceptible for RSV infection,accounting for 90.0% (197/219) of the total positive samples. During the outbreak of influenza A H1N1 in November 2009, the positive rate of RSW was 3.0% (3/100), lower than that in the same month of 2008, 2010 and 2011,which were separately 18.2% (6/33), 10.8% (10/93) and 10.0% (4/40). The difference showed statistical significance (χ(2) = 8.450, P < 0.05). During the outbreak of influenza A (H1N1) in January 2011,the positive rate of RSV was 5.7% (3/53), lower than those in the same month of 2009, 2010 and 2012, which was separately 21.7% (5/23), 28.6% (22/77) and 16.0% (8/50). The difference showed statistical significance (χ(2) = 11.233,P < 0.05). During the period of less influenza happened in September 2011, the RSV positive rate was 25.0% (10/40), higher than those in the same month of 2008, 2009 and 2010, which was separately 11.4% (4/35), 1.7% (2/118) and 0.0% (0/109). The difference showed statistical significance (χ(2) = 32.521, P < 0.01). Both influenza virus and RSV were important etiological agents of ILI of children in Wuhan. The characteristics of seasonal and age distributions of the two viruses were notably different; meanwhile, a certain inhibitional effect of influenza virus on RSV could be observed.

High human bocavirus viral load is associated with disease severity in children under five years of age.

Human bocavirus (HBoV) is a parvovirus and detected worldwide in lower respiratory tract infections (LRTIs), but its pathogenic role in respiratory illness is still debatable due to high incidence of co-infection with other respiratory viruses. To determine the prevalence of HBoV infection in patients with LRTI in Shanghai and its correlation with disease severity, we performed a 3-year prospective study of HBoV in healthy controls, outpatients and inpatients under five years of age with X-ray diagnosed LRTIs. Nasopharyngeal aspirates were tested by PCR for common respiratory viruses and by real time PCR for HBoV subtypes 1–4. Nasopharyngeal swabs from healthy controls and serum samples and stools from inpatients were also tested for HBoV1-4 by real time PCR. Viral loads were determined by quantitative real time PCR in all HBoV positive samples. HBoV1 was detected in 7.0% of inpatients, with annual rates of 5.1%, 8.0% and 4.8% in 2010, 2011 and 2012, respectively. Respiratory syncytial virus (RSV) subtype A was the most frequent co-infection detected; HBoV1 and RSVA appeared to co-circulate with similar seasonal variations. High HBoV viral loads (>106 copies/ml) were significantly more frequent in inpatients and outpatients than in healthy controls. There was a direct correlation of high viral load with increasing disease severity in patients co-infected with HBoV1 and at least one other respiratory virus. In summary, our data suggest that HBoV1 can cause LRTIs, but symptomatic HBoV infection is only observed in the context of high viral load.

Distribution of Respiratory Syncytial Virus Subtypes A and B Among Infants Presenting to the Emergency Department With Lower Respiratory Tract Infection or Apnea

Respiratory syncytial virus (RSV), a leading viral respiratory pathogen worldwide, has 2 major subtypes, A and B. To describe the temporal and geographic distribution and parameters of disease severity associated with RSV A and B in the United States. A US multicenter active surveillance study was conducted in emergency departments (EDs) during 2 RSV seasons. Infants <1 year of age presenting to the ED with symptoms of lower respiratory tract infection or apnea were enrolled. RSV subtypes were detected in nasal swabs by reverse transcriptase polymerase chain reaction. Of 4248 patients enrolled, 4172 patients were evaluable; 32.4% of patients were positive for any RSV subtype in season 1 and 29.9% in season 2. RSV A and B were detected in each region studied. More patients presented to the ED with RSV A than with RSV B (853 [20.4%] versus 453 [10.9%], respectively); RSV A-positive patients were more likely to be admitted to the hospital or intensive care unit (47.7%, versus RSV B, 35.8%; P < 0.0001); hospitalized RSV A-positive patients were less likely to be prescribed antibiotics (32.4%, versus RSV B, 47.8%; P < 0.001). This is the largest epidemiologic study in EDs reporting trends in RSV subtypes. RSV subtypes A and B were documented in both seasons across all US regions studied and detected in September to May. The results of this study support suggestions from smaller studies that RSV A may be more virulent than RSV B; however, more quantitative assessments of disease severity are needed.

Novel Respiratory Syncytial Virus Subtype ON1 among Children, Cape Town, South Africa, 2012

To the Editor: Human respiratory syncytial virus (RSV) is a common cause of severe acute lower respiratory tract infection in young children, accounting for ≈160,000 deaths/year worldwide (1,2). As part of an RSV nosocomial transmission study, we detected RSV genotype ON1, which was identified during November 2010­–February 2011 as a novel genotype in Ontario, Canada, in samples from children in a tertiary pediatric hospital in Cape Town, South Africa during 2012. The genotype described in Canada was characterized by a 72-nt sequence duplication within the second variable domain of the envelope glycoprotein. The 72-nt duplication within the second variable domain in ON1 was the largest sequence duplication described in this virus (3). RSV is divided into 2 genetically distinct groups, RSV A and B, based on the viral envelope glycoprotein nucleotide sequences (4). Sequence variability in the C-terminal variable domain of the glycoprotein gene is commonly used to determine RSV phylogeny (3,5). To date, 11 RSV A (ON1, GA1–GA7, SAA1, NA1, and NA2) and 17 RSV B (GB1–GB4, SAB1–SAB3, and BA1–BA10) genotypes have been identified (3,6). As part of the aforementioned molecular epidemiology study surveying RSV infection in a pediatric hospital, (University of Cape Town research ethics study no. 305/2012), we sequenced the RSV glycoprotein second variable domain of nucleic acid extracts derived from RSV-positive respiratory secretion samples from 160 young children hospitalized for treatment of respiratory tract infections. The techniques used have been described (7). During January–April, in an area where NA1 was the dominant circulating RSV genotype, 119 (74%) of 160 RSV isolates were RSV A. We noted the presence, albeit at a low incidence, of the novel ON1 genotype cluster (8 viral isolates) (Technical Appendix) in specimens collected during February–April. Children in the RSV ON1-infected cohort were brought to health care facilities during February 24–April 25, 2012 (Figure and Technical Appendix), where they received a diagnosis of brochiolitis or bronchiopneumonia (Technical Appendix). With the exception of 1 patient, child 8, who had been hospitalized before onset of this illness, all ON1 isolates were community acquired. Seven of the 8 ON1 isolates were obtained from infants 90% lived within an 18-km radius of one another. These spatial associations with disease prevalence suggested that the ON1-infected children represented a localized cluster of transmission. Figure Alignment of deduced amino acid sequences for ON1 isolates from South Africa (Patient 1–8, accession nos. {type:entrez-nucleotide-range,attrs:{text:JX885730-JX885737,start_term:JX885730,end_term:JX885737,start_term_id:443908498,end_term_id:443908512}} ... None of the children were infected with HIV, although 3 had antenatal exposure to HIV. Co-infection with adenovirus and rhinovirus was noted in 3 of the patients. Although 3 of the patients were hospitalized for a prolonged period and required ventilatory support, the severity and outcome of the RSV ON1 infections were similar to RSV infections caused by other genotypes among children of the same age. Sequence analyses revealed that ON1 isolates identified in South Africa are essentially identical to those isolated in Canada, possessing characteristic amino acid substitutions at positions E232G, T253K, and P314L that distinguish the genotype from circulating NA1 genotypes (3). However, 7 of 8 ON1 isolates from South Africa possess a unique E308K (position 284 before insertion) amino acid change at the 3′ border of the duplicated gene segment not present in the ON1 isolates identified in Canada (Figure). The conservation of the E308K mutation within ≈90% of the isolates from South Africa that we studied suggests a possible functional role for the positively charged lysine residue. The capacity of the RSV glycoprotein to accommodate large insertions and remain functional was first demonstrated with the RSV B, BA genotype (Buenos Aires, Argentina 1999). This genotype contains a 60-nt duplication in the second variable domain, which, similar to ON1, did not cause serious clinical outcomes (6,8–10). Longitudinal analyses during 12 epidemic seasons (1996–97 through 2007–08) of international RSV subtype distribution revealed that since its initial detection in 1999, BA prevalence has gradually increased to become the dominant RSV group B virus genotype in circulation (10). Because RSV A has traditionally been the dominant RSV type in circulation, if the large insertion in ON1 confers similar selection advantage as seen in BA, the potential dominance of a single ON1 genotype within this group might promote bias on RSV type distribution toward RSV A. The novel ON1 genotype was first described in Ontario, Canada (3). Our subsequent findings in South Africa suggest extensive distribution of this genotype, which was assumed to have arisen before winter 2010–11 (3). To understand whether ON1 in South Africa occurred as a result of importation or natural evolution within locally circulating NA1 genotypes, further research is required. Technical Appendix: Respiratory syncytial virus cases among hospitalized children and epidemiologic observations for patients infected with respiratory syncytial virus ON1 in a hospital pediatric unit, Cape Town, South Africa. Click here to view.(82K, pdf)

Prevalence and clinical characteristics of human respiratory syncytial virus in Chinese adults with acute respiratory tract infection

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract illnesses worldwide. Although the prevalence and clinical manifestations of the two subtypes, RSV‐A and RSV‐B, have been studied in some detail in infants and young children, they have not been determined in adults. To evaluate the prevalence of the RSV subtypes and disease severity between RSV‐A and RSV‐B infections in adults, nasal and throat swabs that were collected from patients ≥15 years old who sought medical care for acute respiratory infections at the Fever Clinic of the Peking Union Medical College Hospital in Beijing, China between May 2005 and April 2010. The samples were tested for RSV infection using PCR and sequencing analysis. RSV was detected in 95 (1%) of the adult patients, of whom 53 (55.8%) were positive for RSV‐A and 42 (44.2%) for RSV‐B. The incidence of RSV infections increased with age (χ2 = 37.17, P = 1.66E−07). Demographic data and clinical manifestations of RSV‐A were similar to those of RSV‐B. Although RSV‐A and RSV‐B co‐circulated during the 2005–2006 and 2008–2009 seasons, RSV‐A was predominant in the 2006–2008 seasons, whereas RSV‐B was predominant in the 2009–2010 season. Upper respiratory tract infections were diagnosed in most RSV‐infected patients (n = 80, 84.2%), and three patients suffered from pulmonary infection. This is the first study to provide data on the prevalence and clinical manifestations of RSV subgroups among Chinese adults with fever and acute illness, over five successive epidemic seasons. J. Med. Virol. 85:348–353, 2013. © 2012 Wiley Periodicals, Inc.

Nitric oxide metabolites as biomarkers for influenza-like acute respiratory infections presenting to the emergency room.

Aims:Nitric oxide (NO) is increased in the respiratory tract in pulmonary infections. The aim was to determine whether nasal wash NO metabolites could serve as biomarkers of viral pathogen and disease severity in children with influenza-like illness (ILI) presenting to the emergency department (ED) during the 2009 influenza A H1N1 pandemic.Methods:Children ≤18 years old presenting to the ED with ILI were eligible. Nasal wash specimens were tested for NO metabolites, nitrate and nitrite, by HPLC and for respiratory viruses by real-time PCR.Results:Eighty-nine patients with ILI were prospectively enrolled during Oct-Dec, 2009. In the entire cohort, nasal wash nitrite was low to undetectable (interquartile range [IQR], 0 - 2 μM), while median nitrate was 3.4 μM (IQR 0-8.6). Rhinovirus (23%), respiratory syncytial virus (RSV) (20%), novel H1N1 (19%), and adenovirus (11%) were the most common viruses found. Children with RSV subtype B-associated ILI had higher nitrate compared to all other viruses combined (P=0.002).Conclusion:Concentration of NO-derived nitrate in nasal secretions in children in the ED is suggestive of viral pathogen causative for ILI, and thus might be of clinical utility. Predictive potential of this putative biomarker for ILI needs further evaluation in sicker patients in a prospective manner.

The Respiratory Syncytial Virus G Protein Conserved Domain Induces a Persistent and Protective Antibody Response in Rodents

Respiratory syncytial virus (RSV) is an important cause of severe upper and lower respiratory disease in infants and in the elderly. There are 2 main RSV subtypes A and B. A recombinant vaccine was designed based on the central domain of the RSV-A attachment G protein which we had previously named G2Na (aa130–230). Here we evaluated immunogenicity, persistence of antibody (Ab) response and protective efficacy induced in rodents by: (i) G2Na fused to DT (Diphtheria toxin) fragments in cotton rats. DT fusion did not potentiate neutralizing Ab responses against RSV-A or cross-reactivity to RSV-B. (ii) G2Nb (aa130–230 of the RSV-B G protein) either fused to, or admixed with G2Na. G2Nb did not induce RSV-B-reactive Ab responses. (iii) G2Na at low doses. Two injections of 3 µg G2Na in Alum were sufficient to induce protective immune responses in mouse lungs, preventing RSV-A and greatly reducing RSV-B infections. In cotton rats, G2Na-induced RSV-reactive Ab and protective immunity against RSV-A challenge that persisted for at least 24 weeks. (iv) injecting RSV primed mice with a single dose of G2Na/Alum or G2Na/PLGA [poly(D,L-lactide-co-glycolide]. Despite the presence of pre-existing RSV-specific Abs, these formulations effectively boosted anti-RSV Ab titres and increased Ab titres persisted for at least 21 weeks. Affinity maturation of these Abs increased from day 28 to day 148. These data indicate that G2Na has potential as a component of an RSV vaccine formulation.

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region

The epidemic pattern of respiratory syncytial virus (RSV) in Croatia is biennial. In order to determine if the circulation of different RSV subtypes affects the outbreak cycle, the aim of the present study was to analyze the epidemic pattern of RSV in children in Croatia (Zagreb region) over a period of 3 consecutive years. The study group consisted of 696 inpatients, aged 0-5 years, who were hospitalized with acute respiratory tract infections caused by RSV, in Zagreb, in the period 1 January 2006-31 December 2008. The virus was identified in nasopharyngeal secretions using direct immunofluorescence. The virus subtype was determined on real-time polymerase chain reaction. Of 696 RSV infections identified in children, subtype A virus caused 374 infections, and subtype B, 318. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by four epidemic waves of RSV infections: the first, smaller, in the spring of 2006; the second, larger, in December 2006/January 2007; the third in spring 2008, followed by a fourth outbreak beginning in November of 2008. The biennial virus cycles were persistent although the predominant RSV subtype in the first two epidemic waves was subtype B, and in the second two it was subtype A. Over a 3 year period of observation, the biennial RSV cycle in Croatia cannot be explained by a difference in the predominant circulating subtype of RSV. Other unknown factors account for the biennial cycle of RSV epidemics in Croatia.

Do Circulating RSV-Genotypes Affect Established Biennial Epidemic Periodicity in Zagreb Region?

Objective: The biennial epidemic pattern of respiratory syncytial virus (RSV) circulation in Croatia has been preserved and could not be related to climatic factors and the predominant RSV subtypes. The possibility that the circulation of different RSV genotypes affect the outbreak cycle in children in Croatia (Zagreb region) over a period of 3 consecutive years was explored in the paper. Methods: The study group consisted of inpatients, aged 0-10 years, who were hospi- talized with acute respiratory tract infections caused by RSV, in Zagreb, over the period from 1 January 2006 to 31 De- cember 2008. The virus was identified in the nasopharyngeal secretion using direct immunofluorescence method. The virus subtype and genotype was determined by real-time PCR and sequence analysis, respectively. Results: RSV infec- tions identified in 731 children. RSV subtype A caused 399 infections, and subtype B 332. Two subtype A genotypes (NA1 and GA5) and three subtype B genotypes (BA7, BA9 and BA10) were found. During persistent RSV biennial cycles namely four succeeding outbreaks, the new genotype from the previous smaller outbreak persevered into the up- coming larger outbreak. Conclusion: Our molecular-epidemiology study of RSV subtypes and genotypes during calen- dar months demonstrates that the biennial RSV cycle cannot be fully explained by the dynamic of the predominant cir- culating genotype of RSV. Other unknown factors account for the biennial cycle of RSV epidemics in Croatia.).

Viral epidemiology of acute exacerbations of chronic obstructive pulmonary disease

The role of viruses in Acute Exacerbations of Chronic Obstructive Pulmonary Disease (AECOPD) needs further elucidation. The aim of the present study was to evaluate the molecular epidemiology of viral pathogens in AECOPD.Patients presenting to the Emergency Room with AECOPD needing hospitalization were recruited. Oropharyngeal and sputum samples were collected in order to perform microarrays-based viral testing for the detection of respiratory viruses.A total of 200 (100%) patients were analyzed and from them in 107 (53.5%) a virus was detected. The commonest identified viruses were the human Respiratory Syncytial Virus (subtypes A and B) (40.5%), influenza virus (subtypes A, B, C) (11%), rhinovirus (8%) and human Parainfluenza Virus (subtypes A and B) (7.5%). A bacterial pathogen was isolated in 27 (14%) patients and a dual infection due to a bacterial and a viral pathogen was recognised in 14/107 patients. Patients with AECOPD and a viral infection had a lengthier hospital stay (9.2 ± 4.6 vs 7.6 ± 4.3, p < 0.01) while the severity of the disease was no related with significant differences among the groups of the study population.In conclusion, the isolation of a virus was strongly associated with AECOPD in the examined population. The stage of COPD appeared to have no relation with the frequency of the isolated viruses while dual infection with a viral and a bacterial pathogen was not rare.

Replacement of Previously Circulating Respiratory Syncytial Virus Subtype B Strains with the BA Genotype in South Africa

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis and pneumonia in infants, the immunocompromised, and the elderly in both developed and developing countries. Reinfections are common, and G protein variability is one mechanism to overcome herd immunity. This is illustrated by the appearance of the BA genotype with a 60-nucleotide duplication dominating the subtype B genotypes in epidemics worldwide. To investigate the evolution of subtype B in South Africa since 2002, the genetic variability of the G protein was analyzed in all recent strains isolated over 4 years (2006 to 2009) in South African hospitals. Bayesian analysis revealed a replacement of all subtype B genotypes previously identified in South Africa with the BA genotype since 2006, while subtype A genotypes identified in previous years are still circulating. Compared to BA strains from other countries, the evolutionary rate of the South African BA genotype was shown to be 2.305 × 10(-3) nucleotide substitutions/site/year and drift was evident. The most recent common ancestor (MRCA) of the South African BA viruses was determined to date back to 1996. All South African BA isolates clustered with the BA-IV subgenotype, and the appearance of new subgenotypes within this branch may occur if drift continues. Sequencing of the complete G protein of selected South African strains revealed an additional 6-nucleotide deletion. Acquisition of the 60-nucleotide duplication appeared to have improved the fitness of this virus, and more recent subtype B strains may need to be included in experimental vaccines to evaluate their efficacy in the current setting of evolved circulating strains.