OBJECTIVETo investigate the status of enterovirus (EV) infection in children with acute lower respiratory tract infection (ALRTI).

METHODSA total of 404 samples (with odd numbers) of nasopharyngeal aspirates were collected from the children who were hospitalized in the Children's Medical Center, Hunan Provincial People's Hospital due to ALRTI between September 2007 and April 2008. The conserved sequence in the 5'-noncoding region of EV was used to design the primer, and nested RT-PCR was performed to detect EV in the samples.

RESULTSOf the 404 samples, 19 (4.7%) were EV-positive, and mostly taken from children under 3 years of age (95%); there was no significant difference in the detection rate between male and female children. Of the EV-positive children, 13 (68%) were clinically diagnosed with bronchial pneumonia, and 6 (32%) with bronchiolitis; 90% of them showed symptoms of fever, 84% had a cough, 63% had asthma, and 63% had complications mainly including diarrhea (6 cases), granulocytopenia (4 cases), and acute respiratory distress syndrome (2 cases). In addition, 26% of the EV-positive children had leukocyte disorder, more than half had liver dysfunction, and a few had myocardial involvement.

CONCLUSIONSEV is a pathogen that should not be neglected in children with ALRTI. For these children, close attention should be paid to the epidemiological status and clinical features of EV infection, and blood routine examination, liver function test and myocardial enzyme assay should be carried out periodically to improve prognosis.

Acute Disease ; Child ; Child, Preschool ; Enterovirus Infections ; epidemiology ; Female ; Humans ; Infant ; Male ; Nasopharynx ; virology ; Respiratory Tract Infections ; virology

We designed specific primers and fluorescence-labeled probes to develop real-time and conventional RT-PCR assays for detection of human coronavirus NL63 or HKU1. Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The detection limit of the real-time RT-PCR assays was 10 RNA copies per reaction mixture. No cross-reactivity was observed between RNA samples derived from designed HCoV and other HCoV or human metapneumovirus. A total of 158 nasopharyngeal swab specimens collected from adult patients with acute respiratory tract infection in Beijing were screened for the presence of human coronavirus NL63 and HKU1 by using real-time RT-PCR and conventional RT-PCR method. The fluorescence quantitative RT-PCR method detected six specimens positive for human coronavirus NL63, five specimens positive for human coronavirus HKU1; and conventional RT-PCR method detected three HCoV-NL63 positive and three HCoV-HKU1 positive, respectively. The convention RT-PCR products of positive samples were obtained and sequence analysis confirmed the reliability of the above methods. In summary, the real-time RT-PCR assay for HCoV- NL63 or HKU1 was more sensitive than conventional RT-PCR and with less time (less than 4 hours) for completion. It may be suitable for molecular epidemiological surveillance and clinical diagnosis for human coronavirus NL63 and HKU1.
Coronavirus ; classification ; genetics ; isolation & purification ; Humans ; Nasopharynx ; virology ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

OBJECTIVETo explore the viral etiology of acute low respiratory tract infection (ALRTI) among hospitalized children in Changsha of Hunan Province of China.

METHODSNasopharyngeal aspirates were collected from 1165 hospitalized children with ALRTI in Changsha from September 2007 to August 2008. Respiratory syncytin virus (RSV), human rhinovirus (HRV), influenza virus A (IFVA), influenza virus B (IFVB), parainfluenza 1-3 (PIV 1-3), human metapneumovirus (hMPV), human coronaviruses NL63 (HCoV-NL63), and human coronaviruses HKU1 (HCoV-HKU1) were detected by reverse transcription polymerase chain reaction (RT-PCR). Adenovirus (ADV) and human bocavirus (HBoV) were detected by standard polymerase chain reaction (PCR). WU polyomaviruses (WUPyV) and KI polyomaviruses(KIPyV) were detected by nested PCR. The positive samples further underwent genetic sequencing.

RESULTSAmong the 1165 nasopharyngeal aspirates, viruses were detected in 871 samples (74.76%), among which RSV (27.03%) was the most common virus, followed by HRV (17.33%), PIV3 (13.73%), HBoV (8.67%) and hMPV (6.52%). The overall positive rate of viral detection showed no significant differences between males and females (X2=2.241, P=0.134), whereas the positive rates of PIV3, hMPV, and HBoV in males were higher than in females. The positive rate of viral detection showed significant differences among different age groups (X2=10.934, P=0.027), and the highest positive rate was noted in the age group of 6 months to 1 year. Furthermore, the overall positive rate of viral detection showed a significant difference in term of seasonal distribution, with a peak prevalence in winter.

CONCLUSIONSVirues predominate in the etiology of pediatric ALRTI in Changsha, and RSV, HRV and PIV3 are the main viruses for ALRTI. HBoV and hMPV have become increasingly important. Viral infection-associated ALRTI shows a prevail in the age group of 6 months to 1 year as well as in winter.

Adolescent ; Age Distribution ; Child ; Child, Hospitalized ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Nasopharynx ; virology ; Respiratory Tract Infections ; etiology ; virology ; Seasons ; Sex Distribution ; Viruses ; isolation & purification

OBJECTIVETo observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization.

METHODSThe morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining.

RESULTSMorphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture.

CONCLUSIONSNasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells. Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.

Cell Transformation, Viral ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Epithelial Cells ; physiology ; virology ; Herpesvirus 4, Human ; physiology ; Humans ; Nasopharynx ; cytology ; virology ; Tetradecanoylphorbol Acetate ; pharmacology

Human metapneumovirus (HMPV) shares clinical and epidemiological characteristics with well-known respiratory syncytial virus (RSV). The aim of this study was to investigate the clinical and epidemiological differences between HMPV- and RSV-induced wheezing illnesses. A total of 1,008 nasopharyngeal aspirate specimens was collected from 1,008 pediatric patients hospitalized with acute respiratory tract infection at Inje University Sanggye Paik Hospital from December 2003 to April 2008, and tested for seven common respiratory viruses. Conditions classified as wheezing illness were bronchiolitis, reactive airways disease, and bronchial asthma. HMPV caused a significantly lower proportion of wheezing illness when compared to RSV (48.1% vs. 82.2%, P<0.05). HMPV-induced wheezing illness occurred predominantly in older patients when compared to RSV patients (P<0.001). RSV infections peaked in the fall and winter followed by peaks of HMPV infection in winter and spring. Eosinophil counts were significantly higher (P<0.01) in RSV patients when compared to HMPV patients. These results show that human metapneumovirus patients exhibit several different clinical and epidemiological characteristics, such as higher proportion of wheezing illness, age and seasonal incidence, and eosinophil counts, when compared to RSV patients.
Bronchiolitis/physiopathology/virology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Korea/epidemiology ; Male ; Metapneumovirus/pathogenicity ; Nasopharynx/virology ; Paramyxoviridae Infections/*epidemiology/*physiopathology/virology ; Respiratory Sounds/*physiopathology ; Respiratory Syncytial Virus Infections/*epidemiology/*physiopathology/virology ; Respiratory Syncytial Viruses/pathogenicity ; Retrospective Studies ; Seasons

OBJECTIVETo investigate the Epstein-Barr virus (EBV) BamH I "f" variant in primary nasopharyngeal carcinoma (NPC) and its metastases in lymph nodes (LN).

METHODSIn situ hybridization was used to detect EBV-encoded small RNA (EBER) expression in 21 paired paraffin-embedded tissue from primary NPC and their lymph node metastases and 22 primary NPC without lymph node metastasis. PCR and restriction fragment length polymorphism (RFLP) assay were used to detect EBV BamH I "f" variant in all cases of NPCs, lymph node metastases and 50 cases of chronic inflammation of nasopharynx from Canton.

RESULTSAll cases of NPCs and their lymph node metastases showed EBER expression, indicating a high EBV-positive rate in Cantonese NPC patients. EBV BamH I "f" variant was found in 11 cases (52.4%, 11/21) of primary NPCs with LN metastasis, 12 cases (57.1%, 12/21) of the LN metastases, and 18 cases (81.8%, 18/22) of primary NPCs without LN metastasis. However, of the 50 cases of chronic inflammation of nasopharynx, only one case (2.1%, 1/47) demonstrated BamH I "f" variant. The frequency of BamH I "f" variant in NPC was therefore dramatically higher than that in chronic inflammation of nasopharynx. It is of note that atypical hyperplasia was observed in a few epithelial cells from the case of chronic inflammation of nasopharynx expressing BamH I "f" variant.

CONCLUSIONSThe frequency of EBV BamH I "f" variant in NPC is significantly higher than that in chronic inflammation of nasopharynx. It is the first demonstration that the BamH I "f" variant is also present in the LN metastases of NPC. The frequency of BamH I "f" variant in metastatic NPC of the lymph node is almost equal to that of primary NPCs.

Epithelial Cells ; drug effects ; Epstein-Barr Virus Infections ; classification ; complications ; virology ; Herpesvirus 4, Human ; classification ; genetics ; Humans ; In Situ Hybridization ; Lymph Nodes ; drug effects ; pathology ; virology ; Lymphatic Metastasis ; physiopathology ; Nasopharyngeal Neoplasms ; genetics ; pathology ; virology ; Nasopharynx ; virology ; RNA, Viral ; analysis ; pharmacology

OBJECTIVETo explore the causative role of human bocavirus (HBOV) played in acute respiratory infection and diarrhea in children, a case-control study was prospectively conducted to investigate HBOV detection in symptomatic children with acute respiratory tract infection, diarrhea and asymptomatic children.

METHODBetween Oct. and Dec. of 2008, 436 nasopharyngeal aspirates (NPA) from hospitalized children with acute respiratory infection and 150 NPA from asymptomatic children undergoing cardiac operations were consecutively collected. During the same time, 220 stool samples were taken from outpatients with acute watery diarrhea and 200 control specimens were obtained from children without diarrhea. HBOV was screened in all samples by real-time PCR method. HBOV-positive respiratory samples were tested for other 9 common respiratory viruses and Mycoplasma pneumoniae. HBOV-positive fecal samples were also tested for common enteric viruses causing diarrhea.

RESULTHBOV was detected in NPA samples from 45 (10.3%) of 436 symptomatic patients and from 1(0.7%) of 150 asymptomatic control children. There was a statistically significant difference in the detection rates of HBOV between the symptomatic group and the asymptomatic group (P < 0.001). HBOV co-existence with other respiratory pathogens occurred in 44.7% (20/45) of NPA from symptomatic patients. HBOV was detected in 10.3% (43/417) children with community-acquired respiratory infection and 10.5% (2/19) children with nosocomial respiratory infection. Children with HBOV infection were 1.3 to 72 months of age (mean: 18.3 ± 13.6 months). HBOV was found positive in 6 (2.7%) of 220 stool samples from diarrheal outpatients and in 4 (2%) of 200 control samples. All children with HBOV positive detection in the stool samples were less than 4 years old. No statistical significance was found (P > 0.05) in HBOV between diarrhea patients and asymptomatic ones. In addition, 5 of 6 HBOV-positive fecal specimens from children with diarrhea were found co-infected with rotavirus.

CONCLUSIONThis study supports that HBOV is related to acute respiratory infection in children and HBOV infection usually occurs in infants and young children. However, further study is needed to clarify if HBOV plays a pathogenic role in diarrhea in children.

Adolescent ; Case-Control Studies ; Child ; Child, Preschool ; Diarrhea ; virology ; Feces ; virology ; Female ; Human bocavirus ; isolation & purification ; Humans ; Infant ; Male ; Nasopharynx ; virology ; Parvoviridae Infections ; Prospective Studies ; Respiratory Tract Infections ; virology

BACKGROUND: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. METHODS: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0x10(6) copies/mL as the threshold value of viral load. RESULTS: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78x10(5) copies/mL vs. 1.94x10(4) copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. CONCLUSIONS: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA, Viral/analysis ; Female ; Human bocavirus/*isolation & purification ; Humans ; Infant ; Male ; Middle Aged ; Nasopharynx/virology ; Parvoviridae Infections/diagnosis/*epidemiology/virology ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections/diagnosis/*epidemiology/virology ; Viral Load

BACKGROUND: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. METHODS: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0x10(6) copies/mL as the threshold value of viral load. RESULTS: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78x10(5) copies/mL vs. 1.94x10(4) copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. CONCLUSIONS: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA, Viral/analysis ; Female ; Human bocavirus/*isolation & purification ; Humans ; Infant ; Male ; Middle Aged ; Nasopharynx/virology ; Parvoviridae Infections/diagnosis/*epidemiology/virology ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections/diagnosis/*epidemiology/virology ; Viral Load