Genomic Cloning Of The Human Bocavirus (HBoV1)and Transcription And Translation Mechanism Of Viral Genome

Respiratory viruses are the major pathogens of acute lower respiratory tract infection (LRTI) in infants and young children. To date, pathogenesis of60%respiratory diseases has not been identified yet. In2005, Allander et al. first isolated a novel parvovirus isolated from hospitalized children with pneumonia and named this virus the human bocavirus (HBoV). HBoV is a linear single-stranded DNA virus without envelop and classified in the Bocaviurs genus of the Parvovirinae subfamily within the Parvoviridae family. Three other new species of human Bocavirus, HBoV2,3and4, have recently been isolated from fecal specimens. No infectious clone or efficient cell culture system has been described so far to support the replication of the virus. Most studies of HBoV were focused on epidemiology but not pathogenesis of the virus. In this study, HBoV1was detected from nasopharyngeal aspirates collected from children with lower respiratory tract infections. The recombinant plasmid and baculovirus containing the nearly full length genome of HBoV1were successfully constructed. Using these constructs, we studied the mechanism of transcription and expression strategy of the viral genome, activity of the HBoV1promoter and the regulation of the nonstructural proteins on the promoter activity. The results provided us with pro-fundamental basis for further studies on the HBoV1infection pathway and viral pathogenesis. The main results of this study are summarized as follows:We screened941nasopharyngeal aspirates collected from hospitalized children with lower respiratory tract infections in the Children’s Hospital of Hubei province from October9,2007to March20,2009. The conserved region of HBoV NP1gene was used as the detection target by PCR. Our results showed that33of941samples (3.51%) were detected positive for HBoV;24(72.7%) HBoV-positive samples were from children under12months. Furthermore, 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 SKⅡ vector to generate the plasmid WHL-1(GenBank Acession NO. GU139423). The whole genome of WHL-1was sequenced and compared with those in GenBank. Phylogenetic analyses suggested that WHL-1shares high homology with other bocavirus isolates whose genotype belongs to was HBoV1. However, the both termini of HBoV1have not been obtained.We constructed the both EGFP and luciferase reporter gene vectors under the control of the HBoV unique promoter, respectively. The data of fluorescent microscopy observation of the EGFP and luciferase reporter assays demonstrated that the promoter of HBoV1was highly active in most mammalian cell lines transfected by recombinant vectors. Moreover, the activity of HBoV1promoter was4-5folds higher than that of the Cytomegalovirus (CMV) promoter in293T cells. The region from1-95nt made no difference to the basal activity of the promoter and the putative transcription factor binding region located from96to145nt. In addition, we found that the transcriptional activity of this promoter could be transactivated by the viral nonstructural protein NS1in293T and HeLa cells, suggesting that the NS1protein may be play an important role in virus genome transcription.We designed two specific NP1primers to analyze mRNAs of NP1gene by RT-PCR. All mRNAs were transcribed from a single promoter located at the left side of the HBoV1genome and NP1RT-PCR product displayed two fragments:one about980bp and another about1100bp. Sequencing of the fragments revealed that the two transcripts which presumably encode the NP1protein were alternative spliced. In mRNA1, only one splicing occurred (donor site241nt and acceptor site2236nt), whereas in mRNA2, splicing occurred twice (donor site241nt and acceptor site2044nt; donor site2164nt and acceptor site2236nt). The sequence between positions2044nt and2164nt is retained in the mRNA2. We next determined the3’terminal sequence of the NP1mRNA transcripts by3’-RACE. Sequence results showed that these two NP1PCR fragments of approximately790bp and940bp were properly annealed to the polyadenosine tails and the mRNAs were polyadenylated at two different positions proximal to the3’terminus of the NP1gene (position3268nt and3403nt), which corresponded to two polyadenylation signals situated at position3233nt and3389nt, respectively. Southern Blot analysis showed that no viral DNA replication was detected in293T cells transfected with pWHL-1plasmid, suggesting that ITRs were essential for viral DNA replication during infection cycle. The HBoV1promoter was an early promoter and the NP1gene was transcriped under the control of this promoter. The NP1protein was detected in pWHL-1transfected cells and localized in the nucleus.We developed two recombinant baculoviruses:Bac-Bov-EGFP containing the reporter enhanced green fluorescent protein (EGFP) gene under the HBoVl promoter control, and Bac-HBoV1containing the whole HBoV1genome without both termini. A number of mammalian cells transduced by Bac-Bov-EGFP expressed the transgene with high efficiency, suggesting that baculovirus-mediated gene transfer was more efficient than the plasmid-based vector pGL-pboca-EGFP which contains the HBoV promoter and EGFP reporter gene tranfected by Lipofectamine into the cell lines such as human lung epithelial A549cells and HeLa cells. A HBoV1recombinant baculovirus has been shown to efficiently deliver the HBoV1genome into A549cells resulting in HBoV1transcription and translation. In summary, this system provides a useful tool for the analysis of the transcription and translation not only for HBoV1but also for other viruses without permissive cell culture system.