Beijing Regional Human Bocavirus Survey Of The Prevalence And Epitopes

New emerging infectious diseases damage human health seriously, affect social stabilization and threaten state security, which has earned global attention. Human bocavirus (HBoVs), firstly identified in 2005, has been reconized as a potential pathogen of respiratory infection. Since its first identification, four species of HBoVs (HBoV1-4) have been reported. However, the pathogenicity of HBoV species remains unclear. At present, mostly prevalence data were about HBoV1 and HBoV2, while the data about HBoV3 and HBoV4 were sparse. Prevalence of the four species HBoVs in the population has not been fully assessed. For the high amino acids homology between HBoV 1,2 and 3 species, serological cross-reactivities among the three species of HBoVs have also not been addressed, which reasied difficulties in the development and evaluation of the immunological assays for HBoVs.In the present study, the prevalence of HBoV1-4 in children with acut lower respiratory tract infection (ALRTIs) and acute gastroenteritis (AGE) and the seroprevalence of HBoV1,-2 and-3 in healthy population were investigated. Moreover, the cross-reactivities between different HBoV species and poosible epitopes of HBoVs VP2 were investigated. The main result were displayed as follow:1. Prevalence of HBoVl-4 in children suffered from ALRTIs and AGE in BeijingNasopharyngeal aspirates (NPAs) were collected from 1,238 hospitalized children with acute LRTIs at Beijing Children's Hospital between March 2008 and July 2010. These samples were assayed for HBoVs by a universal nested PCR. The results showed that of all the ALRTIs specimens tested,11.4% were positive for HBoVs, with 10.6% positive for HBoV1,0.4% for HBoV2 and 0.4% for HBoV3. No specimens were positive for HBoV4. Of patients with HBoVs,93.6% were≤5 years old. Similar to previous reports, other Additional respiratory pathogens were detected in 85.1% patients, The seasonal distribution of HBoVs is not obvious during the study period.Stool specimens were collected from 366 outpatients with AGE at Beijing Children's Hospitral between March 2006 and November 2007 and the presence of HBoVs in these samples were also evaluated by the aforementioned method. The results showed that of all the stool specimens tested, the positive rate of HBoVs were 12%, the distribution ages of positive sample from 2 to 24 months of age (median age 9 months). Of those specimens,2.5% were positive for HBoV1,9.0% for HBoV2 and 0.5% for HBoV3, HBoV4 was not detected. Notably,79.5% of HBoVs-positive samples were co-detected with other viral pathogens. Analysis of the HBoV3 genome sequence indicates that HBoV3 may be a recombinant derived from HBoV1 and HBoV2 located in the upstream of NS1 and VP1/2 reading frame, or from HBoV1 and HBoV4.2. Seroprovalence of HBoV1-3 in healthy population in BeijingTo evaluate the prevalence of HBoV in human population, we expressed virus-like particles (VLPs) of HBoV1,-2, and-3 species using High five cells and purified these VLPs by CsCl gradient centrifugation. After verification by electron microscopy and Western blot analysis, these VLPs were used as antigens for setting up ELISA methods to detect specific IgG antibodies against HBoV1, HBoV2 and HBoV3 in serum samples collected from 642 healthy people in different age groups respectively from Beijing. Ubiquitous infection of HBoV 1 was found in healthy population. In the children aged from 6 months to 5 years old, the seropositive rate of HBoV1 was higher than that of HBoV2 and HBoV3. The seropositive rates of HBoV1,-2, and-3 increased with age. In individuals over 20 years old, the seropositive rate of HBoV1,-2, and-3 reached almost 100%. In contrast, the antibodies titer of HBoV2, and-3 were not obviously boosted with age and kept at a relatively low level compared with HBoV1. Taken together, our data indicate a differential prevalence of HBoV species in human. Depite the prevalence of HBoV 1-3 were detected, HBoV1 may be dominant in the HBoV epidemic. Moreover, we also demonstrated that ELISA detection for HBoV1 IgM is effective for the early detection of HBoV infection.3. Identification of epitopes in VP2 protein of HBoV1,2, and-3 using phage display peptide librarySerological cross-reactivities among HBoV1,-2,-3 were found by using HBoV VLPs react to murine sera against HBoV VP2 protein by Western blot and ELISA assays. To identify specific antigens for HBoVs serology diagnosis, an HBoV VP2 gene phage display random library was constructed. The HBoV VP2 gene phage peptide display library was selected by antisera from immunized mice with recombinant HBoV1,-2, and-3 VP2 protein and positive human sera. Positive clones were idedified by ELISA and further analyzed by gene sequencing. Two regions harboring possible epitopes that are conserved between HBoV1,-2, and-3 and one region harboring possible HBoV3 specific epitope were identified.In conclusion, we report the first detection of HBoV3 DNA in the respiratory tract samples. Diffrential prevalence was found among different HBoVs by seroprevalence investigation. Several possible common epitope regions and one possible specific epitope region in HBoV1,2,3 were first identified. This study provides basis for evaluatintg the epidemic and pathogenesis and for developing immunological assays for HBoVs.