Genome cloning of human bocavirus (HBoV1) and analysis of viral promoter activity

Abstract:

Human bocavirus (HBoV) is a recently discovered parvovirus, which is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in human. In the present study, we screened 941 nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections from October 9, 2007 to March 20, 2009 in the Children’s Hospital of Hubei Province. Our results showed that 33 of 941 samples (3.51%) were detected positive for HBoV. To obtain a full-length HBoV clone, three segments which covered the nearly full-length genome were amplified by PCR from HBoV positive samples separately and cloned into pBluescript SKⅡvector, and the resulting plasmid was designated as pWHL-1 (GenBank Acession No. GU139423). We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter, respectively. Our data demonstrated that the HBoV promoter exhibited very high activity in all mammalian cells tested by fluorescent microscopy observation of the EGFP and luciferase activity assay and its strength was 4?5 fold higher compared to that of the CMV promoter. This work provided an excellent tool for further study of the mechanism of transcription and expression of the viral genome.