OBJECTIVEHuman parainfluenza virus (HPIV) types 1, 2 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections. In this study, a real-time RT-PCR was developed using multiplex primers-probe (HPIV-1, 2, 3) for the simultaneous detection of both HPIV1, HPIV2 and HPIV3 genomes.

METHODSOptimal primers and probes were designed using specialized software. The conditions for multiplex real-time RT-PCR had been optimized. The synthesis of RNA standards of HPIV1, 2, 3 were used a T7 RNA polymerase. Check the specificity sensitivities and stability of one step RT-PCR assay.

RESULTSObtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of 10 copies for HPIV1, 100 copies for HPIV2 and 100 copies for HPIV3.

CONCLUSIONThe assays demonstrates an improved sensitivity and scope of detecting HPIV1, 2, 3 viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the pre-diagnosis and respiratory virus pathogenesis.

Humans ; Oligonucleotides ; genetics ; Parainfluenza Virus 1, Human ; isolation & purification ; Parainfluenza Virus 2, Human ; isolation & purification ; Parainfluenza Virus 3, Human ; isolation & purification ; Real-Time Polymerase Chain Reaction ; methods

Human parainfluenza viruses (HPIVs) is one of the main causes of acute respiratory tract infections in children. HPIVs have been grouped into four serotypes (HPIV1~HPIV4) according to serological and genetic variation. Different serotypes of HPIVs have diverse clinical disease spectrum, epidemic characteristics and disease burden. Based on the nucleotide variation in structural protein genes, HPIVs can be further divided into distinct genotypes and subtypes with diverse temporal and spatial distribution features. The standard molecular typing methods are helpful to clarify the gene evolution and transmission patterns of HPIVs in the process of population transmission. However, the development of molecular epidemiology of HPIVs has been hindered by the lack of a standardized molecular typing method worldwide. Therefore, this study reviewed the viral characteristics, genome structure, existing genotyping methods and evolution of HPIVs, and screened the reference strains for molecular typing, so as to improve the understanding of gene characteristics and molecular typing of HPIVs, and provide an important scientific basis for the monitoring and research of molecular epidemiology of HPIVs in China.
Child ; Humans ; Molecular Typing ; Parainfluenza Virus 1, Human/genetics* ; Parainfluenza Virus 2, Human/genetics* ; Parainfluenza Virus 3, Human/genetics* ; Paramyxoviridae Infections/epidemiology* ; Respiratory Tract Infections/epidemiology*

PURPOSE: Croup, a common childhood respiratory illness with various severities, has many unanswered questions. Laryngotracheobronchopneumonitis (LTBP) is a disease entity considered to be an extension of croup to the lower respiratory tract. The object of this study was to compare epidemiology, clinical characteristics, and viral etiologic spectrum between croup and LTBP. METHODS: Patients hospitalized with croup at Gachon University Gil Hospital from January 2010 to April 2016 were recruited. LTBP was defined as pneumonia confirmed on radiographs of patients with croup. Clinical findings and demographic data were reviewed of patients whose nasopharyngeal swabs were done for viral analysis. RESULTS: A total of 371 patients with only croup and 63 patients with LTBP were included. Croup was found to be significantly associated with parainfluenza virus type 1 (P=0.006). LTBP was related to parainfluenza virus type 3, respiratory syncytial virus, and human bocavirus (P=0.001, P=0.030, and P=0.019, respectively). The duration of fever was longer in patients with LTBP than in those with croup (3.87±1.85 days vs. 2.86±1.80 days, P<0.001). CONCLUSION: Specific etiologic viruses might be associated with the progression from croup to LTBP. Pronged fever is also associated with progression from croup to LTBP.
Child* ; Croup* ; Epidemiology ; Fever ; Human bocavirus ; Humans ; Parainfluenza Virus 1, Human ; Parainfluenza Virus 3, Human ; Pneumonia ; Respiratory Syncytial Viruses ; Respiratory System

BACKGROUNDAlthough human parainfluenza virus (HPIV) has been determined as an important viral cause of acute respiratory infections (ARIs) in infants and young children, data on long-term investigation are still lacking to disclose the infection pattern of HPIV in China.

METHODSNasopharyngeal aspirates were collected from 25,773 hospitalized pediatric patients with ARIs from January 2004 through December 2012 for respiratory virus screen by direct immuno-fluorescence assay.

RESULTSOut of these specimens, 1675 (6.50%, 1675/25,773) showed HPIV positive, including 261 (1.01%, 261/25,773) for HPIV1, 28 (0.11%, 28/25,773) for HPIV2, and 1388 (5.39%, 1388/25,773) for HPIV3, 2 of the samples were positive for both HPIV1 and HPIV3, and 36 were co-detected with other viruses. The positive rates of HPIVs were higher in those younger than 3 years old. HPIV3 was detected from all age groups, predominantly from patients under 3 years of age, and the highest frequency was found in those 6 months to 1-year old (352/4077, 8.63%). HPIV3 was the dominant type in each of the years detected between May and July. HPIV1 showed a peak in every odd year, mainly in August or September. HPIV was detected most frequently from patients with upper respiratory infection (12.49%, 157/1257), followed by bronchitis (11.13%, 176/2479), asthma (9.31%, 43/462), bronchiolitis (5.91%, 150/2536), pneumonia (6.06%, 1034/17,068), and those with underlying diseases (1.0%, 15/1506). HPIV3 is the dominant type in these six disease groups referred above, especially in the asthma group.

CONCLUSIONSHPIV is one of the important viral causes of ARIs in infants and young children in Beijing based on the data from the hospitalized children covering a 9-year term. HPIV3 is the predominant type in all these years and in most of the disease groups. HPIVs with different types show different seasonality.

Beijing ; epidemiology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Parainfluenza Virus 1, Human ; pathogenicity ; Parainfluenza Virus 3, Human ; pathogenicity ; Respirovirus ; pathogenicity ; Respirovirus Infections ; diagnosis ; virology

PURPOSE: Croup is a common respiratory disease in children. The aim of this study was to analyze the epidemiology, etiology, and seasonal variations of respiratory virus infections in children with croup. METHODS: From October 2009 to September 2017, children admitted with croup to Gachon University Gil Medical Center under the age of 18 years were enrolled in this study. We retrospectively reviewed patients' medical records. RESULTS: A total of 1,053 of 27,330 patients (3.9%) infected with lower respiratory infections were diagnosed as having croup. In the age distribution, croup was most common (50.0%) in children aged 1 to <2 years. There were 2 peaks, the major in summer (July to August) and the minor in spring (March to May). Parainfluenza virus type 1 (15.8%) was most prevalent and coincided with the summer peaks of croup. Influenza virus type B and parainfluenza virus type 3 were the most frequent etiologic agents in a spring peak of croup. Although parainfluenza virus type 1 was predominant of all ages, human coronavirus was a significant cause of croup in children younger than 1 year, whereas influenza virus played an important role in children above the age of 3 years. CONCLUSION: Seasonality and epidemiology of croup varied with age and regions. Two peaks of seasonal fluctuation were in summer and spring, which were related to the seasonality of respiratory viruses in croup. These results may be helpful in planning clinical and research needs.
Age Distribution ; Child ; Coronavirus ; Croup* ; Epidemiology ; Humans ; Medical Records ; Orthomyxoviridae ; Parainfluenza Virus 1, Human ; Parainfluenza Virus 3, Human ; Respiratory System* ; Respiratory Tract Infections* ; Retrospective Studies ; Seasons*

OBJECTIVETo understand the relationship of parainfluenza virus (PIV) and acute respiratory infections in infants and young children in Beijing, occurred in recent years.

METHODS3141 throat swab/nasopharyngeal aspirate specimens were collected from infants and young children with acute respiratory tract infections in Beijing from Jan 2001 to Dec 2003. All of these 3141 specimens were inoculated into MDCK cells for influenza virus and PIV isolation, since PIV had been isolated in MDCK cells in this laboratory from preliminary studies. Out of 3141 specimens, 702 were inoculated into MDCK as well as Vero cells to compare the sensitivity on virus isolation of these cell lines by micro plate method. Growth of PIV in cell culture were identified by haemoagglutination test and indirect immunofluorescent assay.

RESULTSThe PIV positive cases in Vero cells were also positive in MDCK cells, indicating that the sensitivity for PIV isolation in MDCK was equal to Vero cells. Out of these 3141 specimens, 94 (3.0%) were PIV positive, including 35 (35/1191, 2.9%) of PIV1, 11 (11/1191, 0.9%) of PIV3 in upper respiratory tract infections; 15 (15/1634, 0.9%) of PIV1, 24 (24/1634, 1.5%) of PIV3 in lower respiratory tract infections; 3 (3/207, 1.4%) of PIV in asthma; 1 (1/38) of PIV in patients with fever; 5 (5/71) of PIV in others. Data indicated that among upper respiratory tract infections caused by PIV, PIV1 was more commonly seen than PIV3.

CONCLUSIONMDCK cells could be used for PIV isolation from clinical samples while PIV was one of the important pathogenic viruses causing acute respiratory tract infections in infants and young children in Beijing for the recente years.

Acute Disease ; Animals ; Cell Line ; Cercopithecus aethiops ; Child, Preschool ; China ; Humans ; Infant ; Parainfluenza Virus 1, Human ; isolation & purification ; pathogenicity ; Parainfluenza Virus 3, Human ; isolation & purification ; pathogenicity ; Paramyxoviridae Infections ; diagnosis ; Respiratory Tract Infections ; virology ; Sensitivity and Specificity ; Vero Cells ; Virus Cultivation ; methods

PURPOSE: Acute lower respiratory tract infections (ALRI) are one of the most common causes of morbidity in children. Most infections are known to be caused by virus and bacteria, greater percentage caused by virus than bacteria. This study was aimed to define the viral etiologic agents, age distribution, clinical manifestations, and seasonal occurrences of viral ALRI in Korean children, during 1996 and 2002. METHODS: A total of 4, 311 patients who had been hospitalized for ALRI at Samsung Medical Center, from March 1996 to September 2002, were studied. Nasopharyngeal aspirates were obtained for virus culture. Respiratory viruses were identified by indirect immunofluorescent staining. RESULTS: One or more viral agents were isolated in 14.8% (639 cases). The pathogens identified were RSV (21.8%), influenza virus type A (21.3%), adenovirus (20.7%), parainfluenza virus type 3 and 1 (17.4%, 8.3%), influenza virus type B (7.4%). The clinical patterns of viral ALRI were pneumonia (49%), bronchiolitis (22%), tracheobronchitis (15%) and croup (14%). The occurrence of viral ALRI was highest in the 1st year of life. Pneumonia was developed mostly by adenovirus. The most frequent cause of bronchiolitis was RSV. Croup was frequently caused by parainfluenza and influenza virus. Infections with influenza virus type A, B, parainfluenza virus type 1, 3, and RSV occurred in epidemics, whereas adenovirus was isolated throughout the study period. CONCLUSION: These data expand our understanding of the etiology of ALRI among pediatric inpatients in Seoul, Korea and may contribute to the prevention and control of viral respiratory tract infection.
Adenoviridae ; Age Distribution ; Bacteria ; Bronchiolitis ; Child ; Child, Hospitalized* ; Croup ; Epidemiologic Studies* ; Humans ; Inpatients ; Korea* ; Orthomyxoviridae ; Parainfluenza Virus 1, Human ; Parainfluenza Virus 3, Human ; Paramyxoviridae Infections ; Pneumonia ; Respiratory System* ; Respiratory Tract Infections* ; Seasons ; Seoul*

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.
Glycosylation ; HN Protein ; chemistry ; genetics ; metabolism ; Humans ; Mutation ; Parainfluenza Virus 3, Human ; chemistry ; enzymology ; genetics ; physiology ; Protein Binding ; Receptors, Virus ; metabolism ; Respirovirus Infections ; metabolism ; virology ; Virus Internalization

PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.
Adenoviridae ; Child ; Child, Hospitalized ; Epidemiologic Studies ; Fluorescent Antibody Technique ; Humans ; Influenza, Human ; Korea ; Metapneumovirus ; Orthomyxoviridae ; Parainfluenza Virus 3, Human ; Pediatrics ; Respiratory Tract Infections ; Rhinovirus ; Viruses

PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.
Adenoviridae ; Child ; Child, Hospitalized ; Epidemiologic Studies ; Fluorescent Antibody Technique ; Humans ; Influenza, Human ; Korea ; Metapneumovirus ; Orthomyxoviridae ; Parainfluenza Virus 3, Human ; Pediatrics ; Respiratory Tract Infections ; Rhinovirus ; Viruses