Investigation On Human Bocavirus-a Novel Parvovirus Associated With Acute Respiratory Infections In Pediatric Patients In Beijing

Parvovirus-like sequences were found in respiratory tract samples in a retrospective clinical study described by Tobias Allander and co-workers at the Karolinska University Hospital,Stockholm,Sweden in September 2005.The deduced amino acid sequences were conserved with bovine parvovirus(BPV) and canine minute virus(MVC),which were related members of the Parvoviridae family,subfamily Parvovirinae,genus Bocavirus,but less conserved with human parvovirus B19.Therefore the newly found respiratory virus was named human Bocavirus(HBoV).The fact that HBoV was detected most frequently from pediatric patients with acute respiratory tract infections (ARTIs) suggested that HBoV may be one of the previously unknown cusative agents of acute respiratory infections..Since the first report,the worldwide presence of HBoV in children with ARTIs has been presented by over 40 studies.However,the evidence for an association between HBoV and respiratory tract disease is incomplete.And an unusually high number of coinfections were found where HBoV occurs simultaneously with other viruses,making the association of HBoV with disease more complex.Moreover,Koch's revised postulates cannot be applied to HBoV,since neither a method for HBoV culture nor an animal model of infection has been established.Current knowledge of HBoV infection suggests that the virus is sometimes a passenger and sometimes a pathogen in acute respiratory tract disease.A better understanding of the natural course of HBoV infection will be necessary before any clinical questions can be comprehensively addressed.In order to investigate if the novel parvovirus,human bocavirus is related to acute respiratory infections in pediatric patients in Beijing,728 specimens collected from pediatric patients with acute respiratory infections during Nov.2003 to Oct.2004 were selected to test this virus.These 728 specimens included 185 throat swabs from outpatient department and 543 nasopharyngial aspirates from those hospitalized and had been tested for common respiratory viruses by virus isolation and/or indirect immuno -fluorescent assay.Polymerase chain reaction(PCR) was performed to detect HBoV from these specimens using primers designed from the published sequences of NP1 gene from HBoV.Then PCR products with expected molecular weight were selected randomly for sequencing and then sequences from those PCR products were compared with the sequences in the GenBank.Among these 728 specimens detected,17(2.3%) including one coinfected with adenovirus showed expected PCR products,suggest these were HBoV positive.Five out of these 17 amplicons were used for sequence analysis.When compared to the sequences of the prototype strains ST1 and ST2,these 5 amplicons shared high nucleotide sequence homology with ST1,ST2(99.2%to 99.4%) and among themselves(99.7%to 100%), which confirmed these 17 specimens with expected PCR products were HBoV positive. The highest positive rate was among specimens collected from patients with bronchiolitis (7.3%,6/82),followed by patients diagnosed as bronchitis(4.2%,3/69).Age distribution of the children with HBoV positive was 5 months to 5 years,especially those younger than 1 year of age.The positive rate for HBoV detection was 18.8%among those aged from 8 to 9 months(3/16) and 11.8%(2/17) among those aged from 9 to 10 months.Neither of 69 children older than 5 years nor 211 infants younger than 5 months was positive for HBoV.These data suggest that some of acute respiratory infections in pediatric patients in Beijing are related to human bocavirus,which is most likely the second known parvovirus species being pathogenic to humans.Infants and young children aged 5 months to 5 years,especially those younger than 1 year,are more likely to be infected by HBoV.Then,in order to characterize the genomic sequences of human bocavirus(HBoV) detected from clinical specimens from children in Beijing,and to predict the secondary structures and other characters of HBoV nonstructure protein NS1,nucleioprotein NP-1, major structure proteins VP1 and VP2,the coding domain sequences NS1,NP-1,VP1 and VP2 and the 3'-terminal sequence of HBoV were amplified from 2 clinical specimens BJ3064,BJ3722 after these samples were determined as HBoV positive by PCR amplification of partial NP1 gene.The PCR amplicons were sequenced,and genomic sequence analysis was performed by comparison with the sequences of the prototype strains ST1,ST2,and BPV,MVC,B19.And the secondary structures and other characters of HBoV NS1,NP 1,VP1 and VP2 were predicted by bioinformatics.The genomic sequences from HBoV BJ3064 and BJ3722,with accession number in the Genbank DQ988933(BJ3064) and DQ988934(BJ3722),were 5299 bases and 5300 bases,respectively,with four major coding domain sequences(CDS) coding for proteins NS1,NP-1,VP1 and VP2.The genomic sequence analysis indicated that BJ3064 and BJ3722 share high homology(99.9%) between themselves,as well as with those of ST1 and ST2(＞99%),but showed lower homology with BPV and MVC (＜45%),only about 5%with B19.Phylogenetic analysis of genomic sequence showed that BJ3064 and BJ3722 were more closely related to ST2 than to ST1.The main secondary structure of these four proteins is random coil,and there are some domains which may be important for binding to genome or virion construction.So,the genomic sequence of human bocavirus detected from clinical specimens from children in Beijing is closely related to the prototype strains described by the scientists in Sweden.And these results predicted by bioinformatics may be helpful for the further proteomic study on HBoV in future.To find out the importance of human bocavirus(HBoV) as an infectious agent for population in Beijing,China,seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen.At first,the VP2 encoding gene of HBoV was amplified from HBoV positive clinical specimen BJ3722 by PCR with the primer pairs designed on basis of the sequences of VP2 of HBov followed by cloning of the gene fragment into pUCm-T.The VP2 gene was sub-cloned into pET30b(+) vector, a prokaryotic expression vector,from recombinant plasmid pUCm-T-VP2.The recombinant plasmid pET30b-VP2 identified by PCR,dual-enzyme digestion with Hindâ…¢and Xhoâ… and sequencing was transformed into E.coli BL21(DE3) and expressed under different temperature using inducer IPTG.Target protein was characterized by SDS-PAGE and Commasie blue staining.The protein was purified by affinity chromatography with Ni²⁺ column,and its specific antigenicity was determined by Western-Blot using monoclonal antibodies against His,hyper-immune serum against HBoV VP2 peptide,mouse anti-human parvovirus B19 VP2 IgG and human sera.Then 677 serum specimens collected from children and adults aged from birth to over 60 years during April 1996 to March 1997 and another batch of 141 serum specimens collected from adults aged from 20 years to over 60 years in August,2005 were tested for antibody against HBoV by Western Blot.The correctness of the open reading-frame of recombinant plasmid pET30b-VP2 was confirmed by PCR,dual-enzyme digestion and sequencing.The fusion protein 6oHis-VP2 was produced in a larger quantity at 25℃than at 30℃and 37℃by using inducer IPTG(1mM) for 19 hr and most of protein was expressed in inclusion body.An expected protein band of approximate 68 kDa was revealed by SDS-PAGE and can be purified by affinity chromatography of Ni²⁺ column.Western blot analysis indicated that the target protein was specifically binding to monoclonal antibodies against His, hyper-immune serum against VP2 peptide from HBov as well as some of the tested human sera.Therefore,the recombinant expression plasmid pET30b-VP2 was constructed successfully.The VP2 gene was expressed in prokaryotic system in high yield under certain conditions.And the expressed VP2 protein without cross-reactivity with parvovirus B19 was of specific for antigenicity,so was used as antigen to determine the prevalence of serum antibodies against HBoV in Beijing population to learn about the importance of HBoV infections.Two batches of serum specimens collected in different periods were tested by Western Blot for antibody against HBoV using recombinant VP2 as antigen.Out of 677 serum specimens collected during April 1996 to March 1997,400(59.1%) were positive.About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV IgG,and antibody positive rates were decreased in age groups of 1 and 2 months(41.4%and 31.3%, respectively) then increased in the following ages from 6 months to 7 years old(from 45.6%to 69.7%).The antibody positive rates maintained at a relatively constant level (about 70%) in the age groups from 7 years old to 40 and became lower(61.8%to 62.8%) in groups over age 50.For another batch of 141 serum specimens collected in August, 2005 from adults aged from 20 years to over 60 years,the antibody positive rate was 78.7% (111/141).Comparison of the sero-prevalence profile of the age groups for serum specimens collected during 1996-1997 to those collected in 2005 indicated that the sero-positive rate of the specimens collected in 2005 was higer than that of the corresponding age groups collected in the period of 1996-1997.The data from this study suggest that human bocavirus is a newly discovered virus rather than a new virus and has been circulating in Beijing population at least over 10 years,and higher antibody positive rate was shown in the serum specimens collected in 2005.Most of children had been exposed to HBoV by age of 7 years.Infants under 6 months were susceptible to this virus.The parvoviruses,expression of the structural proteins in insect cells leads to spontaneous self-assembly to particles,VP2 was specifically required for self-assembly, and either VP1 or VP3 could be omitted.In the study,the major capsid gene VP2 of HBoV was expressed in insect cells.At first,the full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector(pFastBac1) to get the recombinant Bacmid,and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells.Then the recombinant VP2 protein with molecular weight of approximately 61 kDa was recognized by SDS-PAGE using Comassie-blue and Western-blot using hyper-immune serum against VP2 of HBoV from rabbit,indicated that expressed VP2 has specific antigenicity.The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titer to infect insect cells at a multiplicity of infection(MOI) of 0.5. After 7-10 days or 4-5 days post infection,the supernatants of culture or the cell lysates treated with lysing solution were harvested,and ultracentrifuged twice with 40%sucrose cushion to get purified virus-like particles(VLPs),which were followed by Western -blot and IFA for VLPs' composition and specificity,by electron microscopy for VLPs' morphologic structure.The presence of VP2 in VLPs was demonstrated by Western-blot and IFA either in the supernatants of culture or the cells lysates,and the expression of VP2 in insect cells led to the formation of VLPs which have the typical icosahedral appearance of parvoviruses with a diameter of approximately 20nm.In conclusion,the present study indicates that some of acute respiratory infections in pediatric patients in Beijing are related to human bocavirus which enlarges the types of viral etiology in pediatric patients,and the human bocavirus(HBoV) detected from clinical specimens from children in Beijing is closely related to the prototype strains described by the scientists in Sweden.Seroprevalence study revealed that HBoV is a newly discovered virus rather than a new virus and has been circulating in Beijing population at least over 10 years,and the expressing of VP2 from HBoV positive clinical specimens from Beijing and successful constructing VLPs in insect cells may contribute to the aim of prevention and control against HBoV infection in China.