Real-Time Quantitative PCR Detection of Four Human Bocaviruses�
Kalle Kantola,1* Mohammadreza Sadeghi,1 Jenni Antikainen,2 Juha Kirveskari,2
Eric Delwart,3,4 Klaus Hedman,1,2 and Maria Soderlund-Venermo1
Department of Virology, Haartman Institute, University of Helsinki,1 and Helsinki University Central Hospital Laboratory Division,2
Helsinki, Finland, and Blood Systems Research Institute3 and Department of Laboratory Medicine,4 University ofCalifornia, San Francisco, San Francisco, California
Received 1 April 2010/Returned for modification 17 August 2010/Accepted 9 September 2010
Human bocavirus (HBoV) was discovered in 2005 and is associated with respiratory tract symptoms in youngchildren. Three additional members of the genus Bocavirus, HBoV2, -3, and -4, were discovered recently fromfecal specimens, and early results indicate an association between HBoV2 and gastrointestinal disease. In thisstudy, we present an undifferentiating multiplex real-time quantitative PCR assay for the detection of thesenovel viruses. Differentiation of the individual bocavirus species can be subsequently achieved with corre-sponding singleplex PCRs or by sequencing. Both multiplex and singleplex assays were consistently able todetect <10 copies of HBoV1 to -4 plasmid templates/reaction, with dynamic quantification ranges of 8 logs and97% to 102% average reaction efficiencies. These new assays were used to screen stool samples from 250 Finnishpatients (median age, 40 years) that had been sent for diagnosis of gastrointestinal infection. Four patients(1.6%; median age, 1.1 years) were reproducibly positive for HBoV2, and one patient (0.4%; 18 years of age) wasreproducibly positive for HBoV3. The viral DNA loads varied from <103 to 109 copies/ml of stool extract. Noneof the stool samples harbored HBoV1 or HBoV4. The highly conserved sequence of the hydrolysis probe usedin this assay may provide a flexible future platform for the quantification of additional, hitherto-unknownhuman bocaviruses that might later be discovered. Our results support earlier findings that HBoV2 is arelatively common pathogen in the stools of diarrheic young children, yet does not often occur in the stools ofadults.
During recent years, the number of bocaviruses known hasincreased rapidly. In addition to human parvovirus B19, thefamily Parvoviridae now also includes another pathogen mem-ber, human bocavirus (HBoV), first detected in pediatricrespiratory secretions in 2005 (2). Several studies have shownan association of HBoV with acute respiratory tract symptomsin young children (1, 8, 14, 16). Three other human bocavi-ruses, designated HBoV2, HBoV3, and HBoV4, were recentlyreported in quick succession to have been detected in humanfeces (4, 12, 13) as well as sewage samples (5). Subsequentstudies have indicated that HBoV2 occurs rarely in respiratorysecretions but fairly commonly in stools of children, and it islikely to circulate globally (4, 6, 7, 10, 13, 19, 21). To date, twopublications have provided evidence for an association ofHBoV2 with gastrointestinal disease (4, 7). HBoV3 andHBoV4 were discovered so recently that little is known of theirepidemiology, transmission, or pathogenicity (4, 6, 12, 19).However, the pathobiological characteristics of HBoV2 to -4could conceivably be expected to be more similar to each otherthan to those of HBoV1, due to significantly closer phylogenicrelationships (12).
While high-performance quantitative PCR (qPCR) assaysare available for HBoV1 detection (15, 18), HBoV2 andHBoV3 have so far been detected by qualitative PCR. Inaddition to allowing for quantification of the target DNA,
real-time qPCR has many other advantages, including a lowerrisk of contamination, decreased hands-on time, and betterspecificity of detection. Moreover, as we have shown serolog-ically, the low-level presence of HBoV1 DNA in the nasophar-ynx is not a reliable indicator of acute HBoV1 infection (11,20). The present study reports the development of multiplexand singleplex qPCR assays for sensitive real-time detection ofall four human bocaviruses (HBoV1 to -4) in clinical samples.These new methods were validated using plasmids with clonedHBoV1 to -4 DNAs and stool samples from patients with orwithout a recent travel history. Furthermore, we wanted toinvestigate whether traveling beyond Northern Europe wouldresult in elevated infection rates by these novel viruses, whichmight have regional differences in occurrence.
MATERIALS AND METHODS
Clinical specimens. Stool samples were obtained from 250 Finnish patients,100 with and 150 without a history of travel outside Northern Europe within 1month before sample acquisition. The median ages were 30.5 years (range, �1 to74 years) and 52.5 years (range, �1 to 95 years), respectively. The destinationsfor the 100 travelers were Europe for 54%, Asia for 29%, Africa for 9%, SouthAmerica for 2%, and unknown for 2% of travelers. Of the 250 subjects, 33 werechildren aged 0 to 10 years, of whom 12 (36%) had traveled abroad. All sampleshad been sent to the Helsinki University Central Hospital Laboratory Divisionfor diagnosis of enteric pathogens, as suggested by symptoms of diarrhea. Allsamples were cultured for enteropathogenic bacteria, with emphasis on Salmo-nella, Yersinia, Shigella, and Campylobacter. In addition, several samples werestudied for enteric parasites and viruses. All samples had further been tested fordiarrheagenic Escherichia coli strains by PCR (3).
For PCR, stool swabs were suspended in 200 �l of Tris-EDTA buffer andboiled for 15 min, and the nucleic acids were purified with a NucliSENS kit usingthe easyMAG platform (bioMerieux, Lyon, France) and eluted in the elutionmedium to a volume of 25 �l. The median DNA concentration of the fecal
* Corresponding author. Mailing address: Department of Virology,Haartman Institute, Box 21, FIN-00014 University of Helsinki, Hel-sinki Finland. Phone: 358-40-1727444. Fax: 358-9-19126491. E-mail:[email protected].
� Published ahead of print on 15 September 2010.
4044
extracts was 40 ng/�l (25th percentile, 20 ng/�l; 75th percentile, 77 ng/�l; range,2 to 200 ng/�l). Potential PCR inhibition had been studied with 80 of the 250DNA extracts by DNA spiking (17). None of the spiked samples were inhibitory.To further evaluate our qPCR assay, we analyzed three stool DNA extractsshown by qualitative PCR to contain HBoV2, HBoV3, or HBoV4 DNA (12).
Plasmids. The performances of the qPCR assays were studied with plasmidscontaining the joint region of the left-hand untranslated region (UTR) and theNS1 gene of the human bocavirus 1 to 4 genomes, corresponding in HBoV1 tonucleotides (nt) 98 to 388 (GenBank accession number EU984245). The corre-sponding regions of HBoV2 (FJ170279) and HBoV3 (EU918736) were synthe-sized and cloned into pUC57 by Genscript (NJ), and that of HBoV4 was syn-thesized and cloned into pJet1.2 (Fermentas, Burlington, Canada) in Helsinki,Finland. The near-full-length HBoV1 clone, pST2, was a kind gift from TobiasAllander (2). Plasmid DNA concentrations were determined by an ND-1000spectrophotometer (Nanodrop, Wilmington, DE). Each plasmid was dilutedserially from 0.5 � 108 to 0.5 � 101 copies/�l in 10 mM Tris-EDTA buffer,aliquoted, and stored at �20°C until use for generation of standard curves forDNA quantification.
Of note, the HBoV2 and HBoV4 primer binding sites in the HBoV2 andHBoV4 plasmids are identical except for a single nucleotide mismatch in thesense primer (Fig. 1). This dissimilarity was taken into account by the use of adegenerate nucleotide (C�T; IUPAC code Y). The HBoV2 and HBoV4 plas-mids were indistinguishable with respect to assay sensitivity and reproducibility.Moreover, Mfold (22) secondary structure predictions showed no major differ-ence between the amplified regions of the two sequences (data not shown).Consequently, the results reported herein for HBoV2 and HBoV4 are desig-nated HBoV2/4 results and were acquired using only the HBoV2 plasmid unlessotherwise stated.
Primers and hydrolysis probe. Conserved and variable regions of the HBoV1to -4 genomes were identified by aligning nine HBoV1 sequences, nine HBoV2sequences, four HBoV3 sequences, and one HBoV4 sequence (Fig. 1). TheHBoV1 and -2 strains were selected from a wide variety of geographical locations(10 countries) to take into account intraspecies variation. For HBoV3 and -4, weused all publicly available near-full-length genomic sequences. Primer selec-tion was done with the aid of AlleleId 6.01 software (Premier Biosoft Inter-national, CA) for maximum intraspecies similarity and, when appropriate,maximum interspecies heterology. The primers are as follows (with GenBankaccession numbers and primer annealing positions in parentheses): HBoV1F,5�-CCTATATAAGCTGCTGCACTTCCTG-3� (NC_007455; 152 to 177);HBoV1R, 5�-AAGCCATAGTAGACTCACCACAAG-3� (NC_007455; 235 to259); HBoV234F, 5�-GCACTTCCGCATYTCGTCAG-3� (FJ170279; 50 to 70);HBoV3R, 5�-GTGGATTGAAAGCCATAATTTGA-3� (EU918736; 205 to230); and HBoV24R, 5�-AGCAGAAAAGGCCATAGTGTCA-3� (FJ170279;128 to 150). The primer region covers the left-hand untranslated region of thehuman bocavirus genome and the beginning of the NS1 gene. Both of theHBoV1 primers and the antisense HBoV3 primer (HBoV3R) were selectedfrom regions with minimal similarity to the other bocaviruses. The qPCRs forHBoV2, -3, and -4 share a single sense primer (HBoV234F), and the qPCR forHBoV2 and -4 (i.e., the HBoV2/4 qPCR) uses the same reverse primer (HBoV2/4R). The hydrolysis probe sequence 6-carboxyfluorescein (FAM)-5�-CCAGAGATGTTCACTCGCCG-3�-minor groove binder (MGB)-quencher black hole 1(BHQ1) (FJ170279; 85 to 104) was selected from a region fully conservedbetween all four known human bocaviruses. In multiplex format, this combina-tion of primers and hydrolysis probe was designed to detect all published typesof human bocaviruses. In singleplex format, the primers were designed to dis-tinguish between HBoV1, HBoV3, and HBoV2/4. All oligonucleotides weresynthesized by Sigma-Aldrich (Steinheim, Germany). The MGB hydrolysis probewas labeled at the 5� end with the reporter dye FAM, and the 3� end was blocked
with the nonfluorescent quencher BHQ1. Blasting of primers and the probeagainst GenBank sequences did not reveal matching sequences that would plau-sibly result in probe hydrolysis. The amplified template region, together with 100bp of surrounding sequence in both directions, was also analyzed for secondarystructures with Mfold (22). No significant DNA secondary structures were de-tected (data not shown).
Real-time PCR. All PCRs were done in a volume of 25 �l using StratageneMx3005p and approved clear PCR plasticware (Stratagene, CA). The singleplexreactions consisted of 1� TaqMan Universal Master Mix (Applied Biosystems,CA) with AmpErase uracil-N-glycosylase (UNG), 0.6 �M concentrations ofsense and antisense primers, 0.3 �M probe, 2 �l of template, and molecularbiology-grade water for a final volume of 25 �l. The multiplex reactions were setup similarly but with all five primers included. UNG was allowed to degrade anypotential carryover PCR products for 2 min at 50°C before activation of theAmpliTaq Gold polymerase for 10 min at 95°C. The amplification consisted of 40cycles of 15 s at 95°C and 1 min at 60°C. Each run included plasmid andno-template controls. To generate baseline-corrected fluorescence (dR) data,baseline fluorescence was automatically determined by the Mx4000 softwareversion 3.01 (Stratagene) baseline algorithm. The cutoff for quantification cycle(Cq) determination was automatically calculated as 20 times the standard devi-ation of the fluorescence value of the baseline in cycles 5 through 9. Eachfluorescent reporter signal from the FAM channel was measured against theinternal reference dye (ROX) signal to normalize for non-PCR-related fluores-cence fluctuations between samples. Strict laboratory procedures were followedto prevent PCR contamination, including separate spaces for handling of sam-ples, Master Mix ingredients, and plasmid templates. All runs included negativecontrols, which gave negative results for human bocavirus DNA throughout thestudy.
Analytical specificity. Analytical specificities of the real-time qPCR assayswere evaluated with 500 ng human DNA per reaction (i.e., 25 �l) from cultured293T cells and cloned full-length or near-full-length genomes of TT virus(GenBank AY666122), parvovirus B19 genotypes 1 (AY504945), 2 (AY044266)and 3 (AJ249437; a kind gift from Antoine Garbarg-Chenon), and polyomavi-ruses BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) (kind giftsfrom Kristina Dorries). We also tested a partial genome of human parvovirus 4(PARV4) and the late-region genes of KI polyomavirus (KIPyV) (EF127906; akind gift from Tobias Allander). We further tested pooled nucleic acid extractsof human stool containing astrovirus, norovirus, and rotavirus. The stool speci-mens had been found virus positive by diagnostic electron microscopy and/orPCR by the Helsinki University Central Hospital Laboratory Division.
RESULTS
Analytical sensitivity, linearity, and amplification efficiency.Limits of detection (LOD) of the real-time assays were deter-mined with serial dilutions of control plasmids. At 10 copies/reaction (5,000 copies/ml of eluate), 100% of 20 replicateswere positive with the multiplex and all singleplex assays re-gardless of the presence or absence of human genomic DNA at500 ng/reaction. This indicates a robust LOD of �10 copies/reaction. We further tested 8 replicates of 10�2 to 102 nominalplasmid copies/reaction diluted in a pool (n � 140) of stoolDNA extracts that were qPCR negative for human bocavirus.Testing was repeated with another instrument on a differentday. A generalized linear model was fitted to the total observed
FIG. 1. Nucleotide sequence alignment of the HBoV1 to -4 genome segments used for qPCR. Intraspecies sequence variation is underlined andshown as degenerate bases according to IUPAC symbols (R, A�G; Y, C�T). Fully conserved nucleotides are shown without underlining, whereasnucleotide differences between the species are in bold. Arrow symbols show the positions and directions of the corresponding primers and probe.For HBoV2 and HBoV4, direct PCR product identification is possible by PCR product sequencing to distinguish three nucleotide differences(boxed nucleotides).
VOL. 48, 2010 qPCR DETECTION OF FOUR HUMAN BOCAVIRUSES 4045
proportion of positive results and to the nominal numbers oftemplate copies using MATLAB software’s (Mathworks,Natick, MA) GLMFIT command with the probit link function.According to the probit analysis, the assay sensitivities in stoolextracts remained good, ranging from �4 copies/reaction(HBoV2/4) to �9 copies/reaction (HBoV1) for both the mul-tiplex and the singleplex assays (data not shown).
The real-time PCRs were linear over the range of 10 to atleast 108 copies/reaction (Fig. 2). The average reaction effi-ciencies for the singleplex and multiplex reactions were highfor all assays regardless of the presence or absence of 500 ng ofhuman genomic DNA per reaction, ranging from 97% to 102%(Table 1).
Analytical specificity. Analytical specificities of the real-timePCR assays were evaluated with purified human DNA, DNAfrom virus-positive stool samples, and with cloned viral DNAof 9 viruses. All plasmids were tested at 1010 copies/reaction,corresponding to 5 � 1012 copies/ml of original sample. Nei-ther the multiplex nor the singleplex qPCR assays showedobservable amplification with any of the plasmids or clinicalspecimens within the 40 PCR cycles.
Cross-reactivity. Potential cross-amplification of HBoV1 to-4 plasmid templates with the three singleplex assays was stud-ied at high input levels of the plasmids (106 to 109 copies/reaction) with 10 replicates. No cross-amplification was ob-served with any of the assays at �2 � 107 copies/reaction. At1 � 108 copies, the HBoV3 primers showed cross-amplificationof HBoV2 and HBoV4 plasmids, with 9 copies/reaction de-
tected. Similarly, HBoV2 primers showed cross-amplificationof the HBoV3 plasmid at 1 � 109 copies/reaction (14 copies/reaction detected); however, no signal was generated at 2 �108 copies/reaction or less. No cross-amplification with HBoV1singleplex was observed at 2 � 109 copies/reaction of HBoV2,HBoV3, or HBoV4 templates. Copy numbers higher than 2 �109 copies/reaction were not tested for cross-reactivity.
All singleplex assays were able to detect the respective boca-virus species also from mixtures of HBoV1 to -4 templates, inwhich HBoV1 to -4 were simultaneously present in variedquantities. Table 2 shows the calculated and measuredamounts of HBoV1, HBoV2/4, and HBoV3 plasmids.
Reproducibility. Reproducibilities of the multiplex andsingleplex assays were studied by replicate analysis of quanti-fication standards in a single run (intraassay variation) andrepeated runs (interassay variation) using three replicates,each at concentrations of 101 to 108 copies/reaction (Table 3).Of note, the intra- and interassay variations were calculatedwith measured quantities of target DNA rather than from thecorresponding Cq values. Highest intra- and interassay varia-tions were observed at 101 copies/reaction, with mean coeffi-cients of variation (CV) of 40% and 33%, respectively. With103 copies/reaction, the corresponding figures were reduced to3% and 16%, respectively.
qPCR of stool extracts. For clinical evaluation of the qPCRmethods presented here, stool samples sent for diagnosis ofenteric infections from Finnish travelers and nontravelers werescreened for DNA of the four human bocaviruses. Of the 250
FIG. 2. Representative results from the real-time PCR quantification of serial dilutions of HBoV1, HBoV2, HBoV3, and HBoV4 plasmids (101
to 108 copies/reaction) with baseline-corrected fluorescence plotted against cycle number. Each type of plasmid was analyzed with the respectivesingleplex assay (solid curves) and the nondiscriminating multiplex assay (dotted curves). The insets show the corresponding standard curves withthe logarithm of the input copy number (y) plotted against the corresponding Cq values (x), and the square of the correlation coefficient (R2). Thestandard curves for the singleplex (solid line) assays are almost indistinguishable from those of the multiplex (dotted line) assays.
4046 KANTOLA ET AL. J. CLIN. MICROBIOL.
samples, 5 (2%) were found reproducibly positive for humanbocavirus DNA in the initial multiplex screening. Of these 5samples, 4 and 1 were positive with the HBoV2 and HBoV3singleplex assays, respectively, and were further confirmed bysequencing. All four samples with HBoV2 DNA were fromchildren aged �2 years, and the sample with HBoV3 DNA wasfrom an 18-year-old female who had recently traveled to Bul-garia. The HBoV2 prevalence among 250 subjects below 95years of age was only 1.6%; however, it was 10% among the 41children below 18 years of age, 12.5% among the 32 childrenaged below 10 years, and 20% among the 20 infants �2 yearsof age. Further patient characteristics, including travel histo-ries, HBoV DNA loads, and other microbial findings, are listedin Table 4. HBoV1 and HBoV4 DNAs were not detected inany of the 250 samples.
With respect to HBoV1 to -3, the qPCR results were vali-dated by rescreening all the samples with a previously de-scribed quantitative assay for HBoV1 (1) and qualitative PCRassays for HBoV2 (6) and HBoV3 (19). The results of theconfirming HBoV1 qPCR assay were in full accordance with
our original results: all 250 samples were negative for HBoV1DNA. Likewise, the samples that were positive with ourHBoV2 or HBoV3 singleplex assays were also positive with therespective qualitative PCR assays, with results confirmed byPCR product sequencing. The sequences from the qualitativePCRs were 98 to 100% identical to previously publishedHBoV2 and HBoV3 sequences, with no observable regionaldifferences.
The three fecal extracts that had previously been shown tocontain HBoV2, HBoV3, or HBoV4 DNA (12) were all re-producibly positive in the respective singleplexes, but not withthe heterologous assays. Samples with HBoV2 and HBoV3DNA were also positive in the multiplex qPCR. The HBoV4positive sample was not multiplex tested due to the very limitedamount of DNA extract available.
qPCR of serum DNA extracts. To further compare our newHBoV1 singleplex assay with the established qPCR test (1)that we have previously used for HBoV1 quantification (11,20), we examined with both tests 15 serum samples that hadbeen shown to contain different loads of HBoV1 DNA (1).
TABLE 1. Mean Cq values from human bocavirus singleplex assaysa
a Values standard deviations (SD) from three replicate human bocavirus singleplex assays of estimated plasmid DNA copy number are shown. Cq, quantitationcycle; R2, square of correlation coefficient.
b Efficiency (E) was calculated from the average slope of the standard curves using the formula E � 10(�1/slope) � 1.c The slope and y intercept were determined by the formula Cq � y intercept � slope � log(initial DNA quantity).
TABLE 2. DNA quantification from heterologous mixtures of multiple human bocavirus plasmidsa
Mix no.bCalculated no. of copies per mix Measured no. of copies per mix (CV%c)
a NT, no template; CV, coefficient of variation; Cq, quantification cycle.b Each row represents a single mix of templates. For instance, the mix 1 contains an estimated 2 � 103 copies of HBoV2 and 2 � 107 copies of HBoV3 plasmids
but no HBoV1 plasmid.c CV%, CV percentage computed from the difference between the calculated and measured numbers of copies per mix without replicates.
VOL. 48, 2010 qPCR DETECTION OF FOUR HUMAN BOCAVIRUSES 4047
Correlation between these two methods was high despite therelatively low levels of viral DNA (r � 0.95) (Fig. 3).
DISCUSSION
The new human bocaviruses and their possible roles in hu-man disease are currently under intensive investigation. Themethods published so far have relied on qualitative rather thanquantitative PCR, which limits the interpretation of results andslows down sample screening. We present real-time qPCRassays for the detection of all currently known human bocavi-ruses, HBoV1, HBoV2, HBoV3, and HBoV4. The initial mul-tiplex screening indiscriminately detects and gives a good es-timate of the level of human bocavirus DNA in a sample. Forthis approximate quantification, the average standard curveof the three plasmids can be used as the quantification refer-ence. The preliminary screening is followed by singleplexqPCRs of the positive samples to identify and accurately quan-tify the amount of viral DNA.
This rather unusual approach of using primers rather thanhydrolysis probes for template identification has both advan-tages and drawbacks. In addition to lowered probe-relatedcosts, another major advantage is that the new assays pre-sented here can be used with minimal technological require-ments. The only requirement is for the real-time machine to becapable of detecting the FAM signal; no multiplexing capabil-ity with multiple detection channels is required. In general,multiplexing with numerous fluorescent dyes can be a chal-lenge for some laboratories with qPCR equipment with a lim-ited variety of wavelength filters. Furthermore, multiplex dataanalysis may be affected by cross talk if the combination ofchosen fluorophores is suboptimal for the qPCR hardware inuse. Our approach bypasses these technical obstacles. Thehighly conserved probe furthermore may provide a flexible and
low-cost qPCR platform for the quantification of related boca-viruses that might be discovered in the future.
A drawback with our approach is low-level cross-amplifica-tion of HBoV2/4 and HBoV3 bocaviruses in very high copynumbers. This is seen as dual positivity in the singleplex qPCRsand can be overcome by diluting and reanalyzing those sam-ples. This could then lead to false-negative results if the sam-ples simultaneously contained two different human bocavi-ruses, one at very high and the other at very low levels.
Such cases are, however, likely to be very rare and of littleclinical significance.
A remaining difficulty is the separation of the genetically sim-ilar HBoV2 and -4 viruses, which in our approach requires PCRproduct sequencing. The currently available GenBank sequencesshow a three-nucleotide difference in the nonprimer sequence ofour HBoV2 and HBoV4 PCR amplicons (Fig. 1). Whether thesedifferences are conserved or variable will become known in thefuture upon accumulation of HBoV4 sequence data. All in all, thepathogenic respiratory bocavirus HBoV1, which has been foundalso in stool, is by our assays most clearly differentiated from theenteric species HBoV2 to -4, with no cross-reactivity with any ofthe three other human bocaviruses.
The repeatability of our assays may superficially appear tobe low. However, rather than using quantification cycles (Cqs)for calculating coefficients of variance (CV), we calculated CVfrom measured numbers of template copies. The Cq methodsignificantly underestimates the true assay variance (9). Fur-thermore, with our method, the CV values are comparablewhen varied amounts of starting template are used. This is nottrue for CV based on untransformed Cqs, as proportionallyequal variations in observed levels of DNA cause significantlyunequal absolute changes in Cq-derived CV in different regionsof the dynamic range.
TABLE 3. Reproducibility of HBoV1, HBoV2/4, and HBoV3 singleplex assays for different concentrations of the plasmidsa
a Intraassay variation was determined from three replicates within a single run, and interassay variation was determined from three replicates, each performed ondifferent days. CV was calculated from the measured number of template rather than Cqs.
TABLE 4. Characteristics of patients with human bocavirus in stoola
Age (yr) Gender Virus DNA load (copies/ml) Recent travel to: Other finding
a The DNA load is given as copies per ml of fecal extract. Recent travel, a journey outside Northern Europe within 1 month before sample acquisition. EPEC,enteropathogenic E. coli; —, no recent travels or no other findings.
4048 KANTOLA ET AL. J. CLIN. MICROBIOL.
The three most recently identified human bocaviruses werediscovered with stool samples, and HBoV2 has since beenobserved to be present not infrequently in feces of youngchildren (4, 6, 12, 13). Also, the respiratory virus HBoV1 hasbeen detected in stool (6, 18). To test and validate our newHBoV1 to -4 assays with such material, we used fecal samplesfrom 250 patients of diverse ages. Our HBoV2 prevalence was1.6% among all 250 subjects. However, all four HBoV2-posi-tive children were �2 years of age. In this age group of 20children, the HBoV2 prevalence was 20%, comparable to thatof a previous study, which reported a 17% prevalence amongyoung children with gastroenteritis and 8% among healthychildren (4). Our results, together with those of two otherrecent studies (6, 13), suggest that HBoV2 DNA is detectedmainly in the stools of children aged 5 years or less. Theputatively narrow age range of HBoV2 infections should betaken into consideration when interpreting the percentages ofHBoV2-positive subjects. In our study, the only bocavirus-positive adult, a female 18 years of age, harbored HBoV3.Altogether, the relatively low frequency of HBoV1, -3, and -4among the 250 samples of this study are in line with the resultsof Kapoor et al. (12), who found these viruses in stools of only1.4%, 2.8%, and 1.4% of 288 children, respectively, whereasHBoV2 occurred in 23.6% of the pediatric stool samples.However, a higher frequency of HBoV1 than HBoV2 DNA inthe stools of children has also been reported (7). The samestudy also found HBoV2 DNA more frequently in the stools ofadults than in those of children.
We furthermore examined the travel histories of our pa-tients, with the hypothesis that traveling outside Northern Eu-rope might result in first contact with a regional bocavirusspecies and result in an elevated infection rate. This same setof samples has been shown to contain a significantly higherproportion of diarrheagenic E. coli in travelers (39%) than innontravelers (8.7%) (3). However, travelers and nontravelersharbored HBoV2 equally. Our results therefore do not supporta link between travel beyond Northern Europe and increasedinfection risk by the enteric human bocaviruses. Our resultswith infants should, however, be considered with caution, be-
cause we tested only 20 children aged �2 years, resulting in alow statistical power. Furthermore, viral shedding among thesubjects with a recent travel history may have ceased betweenthe time of infection and the time of sample acquisition.
Our real-time qPCRs were highly sensitive and specific, andthey reproducibly detected the genomic sequences of all fourknown human bocaviruses down to �10 copies/reaction. Allother viral, human, or bacterial DNAs in native or plasmidform remained negative. Any putative secondary structures orthe flanking terminal regions of the human bocavirus genomedid not seem to interfere with the results, as our HBoV1 qPCRshowed excellent correlation with a well-established HBoV1qPCR assay that amplifies another genomic region (1). Fur-thermore, no interfering secondary structures were revealed byin silico nucleic acid folding predictions. Last, arguing againstpossible PCR inhibition, we found that (i) the analytical sen-sitivities of the multiplex and singleplex assays remained excel-lent (�10 copies/reaction) in the presence of pooled stoolDNA extract, (ii) all the 80 spiked samples were noninhibitory(17), and (iii) 52 of these stool extracts were found to be PCRpositive for other pathogens (3).
In conclusion, we present sensitive and specific real-timeqPCR assays for the detection and quantification of all fourspecies of human bocaviruses currently known, facilitatingforthcoming studies of their epidemiology and pathobiology.Our findings furthermore support earlier notions on the pref-erential occurrence of HBoV2 in stool specimens from youngchildren as opposed to those from adults.
ACKNOWLEDGMENTS
This study was supported by the Academy of Finland (project1122539), the Helsinki University Central Hospital Research and Ed-ucation Fund and Research and Development Fund, the MedicalSociety of Finland (FLS), and the Sigrid Juselius Foundation (for K.K.,R.S., K.H., and M.S.V.).
We thank Kristina Dorries for the BKV, JCV, and SV40 clones,Antoine Garbarg-Chenon for the B19 genotype 3 clone, Tobias Al-lander for the HBoV1 ST2 and polyomavirus KIPyV clones, and OlliRuuskanen and Tuomas Jartti for the 15 serum samples.
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FIG. 3. qPCR quantification of 15 HBoV1 DNA positive sera witha previously established (1) reference assay (HR) plotted against theHBoV1 singleplex assay (HS) introduced in this study. r, Spearman’srank correlation coefficient.
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