OBJECTIVETo clarify the genetic characteristics of N protein coding region of HRSV isolates from Beijing and GenBank downloaded sequences.

METHODSReverse transciption polymerase chain reaction (RT-PCR) was performed to amplify the N protein gene of 2 A and 2 B subgroups HRSV isolates from Beijing in the year 2004. The RT-PCR products were sequenced for N protein coding region. The sequences of N protein coding region of 4 Beijing isolates and those downloaded from GenBank were compared and analyzed.

RESULTSThe differences in number of nucleotide and deduced amino acid between 2 A Beijing 2004 isolates and prototype strain Long were 36-40 (3.1%-3.4%) and 4 (1.0%). The differences in number of nucleotide and deduced amino acid between 2 B Beijing 2004 isolates and prototype strain CH18537 were 17 (1.4%) and 1 (0.3%). The differences in number of nucleotide and deduced amino acid were 3-172 (0.25%-14.63%) and 0-18 (0-4.6%) respectively between 4 Beijing 2004 isolates and GenBank sequences.

CONCLUSIONN gene is the highly conservative gene in the HRSV genome. The variation between A and B subgroups were widely distributed in the entire gene of N protein, while the variation within the A or B subgroups HRSV was considerably lower.

Humans ; Molecular Sequence Data ; Nucleocapsid Proteins ; chemistry ; genetics ; Phylogeny ; Respiratory Syncytial Virus Infections ; virology ; Respiratory Syncytial Virus, Human ; chemistry ; classification ; genetics ; isolation & purification ; Sequence Homology, Amino Acid

OBJECTIVETo 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.

METHODSA 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.

RESULTSOut 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).

CONCLUSIONBoth 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.

Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Influenza, Human ; epidemiology ; Male ; Orthomyxoviridae ; classification ; isolation & purification ; Respiratory Syncytial Virus Infections ; epidemiology ; Respiratory Syncytial Viruses ; classification ; isolation & purification

OBJECTIVETo investigate the distribution of respiratory viruses on throat swabs in hospitalized children with acute lower respiratory tract infection (ALRTI).

METHODSA total of 5,150 children with ALRTI who were admitted to Hebei Children's Hospital between March 2014 and February 2015 were enrolled to investigate the distribution of respiratory viruses in children with ALRTI. Direct immunofluorescence assay was performed for throat swabs from these children to detect influenza virus A (FA), influenza virus B (FB), adenovirus (ADV), respiratory syncytial virus (RSV), and parainfluenza virus types 1, 2, and 3 (PIV-1, PIV-2, and PIV-3).

RESULTSOf all the 5,150 throat swabs from hospitalized children, 2,155 (41.84%) had positive virus detection results. RSV had the highest detection rate (1,338 cases/25.98%), followed by PIV-3 (439 cases/8.52%) and FA (166 cases/3.22%), and 29 patients had mixed infection with 2 viruses. With the increasing age, the detection rates of viruses tended to decrease (χ2=279.623; P<0.01). The positive rate of RSV increased gradually from September, and reached the peak value (60.09%) in November; the lowest positive rate occurred in June (1.51%). The positive rate of PIV-3 was the highest in May (21.38%) and the lowest in November (1.77%).

CONCLUSIONSThe distribution of viruses in children with ALRTI varies with age and season, with RSV prevalence in autumn and winter and PIV-3 prevalence in spring and summer. RSV is the most common viral pathogen that causes ALRTI in hospitalized children.

Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Orthomyxoviridae ; isolation & purification ; Parainfluenza Virus 3, Human ; isolation & purification ; Respiratory Syncytial Viruses ; isolation & purification ; Respiratory Tract Infections ; virology ; Seasons

OBJECTIVEHuman respiratory syncytial virus (hRSV) is the leading cause of acute upper and lower respiratory tract infections in infants and young children worldwide. Pediatric RSV disease claims more than 1 million lives annually. With the rapid development of specific anti-RSV agents and the spread of respiratory infections, RSV detection techniques with higher sensitivity, specificity and quicker performance are badly needed. This study was designed to develop a real-time polymerase chain reaction (PCR) for detection of RSV in nasopharyngeal aspirates.

METHODS(1) The TaqMan probe and primers of real-time PCR for RSV subgroup A and subgroup B detection were designed from the conserved region in N protein encoding gene, respectively. The sensitivity of real-time PCR was evaluated by using the virus with known amount of PFU. The specificity of real-time PCR for RSV detection was assessed by cross testing 10 isolates of strains A, 10 isolates of strains B, and by testing a variety of other respiratory viruses positive samples. (2) Sixty-one stored RSV positive respiratory samples and 103 nasopharyngeal aspirates were detected by real-time PCR, virus isolation, immunofluorescence assay (IFA), and nested-PCR.

RESULTS(1) The sensitivity of the real-time PCR developed in this study for RSV subgroup A detection was 5.25 pfu, and for subgroup B was 3.75 pfu, the same as that of nested-PCR. (2) No positive results were found in cross testing of other viruses positive specimens. (3) Twenty-seven out of 30 (90%) of RSV A stored samples and 27 out of 31 (87.1%) of RSV B stored samples were positive by the real-time PCR. (4) Thirty-five (34.0%) out of the 103 specimens were found RSV positive by real-time PCR (7 of them were subgroup A and 28 subgroup B); 31 (30.1%) specimens were positive by nested-PCR (6 of them were subgroup A and 25 subgroup B); 22 (21.4%) were found positive for RSV with IFA (5 of them were subgroup A and 17 subgroup B); RSV was isolated from 9 (8.7%) specimens (6 of them were subgroup A and 3 subgroup B). All the specimens found to be negative by real-time PCR were negative by rest of the methods used in this study.

CONCLUSIONThe real time PCR method developed in this project with the TaqMan probe and primers is sensitive and specific for detecting RSV subgroup A and B in nasopharyngeal aspirates.

Child ; DNA, Complementary ; isolation & purification ; Fluorescent Antibody Technique ; Humans ; Nasopharynx ; secretion ; virology ; Polymerase Chain Reaction ; methods ; RNA, Viral ; isolation & purification ; Respiratory Syncytial Virus Infections ; genetics ; Respiratory Syncytial Virus, Human ; genetics ; isolation & purification

OBJECTIVETo establish a rapid, sensitive and specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detecting human respiratory syncytial virus (RSV) in respiratory samples collected from children with acute respiratory infections.

METHODAccording to the conserved matrix gene sequences of respiratory syncytial virus subtypes A and B downloaded from GenBank, primers were designed and RT-LAMP assay was developed to detect RNA of RSV sensitivity of the RT-LAMP method was evaluated by using ten-fold serially diluted in vitro-transcribed matrix RNA fragments from RSV A and RSV B, respectively. Specificity of the RT-LAMP method was tested through cross-reaction with other RNA and DNA viruses. Then 5 RSV strains isolated from clinical specimens using tissue cultures were tested by RT-LAMP assay. A total of 101 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 40 positive for RSV and 61 negative for RSV, were tested by RT-LAMP assay and by RT-nested PCR.

RESULTSensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/µl of RSV A and RSV B RNA, no amplification was shown in RT-LAMP with DNA or cDNA from other viruses in 60 min, revealed that the RT-LAMP assay is highly specific. Five RSV isolates confirmed as 4 RSV A and 1 RSV B previously were detected by RT-LAMP method as positive in 30 min. For those 101 specimens tested, 37 were RSV positive determined by RT-LAMP assay, as well as 35 RSV positive by RT-nested PCR. The total coincidence rate of RT-LAMP assay with DFA and RT-nested PCR in detecting RSV is 95.0%, 94.1% with Kappa value 0.895 and 0.871, respectively.

CONCLUSIONA new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, which should be helpful in rapid detection of RSV from respiratory tract samples of children.

Acute Disease ; Child ; Child, Preschool ; DNA Primers ; Humans ; Infant ; Molecular Diagnostic Techniques ; Nasopharynx ; virology ; Nucleic Acid Amplification Techniques ; RNA, Viral ; isolation & purification ; Respiratory Syncytial Virus Infections ; diagnosis ; Respiratory Syncytial Virus, Human ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo characterize the prevalence and occurrence of subgroups of human respiratory syncytial virus (RSV) in infants and young children with acute respiratory infections (ARI) in Beijing area.

METHODSRSVs were identified from nasopharyngeal aspirates and throat swabs collected from infants and children with ARI who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics during the period of November 2000 to March 2006, by virus isolation in Hep-2 cells and antigen detection by indirect immunofluorescence assay (IFA). RT-PCR was used to differentiate subgroups A and B of RSV from part of the positive specimens.

RESULTSOut of 10 048 specimens including 7176 nasopharyngeal aspirates from inpatients and 2872 throat swabs from outpatients, 2286 (22.8%) were RSV positive. The positive rate for RSV identification were 30.0% (2153/7176) in specimens from the hospitalized patients, which was higher than that from outpatients (4.6%, 133/2872). The youngest of the RSV positive patients was 1 day after birth and the oldest was 15 years of age, with 73.0% younger than 1 year. Among those RSV positives, only 1.6% were older than 5 years. The ratio of male to female who were RSV positive was 2.4:1 (1598:674). The clinical diagnosis for 91.2% (1991) of those RSV positive patients was severe lower respiratory infections including bronchiolitis and pneumonia, whereas in only 8.8% (192) the diagnosis was upper respiratory infections. The data revealed that RSV started to be detected in October each year during the survey period and November to next April was the RSV season. The detection rate declined in May and almost no RSV could be found in summer. Positive rates for RSV detection were 42.3%, 41.0% and 40.5% in the seasons of 2001 - 2002, 2003 - 2004, 2005 - 2006, which were higher than those in seasons of 2000 - 2001 (14.0%), 2002 - 2003 (18.2%), 2004 - 2005 (20.4%). Subtyping of A and B during the surveillance period showed that 73.7% (691/938) were subgroup A and 26.3% (247/938) were subgroup B. Subgroup B was predominant in the 2000 - 2001 and 2004 - 2005 seasons, whereas subgroup A predominated in the 2001 - 2002, 2002 - 2003 and 2003 - 2004 seasons. Almost equal proportions of subgroup A and B appeared in 2005 - 2006 seasons.

CONCLUSIONThe data indicate that RSV is an important etiological agent for lower respiratory infections in infants and young children in winter and spring during the survey period. The pattern of RSV circulation varied alternately with higher rate every other year. The predominant subgroup changed between A and B, and co-circulated in equal proportion in some years.

Adolescent ; Cell Line, Tumor ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Population Surveillance ; Prevalence ; Respiratory Syncytial Virus Infections ; diagnosis ; epidemiology ; Respiratory Syncytial Virus, Human ; genetics ; isolation & purification ; Respiratory Tract Infections ; diagnosis ; epidemiology ; virology ; Seasons

OBJECTIVEAnalysis the viral pathogenic spectrum for patients with fever and respiratory tract infection syndrome in Shaanxi province during 2010 and investigate the molecular epidemiology characteristics of respiratory syncytial virus.

METHODSA total of 208 patients' pharyngeal swabs were collected based on surveillance definition from January 2010 to January 2011 and screened for sixteen human respiratory virus types/subtypes by Qiaxcel-based multiplex reverse transcription-PCR assay, including HRV,HCoV, Flu, HPIV, ADV, HRSV, HMPV and HBoV and investigate molecular epidemiology of HRSV by sequencing and phylogenetic analysis of the C-terminal second hypervariable region of the G gene.

RESULTS109 out of 208 specimens (53%) were positive for one or more viruses. HRSV(42. 2%) was the dominant pathogen detected, followed by Flu(24. 5%), PIV(20%), HRV(13.6%) and ADV( 10.9%),there were also 8 strains of HCoV, 5 strains of HMPV and 3 strains of HBoV detected. The results showed that 22 specimens were positive for two or more viruses, PIV (14/22) was the most frequently detected viral agent among co-infection specimens, and the highest incidence of mixed infection is aged 15-39 years group (P < 0.05). The overall viral detection rate was no related to age. In addition to Flu, HMPV and PIV, other viruses (HRV, HBoV, HCoV, ADV, RSV) mainly infected 0 to 4 years old children. Among 46 HRSV positive specimens, 42 HRSV-A strains clustered into NA1 genotype and two HRSV-B strains clustered into two genotypes, BA9 and GB2.

CONCLUSIONHRSV is the dominate pathogen collected from patients with fever and respiratory tract infection syndrome in Shaanxi and HRSV A is the predominant subtype. For most viruses, infection was most prevalent among children aged <4 years. PIV was the most common pathogen in co-infection.

Adolescent ; Adult ; China ; epidemiology ; Coinfection ; virology ; Female ; Fever ; epidemiology ; virology ; Genotype ; Humans ; Male ; Phylogeny ; Respiratory Syncytial Virus Infections ; epidemiology ; virology ; Respiratory Syncytial Virus, Human ; isolation & purification ; Respiratory Tract Infections ; epidemiology ; virology ; Young Adult

OBJECTIVETo investigate the nonbacterial pathogens in children with acute respiratory infection (ARI) in Nanjing.

METHODSThe presence of Mycoplasma pneumoniae (MP) and Chlamydia trachomatis (CT) was determined by quantitative PCR in the nasopharyngeal samples from 1 592 hospitalized children with ARI. Common respiratory viruses, including respiratory syncytial virus (RSV), adenovirus (ADV), influenza virus types A and B (IVA and IVB), parainfluenza virus types 1, 2, 3(PIV-1, 2, 3) and human metapneumovirus (hMPV), were detected using direct immunofluorescence assay.

RESULTSMP and CT were detected in 25.7% and 2.4% of the 1 592 samples respectively. The overall positive rate of respiratory viruses was 40.9%. Among the viruses, the top detected virus was RSV (61.3%), followed by PIV-3 (6.7%) and hMPV (4.9%). Mixed infection among MP, CT and viruses was observed in 107 cases (6.7%). The infants under 1 year old were susceptible to mix-infection (68/107, 63.6%).

CONCLUSIONSRespiratory virus is the main pathogen responsible for ARI in children from Nanjing. RSV is the most commonly identified virus. MP is also the frequently identified pathogen for ARI in children. Mixed infection is common in infants under 1 year old.

Adenoviruses, Human ; isolation & purification ; Adolescent ; Child ; Child, Preschool ; Chlamydia trachomatis ; isolation & purification ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Metapneumovirus ; isolation & purification ; Mycoplasma pneumoniae ; isolation & purification ; Orthomyxoviridae ; isolation & purification ; Respiratory Syncytial Virus, Human ; isolation & purification ; Respiratory Tract Infections ; microbiology ; virology

Airway Resistance ; Animals ; Bronchial Hyperreactivity ; etiology ; Histamine ; pharmacology ; Lung ; pathology ; virology ; Microscopy, Immunoelectron ; Neurofilament Proteins ; analysis ; Neuronal Plasticity ; Rats ; Rats, Sprague-Dawley ; Respiratory Syncytial Virus Infections ; physiopathology ; Respiratory Syncytial Virus, Human ; isolation & purification

OBJECTIVEViruses are common pathogens of acute lower respiratory tract infection (ALRTI) in children. There are few studies on consecutive monitoring of viral pathogens of ALRTI in a larger cohort during the past several years. The aim of this study was to investigate the viral pathogens of ALRTI in children of different age groups and to outline the epidemic feature of different viruses.

METHOD(1) Totally 1914 (1281 male and 709 female) children with clinical diagnosis of ALRTI during the period of March 2007 to March 2010 were recruited into this study. These patients were hospitalized patients in department of internal medicine or outpatients in emergency department in Beijing Children's Hospital. The patients were divided into four groups, including 1072 patients < 1 year old, 326 patients 1- < 3 years old, 158 patients 3- < 6 years old, 358 patients ≥ 6 years old. One nasopharyngeal aspirate specimen was collected from each patient. Reverse transcription (RT) PCR methods were applied to detect common respiratory viruses including respiratory syncytial virus (RSV), human rhinovirus (HRV), influenza virus type A, B and C (IFA, IFB, IFC), parainfluenza virus (PIV) type 1-4, adenovirus (ADV), enterovirus (EV), human coronavirus (HCOV), human metapneumovirus (HMPV) and human bocavirus (HBOV).

RESULT(1) The total positive rate of viruses was 70.3%. The positive rate was 83.0% (890/1072) in the group of < 1 year old, and 80.1% (261/326) in group of 1- < 3 years old, 60.8% (96/158) in group of 3- < 6 years old and 27.7% (99/358) in group of ≥ 6 years old, respectively. There was a significant difference in the positive rate among different age groups (χ² = 2213.5, P = 0.000). The top three viruses were RSV, HRV and PIV; and the positive rates were 50.9%, 36.2% and 12.0% respectively in group of < 1 year old. (2) The epidemic seasons of RSV and HRV were winter and spring, and PIV infection was epidemic in spring and summer. (3) The detection rates of 2 or more viruses were 38.2%, 36.4%, 30.2% and 15.2% in groups of < 1 year old, 1- < 3 years old, 3- < 6 years old and ≥ 6 years old, respectively. There was a significant difference in the mixed infection rate among different age groups (χ² = 1346.00, P = 0.000).

CONCLUSIONRSV, HRV and PIV were the most predominant pathogens in younger children with ALRTI. Different viral infections had different seasonal features. Mixed infections with two or more viruses were detected in substantial proportion of patients with ALRTI, but further studies are needed to explore the clinical significance of mixed infection with viruses in patients with ALRTI.

Acute Disease ; Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Human bocavirus ; isolation & purification ; Humans ; Infant ; Male ; Parainfluenza Virus 1, Human ; isolation & purification ; Parvoviridae Infections ; epidemiology ; virology ; Respiratory Syncytial Virus Infections ; epidemiology ; virology ; Respiratory Syncytial Virus, Human ; isolation & purification ; Respiratory Tract Diseases ; epidemiology ; virology ; Respirovirus Infections ; epidemiology ; virology