BACKGROUNDHuman metapneumovirus (hMPV) has recently been recognized as a notable respiratory pathogen in children. However, no isolation processes and only a limited understanding of hMPV epidemiology present in Chinese children are documented by far.

METHODSNasopharyngeal aspirates from 86 hospitalized children with acute respiratory tract infections from December 2004 to July 2005 were inoculated onto Vero-E6 cells for hMPV isolation. Total RNA was extracted from infected cells with a cytopathic effect and then subjected to a RT-PCR amplification of the N and F genes of the hMPV. Nucleotide sequences of amplified F gene products were examined using a variation analysis with the MegAlign program and the phylogenetic tree construction using the neighbor-joining algorithm with a Phylip package.

RESULTSSix strains of hMPV were isolated from the samples during winter, spring and summer. The most common symptoms were coughing and cyanosis, and the diagnoses were bronchiolitis, bronchopneumonia, infantile asthma, or upper respiratory tract infection. All isolates were in the A2 genetic sublineage and shared a high percentage of homology with the F gene in the nucleotide (99.8%-100%) and amino acid (99.3%-100%) sequences.

CONCLUSIONSThis report indicates that hMPV is an important viral agent for acute respiratory tract infections present in Chongqing, China. Knowledge of phylogeny and genes will benefit the studies on the treatment and prophylaxis of hMPV infection.

China ; Humans ; Infant ; Infant, Newborn ; Metapneumovirus ; classification ; genetics ; isolation & purification ; Paramyxoviridae Infections ; virology ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). METHODS: From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex(TM). All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients' medical records were reviewed for clinical and demographic features. RESULTS: Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex(TM) positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. CONCLUSIONS: Multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus.
Adolescent ; Child ; Child, Preschool ; Coronavirus/classification/*isolation & purification ; Coronavirus 229E, Human/classification/genetics/isolation & purification ; Coronavirus OC43, Human/classification/genetics/isolation & purification ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Metapneumovirus/classification/genetics/*isolation & purification ; Phylogeny ; Reagent Kits, Diagnostic ; Respiratory Tract Infections/*diagnosis/virology ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Rhinovirus/classification/genetics/*isolation & purification ; Sequence Analysis, DNA