| ObjectivesEstablishing and performing the methods for detecting HRV/HEV from respiratory samples of hospitalized children for acute respiratory tact infection. To obtian the features of epidemiology and genomic charactoristic of HRV/HEV in children with acute respiratory tact infection in Lanzhou and to investigate the association of different HRV/HEV genotypes with acute respiratory tact infection. The method of sequence independent single primer amplification (SISPA) was used to obtain 5 complete genome isolates in Lanzhou So as to establish the foundation progessively for providing the pathological data on children's acute respiratory tact infection and demonstating the molecular and epidemic feature about HRV/HEV.Methods1.Seven hundred and seventy two children with acute respiratory tact infection hospitalized in the First Hospital of Lanzhou University, between Dec 2006-Nov 2007, were enrolled in the study. All of the children were<18 years old.2.Respiratory samples were collected and sent to the National Institute for Viral Disease Control and Prevention, China CDC. HRV/HEV were detected and quantified using real-time reverse transcription PCR (RT-PCR), and then directly genotyped by sequencing the product of a nested RT-PCR targeted VP4/2 region and a RT-PCR targeted 5'-UTR region. Phylogenetic analyses of the nucleotide of VP4/VP2 and 5'-UTR partial gene were conducted with MEGA 4.0 software respectively. Furthemore, every sample was screnning for the viruses of HRSV, IFV, PIV, HMPV, HCoV, AVD, HBoV, WUPyV, KIPyV by PCR or RT-PCR, and sequencing in commercial sequencing company. All the data were analyzed by SPSS 13.0 statistical software.3.The method of Dnase I-SISPA-PCR was used to amplify the hole genome of HRV C. Five isolates that it's nucleotide identity low than all known HRV C complete genome were sequencing. More than 100 clones were pick up for sequencing respectively, and sequenced contigs were assembled in the software Invitrogen Vector NTI contigexpress.ResultsHigh prevalence of HRV/HEV was found in the study. In total,205 out of 772 (26.55%) children with acute respiratory tract infection were found infected with HRV/HEV. Of which for HRV was 24.87%(192/772),for HEV was 1.68%(13/772). The rate of detection of males and females were 27.2%(133/488) and 25.4% (72/284),the ratio of male to female was 1.85:1.The detected rate was no significant difference between male and female (P=0.564>0.05).The median ages of the viruses infected children was 10 months (range from 6days to 166 months),56.1%(115/205) children with the virus infection were under 1 year of age; 24.4%(50/205) children were age from 1 to 3 years; 13.2%(27/205) children were at age from 3 to 5 years; 6.3%(13/205) children were older than 5 years old. Of the 205 HRV/HEV positive samples,36.6%(75/205) samples were fond monoinfected with HRV/HEV,63.4% (130/205) HRV/HEV positive samples were detected coinfected with other respiratory viruses.There are no significant statistical difference between male to female (P=0.92>0.05). Among the co-infected RNA viruses,43.1%(56/130) children were coinfected with HRSV(Human respiratory syncytial virus); 31.5%(41/130) children were coinfected with IFV A(Influenza virus A); 27.7%(36/130) cildren were coinfected with PIV 3(Human parainfluenza virus 3); 19.2%(25/130) children were coinfected with IFV B(Human influnenza virus B); 0.3%(4/130) children were coinfected with HcoV-NL63 (Human coronavirus NL63); 0.2%(4/130) children were coinfected with PIV 2(Human parainfluenza virus 2); 0.2%(3/130) children were coinfected with hMPV(Human metepneumovirus). Among the co-infected DNA viruses,13.9%(18/130) children were coinfected with HBoV (Human bocavirus); 8.5%(11/130) children were coinfected with WUPyV (Human polyomavirus WU); 8.5%(11/130) children were coinfected with KIPyV (Human polyomavirus KI); 0.4%(5/130) children were coinfected with AdV (Human adenovirus). Human respiratory syncytial virus and Human influenza virus and Human parainfluenza virus and human bocavirus are the most common etiological pathogen coinfected with HRV/HEV. Among the bacterium which coinfected with HRV/HEV,9 samples were coinfected with Klebsiella pneumoniae; 7 samples were coinfected with Escherichia coli; 7 samples were coinfected with streptococcus pneumoniae; 7 samples were coinfected with staphylococcus aureus; 5 samples were coinfected with Haemophilus influenzae; 4 samples were coinfected with Haemophilus parainfluenzae; Two reigions of 5'-UTR and VP4/VP2 were used to construct philogenetic trees and reveal that all the samples which selected were well clusted into four group HRV A,HRV B,HRV C and HEV. Comparation of clinical feature between the HRV A to HRV C demonstated that there are no significant difference between two group. Children who infected with HRV C won't caused a more severe disease than HRV A.Conclusions1.HRV/HEV was highly prevalent in children with acute respiratory tact infection in Lanzhou. And HRV may act as an important pathogen in acute respiratory tact infection.2.There are no significant differences between two group of HRV A and HRV C in the aspects of clinical feature, children who infected with HRV C won't caused a more severe disease than HRV A.3.Five complete genome of human rhinovirus C were sequenced. |
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