Detection And Analysis Of HCoV-NL63,HCoV-229E And HBoV In Children With Acute Respiratory Tract Infections In Fuzhou

ObjectiveTo investigate the prevalence and clinical features of the novel human coronavirus NL63 (HCoV-NL63),human coronavirus229E (HCoV-229E) and human bocavirus (HBoV) in children with acute respiratory tract infections in Fuzhou.MethodsA total of 223 nasopharyngeal secretion samples were collected from children who visited Fujian provincial hospital with acute respiratory tract infections during the period of Nov.2006 to May 2007. Of the 223samples, aged 15 days～13years old. 134were from children with upper respiratory tract infections,89were from lower respiratory tract infections.Single Reverse Transcription-Polymerase Chain Reaction(RT-PCR) was used for the detection of 1b gene and 1a gene of HCoV-NL63 ,and NP1 gene of HboV. A nested RT-PCR was used for M gene of HCoV-229E.And A multiplex RT-PCR(mRT-PCR) was established for the detection of 1b gene of HCoV-NL63 , NP1 gene of HBoV, and M gene of HCoV-229E. The PCR products with expected molecular mass were selected ramdomly for DNA sequencing.The DNA sequences from those PCR products were compared with the sequences of reference strain in the GenBank.Homological analysis and phylogenetic analysis were also performed for the sequences of them.Results1.Of the 223 samples, HCoV- NL63 was detected in 3 (1.3%),HCoV-229E in 2(0.9%), HBoV in 9(4.0%).2.Three samples were positive for 1b gene of HCoV-NL63,and also showed positive results on 1a gene.Their nucleotide sequences were consistent with each other.The nucleotide sequence was 97% to 100% homology with that of the reference strains in the GenBank,and the amino acid sequence was consistent with that of the reference strains. The phylogenetic tree based on the nucleotide sequence of the PCR products showed that, the HCoV-NL63 strain had the closely relationship with HCoV-NL63 refrence strain whose accession numbers in Genbank are NC-005831 and AY675541.3. DNA sequencing was conducted in 2 HCoV-229E positive PCR products.Their nucleotide sequences were consistent with each other.The nucleotide sequence was 98% to 99% homology with that of the reference strains in the GenBank,and the amino acid sequence was consistent with that of the reference strains.4. Three out of 9 HBoV PCR products were randomly selected for sequence ana lysis. Their nucleotide sequences were consistent with each other.The nucleotide sequence was 99% to 100% homology with that of the reference strains in the GenBank.And the amino acid sequence was 100% homology with that of the reference strains. The phylogenetic tree showed that, these HBoV strains had the closely relationship with HBoV refrence strain whose accession numbers in Genbank isDQ988934,DQ353697 and DQ000496.5. All of 9 chidren with HBoV positive detection are aged less than 5 years.The HBoV positive rate in lower respiratory tract infections group was 7.9%(7/89),significantly higher than that of 1.5% (2/134) in upper respiratory tract infections group(x2=5.27,P<0.05). one patient infected with HBoV was co-infected with RSV.6. Three positive HCoV-NL63 and two positive HCoV-229E samples were from children with lower respiratory tract infections group. 7. The results of the multiplex PCR were according with that of single PCR for HCoV-NL63 and HBoV,and nested PCR for HCoV-229E in all of the 223 specimens .Conclusions1 . HCoV- NL63,HBoV and HCoV-229E were the important pathogens in children with acute respiratory tract infections in Fuzhou from winter 2006 to spring 2007.2 . Attention should be paied to HCoV- NL63 infections which may cause a severe complication in infants.3 . HBoV mainly causes lower respiratory tract infections,especially in infants aged less than 5 years old. one sample infected with HBoV was co-infected with RSV.4. The multiplex Reverse Transcription-PCR can substitute for single RT-PCR in the assistant diagnosis of respiratory tract infections caused by HCoV- NL63,HBoV and HCoV-229E.