to investigate hbov-2 , we developed new pcr - based detection methods for hbov by using primer sets highly conserved between hbov-1 and hbov-2 and species - specific primers for hbov-2 . large - scale screening of persons in the united kingdom and thailand was performed to compare virus detection frequencies in respiratory and fecal samples . a total of 6,138 respiratory samples from 3,754 persons ( 2,018 male , 1,722 female , 14 sex unknown ) during january 1 , 2007june 30 , 2008 , were obtained from the specialist virology centre ( edinburgh , uk ) . samples comprised 3,065 nasopharyngeal swabs / aspirates ( npas ) and throat swabs ( 83% ) . a total of 386 npas were obtained from 386 persons ( 229 male , 154 female , 3 sex unknown ) in bangkok during february 16 , 2006july 20 , 2008 . a total of 2,500 fecal samples were obtained from patients ( 1,093 male , 1,398 female , and 9 sex unknown ) in edinburgh predominantly with gastroenteritis or other enteric diseases referred for bacteriologic screening during march , june , and september 2008 . a total of 530 fecal samples were obtained predominantly from children ( 179 boys and 138 girls ) < 5 years of age with diarrhea during july 12 , 2007july 25 , 2008 , and a control group without diarrhea ( 116 male , 96 female , 1 sex unknown ) during march 4december 2 , 2007 , in bangkok . dna was extracted from 200-l samples of pooled or individual specimens ( respiratory samples , clarified fecal supernatant ) into 40 l tris - edta buffer as described ( 13 ) . respiratory and fecal samples from edinburgh were screened in pools of 10 ; both sample types from bangkok were screened individually . screening was performed by using nested primers conserved between hbov-1 and hbov-2 in the nucleoprotein ( np)1 gene ( universal primers : outer sense [ position 2589 in dq000496 st2 isolate ( 1 ) ] : 5-ccwatcgtcytsyactgcttyga-3 ; outer antisense : 5-tagcyaagtgtytwbkgtacacatyat-3 ) ; inner sense : 5-rtkstgyggbttctaytggca-3 ; and inner antisense : 5-tacacatcatcccartaaywacat-3 ) . amplication conditions were 94c for 2 min and 35 cycles at 94c for 18 s , 50c for 21 s , and 72c for 1.5 min . all known hbov-1 sequences contain an rsai site between nt 2772 and nt 2773 , resulting in fragments of 46 bp and 91 bp ; this site is absent in hbov-2 ( undigested amplicon length of 237 bp ) . each pool or sample was additionally screened by using hbov2-specific primers located in the nonstructural ( ns)1 gene ( outer sense : 5-aacagatgggcaagcagaac-3 ; outer antisense : 5-aggacaaaggtctccaagagg-3 ; inner sense : 5-aacgattgcagacaacgccttata-3 ; and inner antisense : 5-tccaagaggaaatgagtttgg-3 ; sites matching all known hbov-2 variants and not matching hbov-1 variants are underlined . ) amplification conditions were 95c for 2 min ; 5 cycles at 95c for 45 s , 53c for 1 min , and 72c for min ; and 35 cycles at 95c for 30 s , 51c for 30 s , and 72c for 45 s. positive pools of fecal samples from edinburgh were divided and individual component samples were tested . respiratory and fecal samples from both centers were screened by using universal primers , and positive samples were digested with rsai . undigested amplicons and some predicted hbov-1 fragments ( 46 bp and 91 bp ) were sequenced to confirm virus identity . all samples were additionally screened with hbov-2specific primers ; 16 undigested samples were positive with hbov-2specific primers , and all samples identified as hbov-1 were negative . thus , species - specific primers enabled effective screening of hbov-2 among samples with high frequencies of hbov-1 . hbov - positive fecal samples were generally restricted to children < 5 years of age ( 25 from 30 infected children whose ages were known ) ( figure , panel a ; table ) . median age of children infected with hbov-2 ( 712 months ) was lower than that for those infected with hbov-1 ( 12 years ) . infections with hbov-1 and hbov-2 were observed at low frequencies in older persons ( 2 and 5 of 1,791 persons > 35 years of age , respectively ) . for respiratory samples , hbov-1 infections showed a similar peak incidence among children 12 years of age ( figure , panel b ) , similar to that observed for fecal samples . this age group was most frequently infected in our previous analyses of respiratory samples from edinburgh ( 12 ) . there were no differences in frequencies of hbov-1 or hbov-2 infection between male and female participants . samples from bangkok were divided into those from persons with diarrhea ( 327 ) and asymptomatic controls ( 213 ) ; detection of hbov-1 and hbov-2 was restricted to persons with diarrhea ( n = 12 and 2 , respectively ) . age distribution of study participants with positive fecal ( a ) and respiratory ( b ) sample results for human bocavirus ( hbov ) , subdivided by hbov species . analysis of age distribution of persons with positive respiratory samples was restricted to samples from bangkok , thailand . in contrast to its frequent detection in fecal samples , hbov-2 was not detected in > 6,500 respiratory samples ( table ) . however , high frequencies of hbov-1 were recorded ( 14% among children in bangkok and 3.4% among children in edinburgh ) ; the group from edinburgh contained a substantial number of older children ( 37% > 5 years of age ) . frequency among children < 5 y of age : hbov-1 , 1.4% ; hbov-2 , 3.1% . first , hbov-2 circulates in 3 widely separated areas ( united kingdom , thailand , and pakistan ) and is likely distributed globally . second , infections with hbov-2 show a pattern of infecting young children , most < 1 year of age . third , absence of hbov-2 in respiratory samples suggests a different tissue tropism that may influence its transmission route and ability to infect systemically and establish persistence . determining the biologic basis for such differences will be useful in understanding the pathogenesis of hbov-1related respiratory disease . fourth , at a practical level , absence of hbov-2 in respiratory samples indicates no likely role for this virus in respiratory disease . nevertheless , the unexpectedly diverse human bocavirus group may contain additional variants with potential etiologic roles in respiratory or other diseases . since this study was completed , evidence for an interspecies hbov-1/-2 recombinant associated with acute gastroenteritis has been obtained ; the structural gene region was most closely related to hbov-2 , and ns1/np-1 grouping with hbov-1 ( 14 ) . although this recombinant would have been identified as hbov-1 by using typing assays described in the current study , sequence analysis of hbov-1positive samples in this study and our previous study of respiratory samples from edinburgh and bangkok ( 12,15 ) identified only hbov-1 in the study population , consistent with all other analyses of this sample type worldwide nevertheless , future typing assays should analyze both vp1/2 and ns / np-1 to ensure that this and potentially other interspecies recombinants are identified . investigation of genetic diversity of this group and development of effective screening methods for variants of hbov is required for studies of human disease .
human bocavirus ( hbov ) commonly infects young children and is associated with respiratory disease ; disease associations of the divergent hbov-2 species are unknown . frequent hbov-2 detection in fecal samples indicated widespread circulation in the united kingdom and thailand , but its lack of detection among 6,524 respiratory samples indicates likely differences from hbov-1 in tropism / pathogenesis .