This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by C_t value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 (P < 0.001) and 0.887 (P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 (P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.
Humans ; Respiratory Syncytial Virus Infections/diagnosis* ; Respiratory Syncytial Virus, Human/genetics* ; Nucleic Acid Amplification Techniques ; Real-Time Polymerase Chain Reaction ; Adenoviridae ; Sensitivity and Specificity

Objective: To investigate the relationship between amino acid variations of respiratory syncytial virus (RSV) nonstructural protein (NS) 1 and the clinical characteristics. Method: A retrospective case review was conducted. From December 2018 to January 2020, a total of 81 cases of hospitalized children who were tested only positive for RSV by RT-PCR or PCR at the Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University were included in the study. The NS1 genes of RSV subtype A and subtype B were amplified by PCR and sequenced. The amino acid sequences were analyzed. The Chi-square test and Mann-Whitney rank sum test were used to compare the clinical characteristics and type Ⅰ interferon levels of children with or without NS1 variation in the variation and non-variation groups. Results: Among 81 cases, there were 58 males and 23 females. There were 11 cases in the variation group, the age of onset was 2.0 (1.0, 11.0) months, included 4 cases of subtype A (variant sites were: 2 cases for Lys33Gln, one case for Gly2Asp, Pro67Ser, Leu137Phe, respectively) and 7 cases of subtype B (variant sites were: two cases for Val121Ile, one case for Tyr30Cys, Val65Met, Asn85Ser, Ser118Asn, Asp124Asn, respectively). These variant sites all appeared at a very low frequency 0.08 (0.04, 0.29) % in the NCBI PROTEIN database. There were 70 cases in non-variation group, the onset age was 3.5 (1.0, 7.0) months. The proportion of dyspnea in the variation group was higher than that in the non-variation group (10/11 vs. 47% (33/70), χ²=7.31, P<0.01). Conclusions: There are some variant sites in nonstructural protein NS1 of RSV. Children may be prone to have dyspnea with NS1 variations.
Child ; Male ; Female ; Humans ; Infant ; Respiratory Syncytial Virus Infections ; Amino Acids ; Retrospective Studies ; Respiratory Syncytial Virus, Human/genetics* ; Polymerase Chain Reaction

OBJECTIVETo 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).

METHODNasopharyngeal 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.

RESULTOf 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.

CONCLUSIONThe 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.

Child, Preschool ; China ; epidemiology ; Genotype ; Hospitalization ; Humans ; Infant ; Nasopharynx ; Phylogeny ; Polymerase Chain Reaction ; Respiratory Syncytial Virus Infections ; epidemiology ; Respiratory Syncytial Virus, Human ; genetics ; Respiratory Tract Infections ; epidemiology ; Seasons

This study was performed to characterize respiratory viral infections in pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). Study samples included 402 respiratory specimens obtained from 358 clinical episodes that occurred in the 116 children of the 175 consecutive HSCT cohort at Seoul National University Children's Hospital, Korea from 2007 to 2010. Multiplex reverse-transcription polymerase chain reactions were performed for rhinovirus, respiratory syncytial virus (RSV), parainfluenza viruses (PIVs), adenovirus, human coronavirus (hCoV), influenza viruses and human metapneumovirus. Viruses were identified in 89 clinical episodes that occurred in 58 patients. Among the 89 clinical episodes, frequently detected viruses were rhinovirus in 25 (28.1%), RSV in 23 (25.8%), PIV-3 in 16 (18.0%), adenovirus in 12 (13.5%), and hCoV in 10 (11.2%). Lower respiratory tract infections were diagnosed in 34 (38.2%). Neutropenia was present in 24 (27.0%) episodes and lymphopenia was in 31 (34.8%) episodes. Sixty-three percent of the clinical episodes were hospital-acquired. Three patients died of respiratory failure caused by respiratory viral infections. Respiratory viral infections in pediatric patients who have undergone HSCT are common and are frequently acquired during hospitalization. Continuous monitoring is required to determine the role of respiratory viruses in immunocompromised children and the importance of preventive strategies.
Adenoviridae/genetics/isolation & purification ; Adolescent ; Adult ; Child ; Child, Preschool ; Cohort Studies ; Coronavirus/genetics/isolation & purification ; Female ; *Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/cytology ; Hospitalization ; Humans ; Infant ; Lymphopenia/epidemiology ; Male ; Neutropenia/epidemiology ; Parainfluenza Virus 3, Human/genetics/isolation & purification ; Prevalence ; Respiratory Syncytial Viruses/genetics/isolation & purification ; Respiratory Tract Infections/epidemiology/therapy/*virology ; Reverse Transcriptase Polymerase Chain Reaction ; Rhinovirus/genetics/isolation & purification ; Seasons ; Young Adult

Humans ; Molecular Epidemiology ; Respiratory Syncytial Virus Infections ; epidemiology ; virology ; Respiratory Syncytial Virus, Human ; genetics

To investigate whether influenza virus (IFZ) could up-regulate the expression of mucin 1 (MUC1) which exists in epithelial cells of upper respiratory track to restrict the inflammation, as respiratory syncytial virus (RSV) does. Quantitative RT-PCR and Western Blot were performed to detect the expression level of MUC1 induced by two single-strand RNA viruses in A549 cell lines. HEp-2 and MDCK cells were used respectively to culture RSV and IFZ. At 24h post A549 cells infection with the same titer of RSV or IFZ, the total RNA was harvest, qRT-PCR was then performed to observe the expression level of MUC1 mRNA. Meanwhile, at 24 h and 48 h post A549 cells infection with the same titer of RSV or IFZ, the total protein and supernatant were collected respectively after cell lysis, Western Blot was then used to detect the expression level of MUC1. Results showed that RSV could up-regulate the expression of MUC1 in airway epithelial cells with a significant dose-effect correlation, whereas IFZ could not. This study firstly investigated the differences of the regulation on the expression of MUC1 induced by two single-strand RNA viruses, and demonstrated initially that the mechanism of IFZ self-limiting differed from RSV, which attributed to up-regulation of the expression level of MUC1.
Animals ; Cell Line ; Dogs ; Epithelial Cells ; metabolism ; virology ; Gene Expression Regulation ; Humans ; Influenza, Human ; genetics ; metabolism ; virology ; Madin Darby Canine Kidney Cells ; Mucin-1 ; genetics ; metabolism ; Orthomyxoviridae ; genetics ; physiology ; Respiratory Syncytial Virus Infections ; genetics ; metabolism ; virology ; Respiratory Syncytial Viruses ; genetics ; physiology ; Up-Regulation

To characterize nucleocapsid (N) protein genes of human respiratory syncytial viruses isolated from children in Beijing and express the N genes in E. coli,seven HRSV strains (three subtype A and four subtype B) were isolated from clinical samples of infants and children with acute respiratory infections and visited the Children's Hospital affiliated to Capital Institute of Pediatrics in Beijing during the period of Jan. 2006 to Mar. Full length of N genes from seven HRSV strains were amplified by reverse-transcription PCR (RT-PCR). The seven PCR amplicons were sequenced after cloning into pUCm-T and the sequences were compared with the N genes from HRSVs in GenBank. N gene was amplified from recombinant plasmid pUCm-N9968 by PCR and then sub-cloned into the prokaryotic expression vector pET30a(+) after digestion with EcoR I and Xho I . The pET30a-N9968 was transformed into E. coli BL21 (DE3) and expressed by inducing with IPTG. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. The amplified N genes were 1 176 bp in length and the deduced N proteins were 391 amino acids in length. The nucleotide identities of N genes among these seven strains were 85.4%-99.7% and the de-duced amino acid similarities were 95.4%-100%. The recombinant plasmid pET30a-N9968 had correct open reading frame confirmed by dual-enzyme digestion and sequence analysis. The fusion protein 6 x HisN was produced after inducing by 1 mmol/L IPTG at 37 degrees C. A unique protein band with molecular weight 49 kD was characterized by SDS-PAGE and purified by Ni2+ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein had specific binding reaction to specific monoclonal antibody and human sera, indicating that the expressed protein is of specific antigenicity.
Base Sequence ; Child ; Escherichia coli ; genetics ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; Respiratory Syncytial Virus, Human ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

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

Child ; DNA, Antisense ; genetics ; DNA, Viral ; genetics ; Genetic Therapy ; methods ; Humans ; RNA, Antisense ; genetics ; Respiratory Syncytial Virus Infections ; therapy ; Respiratory Syncytial Virus, Human ; genetics ; pathogenicity

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