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npa samples were collected from 235 children hospitalized with arti at the first hospital of lanzhou university , gansu province , china during december 2007november 2008 .
all patients were < 15 years of age , and informed consent was obtained from their parents .
dna and rna were extracted from the npas by using qiaamp dna and qiaamp viral rna mini kits ( qiagen , beijing , china ) .
the cdna sample was synthesized by using random hexamer primers . a standard reverse transcription 
pcr was used to screen for human rhinovirus , respiratory synctial virus ( rsv ) , influenza virus a , influenza virus b , parainfluenza virus 13 , human metapneumovirus , human coronavirus ( hcov)nl63 , and hcov - hku1 , and pcr method was used to screen for adenovirus ( adv ) ( 711).to screen for hbov , pcr was performed by using primers 188f and 542r , as described by allander et al .
nested pcr was performed with sf1/sr1 and sf2/sr2 primers , which amplified a 455-bp fragment of the partial ns1 gene , as described ( 6 ) .
in addition , we designed hbov2 forward ( sn1 : 5-accagtgggagaaccacaag-3 ) and reverse ( sn2 : 5-ggcatttgtttccatgcttt-3 ) primers , which produced a 563-bp fragment of the np1 gene of hbov2 .
clustalx ( ftp://ftp-igbmc.u-strasbg.fr/pub/clustalx ) was used to align the obtained sequences with sequences available in genbank . in total ,
260 viruses were identified in 196 ( 83.4% ) of the 235 children . using nested pcr , we found 21 positive specimens ; further nucleotide sequence analysis showed that 10 ( 4.3% ) were hbov2 and 11 were hbov ( figure , panel a ) .
all 11 hbov strains detected by using hbov2 nested - pcr were included in the 18 hbov - positive patients as determined by pcr using primers 188f and 542r . of the 10 hbov2-positive patients , 7 ( 70% )
of the 18 hbov - positive patients , 12 ( 66.7% ) displayed co - infections .
there were no statistically significant differences in the hbov2 and hbov detection ( p = 0.119 by  test ) and co - infection ( p = 1.000 by fisher exact test ) rates .
phylogenetic analysis of a ) the partial nonstructural protein 1 ( ns1 ) nucleotide sequences ( 412 bp ) and b ) the partial nucleoprotein 1 ( np1 ) nucleotide sequences ( 256 bp ) of human bocavirus 2 ( hbov2 ) , gansu province , people s republic of china .
the phylogenetic trees were constructed by the neighbor - joining method using mega 3.1 ( www.megasoftware.net ) , and bootstrap values were determined for 1,000 replicates .
human bocavirus ( hbov ) and hbov2 reference sequences are indicated by circles and squares , respectively .
bovine parvovirus ( bpv ) , cytomegalovirus ( cmv ) , and human parvovirus b19 ( b19 ) reference sequences are indicated by inverted triangles .
dq000495 , dq000496 , ef450717 , eu082213 , eu918736 , eu984232 , fj170278 , fj170279 , fj170280 , nc001540 , nc004442 , and nc000883 ) .
of the 10 hbov2-positive patients , 9 were male and 1 was female (   =  1.957 , p = 0.162 ) .
the median age was 8.5 months , and 9/10 ( 90% ) were <3 years old .
of the 18 patients who were hbov positive , 11 were male and 7 were female (  = 0.084 , p = 0.772 )
. the median age of patients was 11.5 months , and 16/18 ( 88.9% ) were <3 years old .
hbov infections were detected in every month except august , with peaks in december ( 3 cases ) and january ( 4 cases ) .
the main diagnoses of the 3 patients with hbov2 monoinfection were acute asthmatic bronchopneumonia , bronchopneumonia , and acute upper respiratory tract infection in 1 patient each .
for the 6 patients with hbov monoinfection , the main diagnoses were acute asthmatic bronchopneumonia ( 4 cases ) and bronchopneumonia ( 2 cases ) .
the clinical signs and symptoms of hbov2 and hbov positive patients included cough , fever , sputum production , crackles , wheezing , rhinorrhea , cyanosis , vomiting , and diarrhea ( table 1 ) . for patients with hbov2 monoinfection ,
the median hospital stay was 11.3 days ( range 423 days ) , and 2 had underlying illnesses ( idiopathic pulmonary hemosiderosis and iron deficiency anemia ) .
the chest radiograph of 1 patient showed upper middle zone air - space shadows . for patients with hbov monoinfection ,
the median hospital stay was 7.8 days ( range 610 days ) , and none had underlying illnesses .
group 1 , hbov2 monoinfection ; group 2 , hbov2 co - infection ; group 3 , hbov1 monoinfection ; group 4 , hbov1 co - infection . 
ten hbov2 ns-1 sequences ( 455 bp ) shared 98%99% and 95%96% nucleotide sequence identity and 99%100% and 98%99% deduced amino acid sequence identity with hbov2 strain pk-2255 ( fj170279 ) and hbov2 strain w153 ( eu082213 ) , respectively ; these sequences also shared 81%82% and 83.3%84.4% nucleotide sequence identity and 90% and 88% deduced amino acid sequence identity with the hbov prototype strain st1 or st2 ( dq000495 and dq000496 ) and human bocavirus 3 strain w471 ( eu918736 ) , respectively .
the 4 hbov2 np-1 sequences shared 98%99% and 97.6%98.3% nucleotide sequence identity and 98%100% and 97%100% deduced amino acid sequence identity with hbov2 strain pk-2255 and hbov2 strain w153 , respectively , and shared 74%78% and 69.7%70.3% nucleotide sequence identity and 69%73% and 58%62% deduced amino acid sequence identity with the prototype strain st1 or st2 and human bocavirus 3 strain w471 , respectively .
the nucleotide and deduced amino acid sequences of ns-1 and np-1 shared high identities ( > 97% ) with the hbov2 and hbov sequences ( table 2 ) .
* hbov , human bocavirus ; ns1 , nonstructural protein 1 ; np1 , nucleoprotein 1 .
using nested pcr and sequencing , we identified hbov2 infections in 10 ( 4.3% ) of 235 npas from children hospitalized with arti .
in hbov2-positive patients , co - infection was high ( 70% ) , with rsv being the most common co - pathogen .
primers sn1 and sn2 were designed to detect the np1 gene in the 10 hbov2-positive patients . however , only 4 gave positive results , which occurred because of the low pcr sensitivity with this pair of primers and because the np1 gene of hbov2 is divergent , as described ( 6 ) .
furthermore , as previous studies ( 6,12 ) pointed out , potential recombination upstream from the np1 gene may explain the lower detection .
phylogenetic analysis showed that the ns-1 region of the hbov2 strain ( lz480 and lz578 ) clustered closely with that of the hbov2 pk-2255 strain ( fj170279 ) , and the np-1 region clustered closely with that of the hbov2 w153 strain ( eu082213 ) , suggesting potential recombination in the hbov2 strains ( figure ) . in addition , 11 hbov sequences were amplified by using nested - pcr for hbov2 . in the future , hbov2-specific primers should be designed to investigate the prevalence of hbov2 and its potential association with disease .
we found no difference in the clinical symptoms or length of hospital stay between the groups with hbov2 and hbov monoinfection , as well as between the groups with hbov2 monoinfection and hbov2 co - infection ( table 1 ) .
statistical analysis indicated that hbov2 and hbov co - infection obviously did not correlate with disease severity ( data not shown ) .
two of 3 patients with hbov2 monoinfection had diarrhea with no vomiting ( table 1 ) , and only 1 of 10 patients who were hbov2 positive vomited .
further investigation is needed to exclude oral or inhaled gastric viruses as possible sources of npa - associated hbov2 .
phylogenetic analysis showed a high degree of similarity between hbov2 sequences found in china and those in other areas ( figure ) .
our results suggest that like hbov , hbov2 is distributed worldwide and may be associated with respiratory and enteric diseases .
additional studies are needed to confirm the association between human bocavirus species ( hbov2 and hbov ) and respiratory tract infections or other diseases .
human bocavirus ( hbov ) and hbov2 , two human bocavirus species , were found in 18 and 10 of 235 nasopharyngeal aspirates , respectively , from children hospitalized with acute respiratory tract infection . 
 our results suggest that , like hbov , hbov2 is distributed worldwide and may be associated with respiratory and enteric diseases .