human bocavirus: passenger or pathogen in acute ... · to early 2007, includes prospectively...

CLINICAL MICROBIOLOGY REVIEWS, Apr. 2008, p. 291–304 Vol. 21, No. 20893-8512/08/$08.00�0 doi:10.1128/CMR.00030-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Bocavirus: Passenger or Pathogen in Acute RespiratoryTract Infections?

Oliver Schildgen,1‡* Andreas Muller,2 Tobias Allander,3 Ian M. Mackay,4,5

Sebastian Volz,2 Bernd Kupfer,2‡ and Arne Simon1

Institute for Virology, University of Bonn, Bonn, Germany1; Children’s Hospital Medical Center, University of Bonn, Bonn, Germany2;Karolinska Institutet, Department of Microbiology Tumor and Cell Biology, Laboratory for Clinical Microbiology,

Karolinska University Hospital, Stockholm, Sweden3; Queensland Paediatric Infectious Diseases Laboratory,Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Brisbane, Australia4; and

Clinical Medical Virology Centre, University of Queensland, Brisbane, Australia5

INTRODUCTION .......................................................................................................................................................291TAXONOMY ...............................................................................................................................................................292BIOLOGY OF BOCAVIRUS.....................................................................................................................................292LABORATORY DIAGNOSIS....................................................................................................................................292HBoV AND RESPIRATORY TRACT DISEASE ....................................................................................................293

Possible Role of Coinfections ................................................................................................................................295EPIDEMIOLOGY.......................................................................................................................................................295

Prevalence of HBoV................................................................................................................................................297Seasonal Distribution of HBoV Detection...........................................................................................................297Transmission ...........................................................................................................................................................298

CLINICAL OBSERVATIONS ...................................................................................................................................299Limitations of Available Studies...........................................................................................................................300Symptoms Presumably Associated with HBoV Infection ..................................................................................300Chest Radiography Findings.................................................................................................................................301HBoV and Acute Wheezing....................................................................................................................................301HBoV in Immunocompromised Patients .............................................................................................................301Laboratory Results for HBoV-Infected Patients.................................................................................................301

TREATMENT AND PREVENTION.........................................................................................................................302CONCLUSIONS AND FURTHER RESEARCH ....................................................................................................302ACKNOWLEDGMENTS ...........................................................................................................................................302REFERENCES ............................................................................................................................................................302

INTRODUCTION

Human bocavirus (HBoV) was first described in September2005 by Tobias Allander and coworkers at the KarolinskaUniversity Hospital, Stockholm, Sweden (2). The finding re-sulted from the intensive investigation of two chronologicallydistinct pools of nasopharyngeal aspirates (NPAs) obtainedfrom mostly pediatric patients with suspected acute respiratorytract infections (ARTIs). Thus, HBoV joined the ranks ofviruses colloquially termed “respiratory viruses,” which aredetected predominantly in patients with infection of the respi-ratory tract. A random PCR-cloning-sequencing approach wasemployed. In the original study, HBoV DNA was subsequentlyidentified in 17 out of 540 NPAs (3.1%). Coincident detectionof another virus occurred for three patients (17.6% of positivepatients), including two instances of human respiratory syncy-tial virus (RSV) and one detection of human adenovirus(AdV) (2). No other viruses were detected in 14 of 17 HBoV-positive symptomatic patients, at a glance suggesting a highoccurrence of sole detections. However, common respiratory

viruses were not sought using PCR, and several other knownrespiratory pathogens, including human rhinoviruses (HRVs)and human coronaviruses (HCoVs), were not sought by anymeans. The fact that HBoV was not detected randomly in thematerial but was detected significantly more often in the ab-sence of other detected viruses nevertheless suggested thatHBoV may be a causative agent of previously unexplainedrespiratory tract disease. All 14 children without codetectionhad been admitted to an inpatient medical treatment centerafter presenting with symptoms of cough and fever during theprevious 1 to 4 days.

Since the first report, the worldwide presence of HBoV inchildren with ARTI has been confirmed by over 40 studies.However, most published studies describe virus prevalence andwere not designed to address the issue of disease association.Thus, to date, the evidence for an association between HBoVand respiratory tract disease is incomplete. The many preva-lence studies have found an unusually high number of coinfec-tions where HBoV occurs simultaneously with other viruses,making the association of HBoV with disease more complex.Moreover, Koch’s revised postulates cannot be applied toHBoV, since neither a method for HBoV culture nor an ani-mal model of infection has been established (26). This situa-tion applies to most newly identified viruses, including HCoV-NL63 (72) and HCoV-HKU1 (82), polyomaviruses KI (2) and

* Corresponding author. Mailing address: Institute for Virology, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Phone: 49-(0)228-28711186. Fax:49-(0)228-28714433. E-mail: [email protected].

‡ These authors contributed equally to this work.

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WU (29), and the HRVs, HRV-QPM, NAT-001, and NAT-045 (51). Many newly identified viruses have probably re-mained undetected until now exactly because of their inabilityto replicate in vitro under standard conditions and may there-fore never fulfill Koch’s postulates. Well-designed clinical stud-ies will be needed to confirm the causative role of a virus for adisease, as proposed by Fredericks and Relman (26). A num-ber of these studies will be required before a causative role forHBoV in respiratory tract disease can be established.

This review includes all HBoV studies published online upto early 2007, includes prospectively collected data from thewinter season from 2005 to 2006 (74), and discusses virologicaland clinical aspects of this newly identified virus.

TAXONOMY

HBoV is a putative member of the family Parvoviridae (sub-family Parvovirinae, genus Bocavirus). Until the identificationof HBoV, human parvovirus B19 (B19V) (subfamily Parvoviri-nae, genus Erythrovirus) had been the only human pathogen inthe family. B19V is the causative agent of fifth disease, hydropsfetalis (53), and aplastic anemia, in particular in patients withpreexisting hematopoietic disease (20, 21, 23, 30, 42, 76).HBoV was classified as a bocavirus based on genomic structureand amino acid sequence similarity shared with the namesakemembers of the genus, bovine parvovirus (13) and canineminute virus (9, 65). Consequently, the first human member ofthis virus genus has been provisionally termed human bocavi-rus (2, 52). Other human parvoviruses of interest include thenewly identified human parvovirus 4 (PARV4), which is cur-rently unclassified, and the five current species of human ad-eno-associated viruses (AAV), which reside in the genus De-pendovirus. PARV4 is detected in human plasma used in themanufacture of medicinal products, but no pathogenic roleshave as yet been demonstrated (28). The AAVs rely on an-other “helper” virus to replicate, usually an AdV, but in theirabsence AAVs integrate in a site-specific manner into thehuman genome.

The International Committee on Virus Taxonomy definesspecies within the genus Bocavirus as probably antigenicallydistinct, with natural infection confined to a single host species.Species are �95% related by nonstructural gene DNA se-quence. To date, studies of HBoV have addressed only themolecular criterion. This is indeed the main criterion, sincethere have been no comparative antigenic studies among anyof the species of this genus. Although humans are assumed tobe the natural host of HBoV, it should be noted that no studieshave investigated lower animals for the presence of HBoV.

BIOLOGY OF BOCAVIRUS

The members of the family Parvoviridae are small, nonen-veloped viruses. They have isometric nucleocapsids with diam-eters of 18 to 26 nm that contain a single molecule of linear,negative-sense or positive-sense, single-stranded DNA. Thecomplete genome has a length of approximately 4,000 to 6,000nucleotides (nt) (1, 2).

The complete genome length of HBoV has not been deter-mined, but at least 5,299 nt were identified in one of thereference strains. It can be assumed from the genome structure

of other parvoviruses that the genomic DNA of bocavirus isflanked by hairpin structures. These structures cannot be de-ciphered by sequencing methods alone; thus, the completesequence of the entire genome will not be available until theflanking structures are elucidated (2).

The genome contains three proposed open reading frames,with two open reading frames putatively encoding the non-structural proteins (NS1 and NP-1) and one encoding two viralcapsid proteins, VP1 and VP2; the VP2 sequence is nestedwithin VP1 (2). The function of the HBoV NS1 protein isunknown, but one could speculate on its role in HBoV DNAreplication, since the related protein in other parvoviruses islikely to be involved in the binding and hydrolysis of nucleosidetriphosphates and to have helicase activity (85). NP-1 is absentfrom other parvoviruses and its function is unknown (2, 65).Phylogenetic analyses have shown that two genetically distinctbut very closely related clusters cocirculate in the UnitedStates, Sweden, Canada, and France (7, 35). As expected, thededuced coding sequence for the structural proteins VP1 andVP2 from different isolates showed high variability comparedto the coding sequences for the nonstructural NS1 and NP-1proteins, reflecting the more immunogenic character of thevirion-associated proteins.

The cells hosting HBoV replication have not been deter-mined. Parvoviruses in general require proliferating cells fortheir replication. Studies of animal bocaviruses suggest infec-tion of respiratory and gut epithelium and lymphatic organs(18, 19). HBoV DNA is present in patients with ARTI andsometimes reaches high copy numbers in respiratory tract se-cretions, consistent with infection of the respiratory epithelium(1). HBoV DNA has also been detected in the sera of patientswith ARTI and in the feces of patients with ARTI and/orgastroenteritis, suggesting the possibility that a range of cellsmay support HBoV replication in vivo (1, 27, 50, 56, 73).

Until recently, HBoV infection could be identified only bythe detection of its nucleotide sequence. In a recent report,Brieu et al. described parvovirus-like particles in HBoV DNA-positive NPAs by electron microscopy (12). Confirmation ofthese findings by immunoelectron microscopy with a (hithertounavailable) HBoV-specific antibody would support the as-sumption that HBoV DNA, at least at high copy numbers, isvirion associated. Antibodies elicited in humans against HBoVstructural proteins have also recently been demonstrated(22, 33).

LABORATORY DIAGNOSIS

To date, the detection of HBoV has been performed pre-dominantly on NPAs and swabs and has been possible onlywith PCR-based methods, since no virus culture method, ani-mal model of infection, or antibody preparation for antigendetection has been available (2). No comparative studies toidentify an optimal sampling site have been reported, and theselection of a sampling site is also hindered by a lack of knowl-edge about the site of HBoV replication. Specimen handlingand storage is infrequently detailed in the published studies.However, the most frequent approach is certainly immediateor batched column-based nucleic acid extraction and PCRtesting of convenient populations by use of patient materialthat has been previously stored after routine microbial testing.

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Oligonucleotide sequences from PCR methods described todate are summarized in Table 1, but as of yet no comparativestudies have identified an optimal gene target or oligonucleo-tide set(s). For diagnostic purposes, more-conserved geneticregions are preferred; thus, primers directed toward the NS1gene should yield the most robust assays. However, the limitedgenetic variability of HBoV allows multiple suitable PCR tar-gets, including the frequently targeted NP1 gene. The use ofreal-time PCR serves to minimize the risk of amplicon car-ryover contamination, reduce the result turnaround time, andadd an extra layer of specificity (if an oligoprobe-based ap-proach is employed) and can prove less costly overall. Differentresearch groups have described real-time PCR assays that per-mit some degree of quantification of the viral load in respira-tory secretions (1, 37, 45, 62). Since there is no way to stan-dardize respiratory tract specimen collection, respiratory virusquantification by PCR is better described as being semiquan-titative (47).

Nevertheless, recent results obtained by “quantitative” real-time PCR suggest that high HBoV viral loads (defined as �104

copies/ml) are frequently present as the sole viral finding forchildren admitted for acute wheezing, while the clinical signif-icance of low to moderate viral loads is uncertain. High viralload in the respiratory tract was frequently associated with thedetection of HBoV DNA in the blood (1). HBoV DNAemiadeclined after the resolution of symptoms, suggesting that highviral load may represent primary infection. Fry et al. (27)compared patients with pneumonia to healthy control subjects.Quantitative results were not reported in detail but, impor-tantly, they found that the healthy controls exclusively had lownumbers of HBoV DNA copies in respiratory specimens, whileboth high and low HBoV DNA loads were found among thosecases with pneumonia. Low to moderate viral load is relativelycommonplace among studies (62, 38, 45), suggesting that alarge proportion of HBoV detection by PCR may representvirus shedding of uncertain clinical relevance. Thus, standard-ized diagnostic tests that can more accurately identify primaryinfections, which are more likely the true symptomatic cases,are a top priority for future HBoV research. The predictivevalue of high virus copy numbers as well as the diagnostic valueof PCR testing of blood samples should be further investi-gated, but if its limitations are kept in mind, quantitative PCRis a useful interim tool for understanding the course of HBoVinfection. Preliminary serological studies have recently beenpublished (22, 33) and it is expected that serology will be a veryuseful diagnostic addition to the study of HBoV infection, as ithas been for B19V (83).

HBoV AND RESPIRATORY TRACT DISEASE

The fact that HBoV is prevalent in samples from patientswith ARTI does not guarantee a causative role for the symp-toms. For example, many viruses are transmitted via the respi-ratory tract without triggering substantial respiratory symp-toms. Establishing the causative role of a specific agent indisease is a thorough process requiring multiple studies (26). Itis particularly difficult to do so in the absence of culture sys-tems and/or animal models, which is a problem common tostudies of other newly identified viruses. Nevertheless, thepathogenicity of newly identified HCoV or HRV strains has

not been a major issue of debate, most likely because of theirgenetic relatedness to other established respiratory pathogens.With HBoV, the situation is different and confounded by sev-eral facts. First, HBoV is not related to a known human respi-ratory pathogen. Second, HBoV may be shed persistently,since other human parvoviruses (B19V, PARV4, and theAAVs) have the capacity for asymptomatic persistence (41, 44,50). Third, HBoV is commonly detected in association withother respiratory viruses which have an established pathogenicpotential. These facts raise the possibility that HBoV detectionin respiratory tract samples simply reflects asymptomatic per-sistence or prolonged viral shedding. Another hypothesis isthat HBoV is reactivated or produces a transient asymptomaticsuperinfection that is triggered by the presence of anotherreplicating respiratory agent. A few studies providing datarelevant to these issues have been published (1, 2, 27, 33, 35,48, 49).

The first description of HBoV by Allander et al. (2) did,as mentioned earlier, include a study indicating a statisticalassociation between the detection of HBoV on one handand the patient suffering from otherwise unexplained ARTIon the other hand. Diagnostics for other viruses was incom-plete. However, simple asymptomatic shedding of HBoVwould still not result in the observed skewed distribution ofHBoV findings.

Manning et al. (49) identified HBoV in stored respiratorytract samples and compared the frequencies of reported symp-toms associated with each of the different agents sought. Of the21 HBoV-positive patients, 20 children had symptoms of ARTIversus 1 asymptomatic child, a situation similar to that for RSVbut different from what was found for AdVs. The most com-mon clinical diagnosis was “lower RTI,” made for 15 patients(72%).

To date, five studies have included control groups of asymp-tomatic children (1, 27, 35, 48, 49). All studies found highlysignificant prevalence differences between individuals withARTI and asymptomatic individuals. Kesebir and coworkersdetected HBoV DNA from 22 of 425 NPAs of symptomaticchildren, while none of the 96 asymptomatic children testedpositive for HBoV (35). Allander et al. found no positivesamong 64 asymptomatic children compared to 49 of 259 (19%)positive samples from children with acute wheezing (1). Un-fortunately, in these studies the type of specimen varies be-tween both groups, with an unknown impact on the efficiencyof collection, nucleic acid extraction, and PCR sensitivity. Inthe study of Allander et al., the asymptomatic children wereslightly older than the children with ARTI (1).

Maggi et al. tested 335 children with ARTI and 51 asymp-tomatic children (30 healthy infants and 21 preadolescenthealthy children) and detected 4.5% positives among the nasalswabs of ARTI cases and no HBoV in nasal swabs of asymp-tomatic children (48). However, the main weakness of thisstudy is that cases and controls were sampled during differentyears, which may have falsely lowered the detection of virus inthe asymptomatic group.

One recently published study by Fry et al. (27), performed inThailand but coordinated by the Centers for Disease Controland Prevention (Atlanta, GA), included nasopharyngeal swabsfrom 1,168 patients with community-acquired pneumonia, 512patients with “influenza-like illness,” and 280 asymptomatic

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TABLE 1. Overview of those published protocols describing oligonucleotide sequences used for PCR detection of HBoV

Study (reference) Detection method Primer name (sequence) Probe sequence and labelinga Targetregion

Allander et al.,2007 (1)

Real-time PCR(LightCycler)

Boca-forward (GGAAGAGACACTGGCAGACAA); Boca-reverse (GGGTGTTCCTGATGATATGAGC)

FAM-CTGCGGCTCCTGCTCCTGTGAT-TAMRA

NP-1

Allander et al.,2005 (2)

PCR 188F (GAGCTCTGTAAGTACTATTAC);542R (CTCTGTGTTGACTGAATACAG)

None NP-1

Arden et al., 2006(5); Sloots etal., 2006 (68)

PCR HBoV 01.2 (TATGGCCAAGGCAATCGTCCAAG); HBoV 02.2 (GCCGCGTGAACATGAGAAACAGA)

None NP1

Bastien et al.,2006 (7)

PCR VP1/VP2F (GCAAACCCATCACTCTCAATGC); VP1/VP2R (GCTCTCTCCTCCCAGTGACAT)

None VP1/2

Bastien et al.,2007 (8)

PCR VP/VP2–1017F (GTGACCACCAAGTACTTAGAACTGG); VP/VP2–1020R (GCTCTCTCCTCCCAGTGACAT)

None VP1/2

Foulonge et al.,2006 (24)


BocaRT1 (CGAAGATGAGCTCAGGGAAT);BocaRT2 (GCTGATTGGGTGTTCCTGAT)

FAM-CACAGGAGCAGGAGCCGCAG-TAMRA

NP1

Sequencing BocaSEQ1 (AAAATGAACTAGCAGATCTTGATG); BocaSEQ4 (GAACTTGTAAGCAGAAGCAAAA); BocaSEQ2 (GTCTGGTTTCCTTTGTATAGGAGT); BocaSEQ3 ( GACCCAACTCCTATACAAAGGAAAC)

None

Kesebir et al.,2006 (35)

PCR GGACCACAGTCATCAGACCCACTACCATCGGGCTG

None VP1

Kleines et al.,2007 (37)


Same as that found in the work of Allander etal., 2005 (2)

GGAAGAGACACTGGCAGACAAC-fluorescein; LC-Red 640-CATCACAGGAGCAGGAGCCG

NP1

Kupfer et al.,2006 (39);Simon et al.,2007 (66)

PCR OS1 (CCCAAGAAACGTCGTCTAAC); OS2(GTGTTGACTGAATACAGTGT)

None NP1

Lin et al., 2007(43)

Real-time PCR(TaqMan)

Forward primer (AGCTTTTGTTGATTCAAGGCTATAATC); reverse primer (TGTTTCCCGAATTGTTTGTTCA)

FAM-TCTAGCCGTTGGTCACGCCCTGTG-TAMRA

NS

Lu et al., 2006(45)

Real-time PCR(iCycler iQ real-time detectionsystem �Bio-Rad�)

Primer, fwd (TGCAGACAACGCYTAGTTGTTT); Primer, rev (CTGTCCCGCCCAAGATACA)

FAM-CCAGGATTGGGTGGAACCTGCAAA-Black_Hole_Quencher

NS1

Primer, fwd (AGAGGCTCGGGCTCATATCA); Primer, rev (AGAGGCTCGGGCTCATATCA)

FAM-AGGAACACCCAATCARCCACCTATCGTCT-Black_Hole_Quencher

2478–2497;2558–2537

Manning et al.,2006 (49)

Nested PCR Outer sense primer (TATGGGTGTGTTAATCATTTGAAYA); outer antisense primer (GTAGATATCGTGRTTRGTKGATAT); innersense primer (AACAAAGGATTTGTWTTYAATGAYTG); inner antisense primer (CCCAAGATACACTTTGCWKGTTCCACCC)

None NS

Outer primers used were CCAGCAAGTCCTCCAAACTCACCTGC and GGAGCTTCAGGATTGGAAGCTCTGTG; inner primers fol-lowed the sequence of the primer sequences188F and 542R

None NP

Qu et al., 2007(59)


TAATGACTGCAGACAACGCCTAG; TGTCCCGCCCAAGATACACT

FAM-TTCCACCCAATCCTGGT-MGB

Neske et al., 2007(56)

Real-time PCR (LightCycler) andphylogeneticanalysis

BoV2466a (TGGACTCCCTTTTCTTTTGTAGGA) targeting NP1 2466–2443 (real-timePCR); BoV3885s (ACAATGACCTCACAGCTGGCGT) (phylogenetic analysis); BoV4287s(CAGCCAGCACAGGCAGAATT) (phyloge-netic analysis); BoV4456a (TCCAAATCCTGCAGCACCTGTG) (phylogenetic analysis);BoV4939a (TGCAGTATGTCTTCTTTCTGGACG) (phylogenetic analysis)

FAM-TGAGCTCAGGGAATATGAAAGACAAGCATCG-TAMRA

NP1; VP2

Regamey et al.,2007 (60)


Primer forward (CACTGGCAGACAACTCATCACA); primer reverse (GATATGAGCCCGAGCCTCTCT)

AGCAGGAGCCGCAGCCCGA NS1

Schenk et al.,2007 (62)


HBoV-UP (AGGAGCAGGAGCCGCAGCC);HBoV-DP (CAGTGCAAGACGATAGGTGGC)

HBoV-P: FAM-ATGAGCCCGAGCCTCT-TAMRA

NP1

Smuts andHardie, 2006(69)

Seminested PCR NP-1 s1 (TAACTGCTCCAGCAAGTCCTCCA); NP-1 as1 (GGAAGCTCTGTGTTGACTGAAT); NP-1 as1 and NP-1 s2 (CTCACCTGCGAGCTCTGTAAGTA)

None NP1

VP s1 (GCACTTCTGTATCAGATGCCTT);VP as1 (CGTGGTATGTAGGCGTGTAG);VP s2 (CTTAGAACTGGTGAGAGCACTG)

None VP1/2

a FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binder (Applied Biosystems).



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individuals. HBoV DNA was detected in only 3 asymptomaticindividuals (1%), whereas 20 out of 512 (3.9%) outpatientswith “influenza-like illness” (according to the WHO definition)and 53 (4.5%) out of 1,168 hospitalized patients with the di-agnosis “pneumonia” tested positive for HBoV (27). For chil-dren aged from 0 to 4 years, the HBoV prevalence was 12%among pneumonia cases and 2% among asymptomatic con-trols. To date, this is the only study from which all groups havebeen sampled in the same way, making these data more robust.Among hospitalized children of �5 years of age with the di-agnosis “pneumonia,” HBoV was the third most commonlydetected virus (12%). Higher prevalence could be confirmedonly for RSV and HRV (27). Viral loads of this study werereported separately (45). Most positive samples among allgroups had low viral loads but, importantly, high loads wereseen only among cases and not among asymptomatic controls.

A main concern regarding studies comparing virus preva-lences in respiratory tract samples of symptomatic and asymp-tomatic individuals is the risk for bias related to respiratorytract sampling. An inflammatory process, regardless of cause,will produce a cell-rich mucoid secretion, easily available forsampling, while asymptomatic individuals have very littlenasopharyngeal secretion at all. Thus, the detection of, e.g., anintracellular persisting virus could very well be enhanced by aninflammatory process regardless of its cause. The main alter-native hypothesis to HBoV being a pathogen is that the virus ispersisting or being shed for long periods from the respiratorytract at copy numbers near the lower limit of PCR detection.Because of these possibilities, comparisons of prevalenceamong symptomatic versus asymptomatic subjects must be in-terpreted with great care unless viral loads are reported.

It is also possible to establish a statistical association be-tween HBoV and disease without using asymptomatic controls.Allander et al. (1) studied patients hospitalized for acutewheezing in Finland and found that the occurrence of HBoV inblood was linked in time with an acute infectious episode andnormally disappeared after recovery. In another statisticalanalysis of the data, HBoV-positive patients with and withoutother pathogens detected in the respiratory tract were com-pared. HBoV was significantly more prevalent in patientswhere no other virus explaining the symptoms was detected.Interestingly, only the cases with high HBoV loads showed thisassociation. Thus, in two ways, internal symptomatic controlscould be used to support a statistical association betweenHBoV and disease in this study. Results were highly significantand at the same time 76% of HBoV-cases were codetectionswith other viruses, showing that frequent codetections are notnecessarily an argument against disease association. The studysuggested that high-load and viremic HBoV infection is asso-ciated with respiratory tract symptoms, while detection of a lowviral load in the nasopharynx alone has uncertain relevance. Itwas hypothesized that these two entities represent primaryinfection and persistence, respectively, each accounting forapproximately half of the HBoV findings in this particularmaterial. This hypothesis has recently been confirmed by ap-plying serology to the same material (33). Further studies areneeded in order to determine the length of possible viral shed-ding or persistence. Regamey et al. detected HBoV DNA inone patient’s respiratory specimen 3 weeks after the acutephase of infection (60).

In summary, several studies have found a statistical associ-ation between HBoV and acute respiratory symptoms, in a waythat is consistent with a causal role. However, accurately es-tablishing a causal relationship will require further studies,since current data also indicate that HBoV does not have acausal role for many of the ARTI cases in which it is detected.The diagnostic value in the individual case of detecting HBoVDNA in the respiratory tract therefore remains unclear.

Possible Role of Coinfections

While the main hypothesis explaining the frequently ob-served HBoV codetections involves some kind of innocuouspersistence or prolonged shedding, a possible role for HBoV asa true copathogen remains uncertain and uninvestigated. Itsfrequent presence alongside other viruses cannot be ques-tioned (Table 2). The results of our University of Bonn studyreported a codetection frequency of 36%. Such high percent-ages have been reported by most studies which have looked forcoinfections, with codetection frequencies of 18% to 90% be-ing reported (2, 27). Manning and coworkers detected one ormore additional viruses in 43% (23/53) of HBoV-positive sam-ples (49). The overall frequency of codetection among HBoV-negative samples that were positive for other viral pathogens inthe same study was 17% (47/271) (P � 0.001). One explanationfor the wide range of results is the nonstandardized diagnosticpanel common to published studies. In addition, differences intest sensitivity have to be considered, in particular in light ofthe high proportion of low-load infections (14, 77). Codetec-tion of another virus with HBoV, usually when the latter is atlow viral load, occurs frequently from patients with ARTI, butHBoV is still rare in asymptomatic individuals. One hypothesisfor this is that detection of innocuous HBoV shedding is en-hanced by airway inflammation caused by another virus, asdiscussed above. Another is that HBoV is involved in thepathogenesis and, in some way, the aggravation of symptoms,so that it is frequently observed in hospitalized patients. Yetanother possibility is that HBoV is a helper virus which aidsother viruses or itself requires the aid of another ongoinginfection for activation or reactivation of replication. There arecurrently no data defining a mechanism by which HBoV couldbe described as either a pathogen or a passenger. To date, itremains uncertain whether codetection with any respiratoryviruses results in more-serious clinical outcomes. This is not aquestion unique to HBoV infections but an important facet ofARTIs in general that must be addressed in the future, per-haps with the aid of animal models.

EPIDEMIOLOGY

Reports suggest that HBoV has worldwide endemicity. Ithas been detected over several years in many countries, includ-ing Sweden, Australia, the United States, Japan, Germany,South Africa, Jordan, France, Canada, Iran, Spain, The Neth-erlands, Korea, Thailand, Switzerland, and China (1, 2, 4–7, 14,15, 24, 25, 27, 34–37, 39, 43, 45, 46, 48–50, 52, 54, 55, 59, 60, 62,66, 68, 69, 73, 74, 77).

Based on phylogenetic analysis of predicted amino acidalignments, HBoV exists worldwide as a single lineage com-posed of two subtly different genotypes, as shown in Fig. 1. The



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greatest variability was observed within the 285-bp portion ofVP1/VP2 (Fig. 1C), whereas significantly lower genetic vari-ability was seen for viral sequences within the NS1 and theNP-1 genes (Fig. 1A and B).

Prevalence of HBoV

The proportion of respiratory specimens from symptomatichospitalized children that contain HBoV sequences has rangedfrom 1.5% to 19% (1, 7) Most children infected with HBoVhave been younger than 24 months (2, 15, 55, 59, 68, 69), butolder children may also be affected (7, 14). For example, 4 of27 (15%) positive specimens in the study of Chung et al. wereobtained from children of �36 months of age (15). Thus, itseems reasonable to include older children into prospectivesurveillance studies. As expected, studies which included onlyhospitalized children and children with wheezing (14) pre-sented a higher illness severity than those studies that analyzedrespiratory specimens from outpatients as well (6, 59, 60). Inorder to determine a more realistic prevalence of HBoV inrespiratory tract samples, future prospective studies shouldalso include appropriately age-matched children and adultsembodying a clinical description of “asymptomatic” as well aspatients presenting with mild illness.

There are few systematic studies including adults, but avail-able studies indicate a very low virus prevalence by PCR in therespiratory tract of adults (2, 7, 27, 49).

Recently, seroepidemiological data were published from Ja-pan by Endo et al. (22). Anti-HBoV antibodies were detectedin 145 of 204 (71.1%) serum samples from people aged from 0month to 41 years from the Hokkaido Prefecture. The sero-prevalence was lowest (5.6%) in the age group from 6 to 8months and highest in the age groups older than 6 years (94.1to 100%). The findings of high antibody prevalence and lowvirus prevalence among individuals older than 6 years are con-sistent with each other and suggest that there may be protec-tive immunity after past infection. Positive antibody titers werealso detected in the age group younger than 6 months, but thisphenomenon is explained by the antibody transfer via the pla-centa to the fetus predominantly in the third trimester ofpregnancy (22).

Seasonal Distribution of HBoV Detection

According to the literature, HBoV DNA-positive ARTIsoccur in children across a range of months. The peak “respi-ratory season” varies from year to year (79). Therefore, it is notfeasible to draw conclusions concerning the epidemiology of anewly identified virus based on snapshot analyses of singleseasons or even multiple respiratory seasons. In accordancewith the University of Bonn’s data, most authors reportingfrom regions with temperate climates have observed a higheroccurrence of HBoV detections during the winter and springmonths (2, 69). Choi et al. (Korea 2000 to 2005) reported arelatively high occurrence of HBoV in the late spring and earlysummer. They did not reveal any obvious correlation tochanges in the parallel RSV season (14). Maggi et al. fromItaly could not confirm a seasonal distribution of the HBoVinfections in their study of hospitalized infants with RTI (48),but they found significant differences between years, with noV

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HBoV detected in any specimen from 2000 to 2002 (n � 43,including 30 specimens from symptomatic infants). Theweakness of most retrospective studies is that more speci-mens are collected during the winter months, because that isthe epidemic season for most viral RTIs. Both more-activesampling and enhanced detection of HBoV by other infec-tions, as discussed above, could therefore lead to false ob-servations of seasonal patterns. It must also be kept in mindthat the numbers reported in most studies probably reflect amix of incidence and carrier prevalence. The true incidenceand seasonality of primary HBoV infection remain un-known.

Transmission

Nothing is known about the routes of HBoV transmission.Because of its sometimes very high copy numbers in respi-ratory tract secretions, aerosol and contact transmission arelikely effective, as they are for other respiratory viruses.Hand-to-hand, hand-to-surface, and self-inoculation routeshave certainly proven to be efficient steps in the transmis-sion of the “common cold.” Since we know that HBoV DNAexists in some capacity within feces, the possibility of fecal-oral transmission must also be considered. Further studiesshould include more testing of stool samples for HBoV to

FIG. 1. Phylogenetic analysis of HBoV. Phylogenetic trees are based on 61 partial NS1 genes (245 nt, corresponding to nt positions 1509to 1753 in the ST1 isolate [accession number DQ000495]) (A), 167 partial NP-1 genes (242 nt, corresponding to nt positions 2340 to 2581in the ST1 isolate) (B), and 133 partial VP1/VP2 genes (285 nt, corresponding to nt positions 4547 to 4831 in the ST1 isolate) (C). Sequenceswere aligned using the program CLUSTAL_X, version 1.83 (71). Phylogenetic relationships of the aligned sequences were inferred from thegenerated alignment by the neighbor-joining method (61). The reliability of the tree topology was evaluated by 500 replicates of bootstrapresampling (84). Phylogenetic trees were visualized using the TREEVIEW software tool (58). Trees show the HBoV sequences used for theanalysis and their individual geographical origins. The numbers in parentheses indicate the numbers of isolates for the respective locations.For reasons of clarity, this figure does not include GenBank accession numbers. Detailed information on GenBank accession numbers ofsequences used for the phylogenetic analysis is available upon request. (D) Phylogenetic placement of HBoV and other members of thegenus Parvovirinae. Bootstrapped (n � 1,000) neighbor-joining tree based on 80% of the complete genomic nucleotide sequence. Bootstrapvalues (%) are indicated at each branching point. B19, erythrovirus B19; PTMPV, pig-tailed macaque parvovirus; RMPV, rhesus macaqueparvovirus; SPV, simian parvovirus; ChPV, chipmunk parvovirus; BPV2 and BPV3, bovine parvovirus 2 and 3; GPV, goose parvovirus;MDPV, Muscovy duck parvovirus; AMDV, Aleutian mink disease virus; PPV, porcine parvovirus; CPV, canine parvovirus; RPV-1a, ratparvovirus-1a; KRPV, Kilham rat parvovirus; MPV1, mouse parvovirus 1; MVM, minute virus of mice; MVC, minute virus of canines.



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confirm the extent and nature of virus DNA shedding andthe capacity of the virus to survive disinfectants (10, 11, 17,31, 38) and permit broader investigations of a possible rolefor HBoV as an enteric pathogen. So far there have been nostudies on the tenacity of the virus or about the effect ofcommonly used hospital-grade disinfectants. Since otherparvoviruses are known to be highly resistant to disinfec-tants (10, 11), such investigations will be important but willrequire an HBoV culture system or animal model of infec-tion.

Kesebir and coworkers reported 3 infants (14%) of 22with presumed nosocomial HBoV infection (35). The in-fected infants were 1, 4, and 6 months of age at the timetheir NPAs were sampled and had been hospitalized sincebirth. Two of the three patients had HBoV-positive NPAswithin a period of 4 days and had been cared for by the samemedical personnel on the same ward. Phylogenetic analysisof the two positives showed identical nucleotide sequencesin both the NP1 and VP1/VP2 gene region; however, be-

cause of the low genetic variability of HBoV, the signifi-cance of such a finding should not be exaggerated. Notably,vertical transmission could not be excluded. Three of 12HBoV-positive children reported in the study of Kleines etal. developed symptoms of ARTI after at least 4 weeks ofhospitalization (37). Since the incubation period of HBoVinfection is unknown, it is not possible to state that this wasnosocomial transmission.

The presence of HBoV DNA in the blood combined withsuspected persistence could have implications for transfusionmedicine, since organs or blood products derived from acutelyinfected donors could be contaminated and serve as a source ofinfection (1, 59). However, unlike PARV4, HBoV was notdetected in plasma pools (28).

CLINICAL OBSERVATIONS

While the role for HBoV in causing any symptoms remainsunclear, studies of the symptoms reported for HBoV-positive

FIG. 1—Continued.



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patients nevertheless provide an important starting point. Be-sides some case reports (39, 62, 66), 23 research study publi-cations were included in this review that contained data aboutsymptoms and outcomes for and radiological findings and lab-oratory results from HBoV-positive hospitalized children (Ta-bles 2 and 3) (1, 2, 6, 7, 14, 15, 24, 25, 27, 34, 35, 37, 43, 46, 49,54, 55, 59, 60, 68, 69, 73, 77). We also added our data, whichwere collected in the winter of 2005/2006 (74).

Limitations of Available Studies

Only a few studies of HBoV have collected clinical dataprospectively (the University of Bonn study presented here[Germany] and the studies of Regamey et al. [Switzerland][60], Monteny et al. [The Netherlands] [54], Allander et al.[Finland] [1], and Fry et al. [Thailand] [27]). In the remainingstudies we cite, laboratory, clinical, and radiological findingshave been acquired retrospectively, similar to many studies ofhuman metapneumovirus (HMPV) infection (78, 81). Onlynonstandardized, research-only, in-house PCR diagnosticshave been employed to date. Because of the obligate use ofPCR, one cannot truly talk about “infection”; rather, eachHBoV DNA-positive specimen should be described as a virus“detection.”

Considering that prolonged shedding of HBoV or reactiva-tion by other infections may account for a remarkable numberof the HBoV detections discussed above, it is a severe limita-tion that diagnostic assays separating these cases from primaryinfections are not yet available. Most published studies havenot taken this into consideration.

A lack of international consensus about the definition ofcertain respiratory diseases is another obstacle to accuratelycharacterizing the clinical outcomes of HBoV infection, just asit is for any respiratory infection. There is no agreement aboutthe definition of obstructive bronchitis, recurrent obstructivebronchitis in infants, bronchiolitis, bronchopneumonia, or lo-

bar pneumonia (3, 70). Six out of the 23 analyzed studies usedthe diagnosis “bronchiolitis” (6, 7, 14, 24, 25, 46, 77). Thepercentages of “bronchiolitis” within the diagnostic spectrumranged from 3.2% to 46% (24, 77). Two studies provideddiffering definitions (6, 14), whereas the remaining publica-tions did not even comment on the clinical criteria. Only 7of 23 studies (1, 2, 6, 27, 35, 37, 46) definitively stipulated aradiological confirmation of the clinical diagnosis “pneumo-nia.” Most studies did not make a distinction between(central) bronchopneumonia and segmental or lobar pneu-monia.

Symptoms Presumably Associated with HBoV Infection

Clinical symptoms most frequently reported in individualswhere HBoV is the only detected virus include cough, rhinor-rhea, and fever, which are also the most common nonspecificsymptoms leading to respiratory viral testing in children. Themost common clinical diagnoses given to HBoV-positive pa-tients, with or without coinfections, include upper RTI, bron-chitis, bronchiolitis, pneumonia, and acute exacerbation ofasthma. This clinical spectrum is in accordance with other viralARTIs, similar to the situation with RSV infections (75) andwith HMPV infections (78, 80). There are no described distinctclinical signs differentiating HBoV-positive infections fromthose ascribed to other viruses (2, 37). This could imply thatHBoV indeed has a clinical picture similar to those seen forother ARTIs or simply that because of the mentioned diag-nostic problems with HBoV many of the studied patients wereactually suffering from other infections. Symptoms seem topersist for 1 to 2 weeks on average (range, 2 days to 3 weeks)(1, 60); Monteny et al. reported a prolonged course of fever(�7 days or recurring) in HBoV-infected patients (54). HBoVhas also been detected in individuals with skin rash, althoughno causal association has been identified (6, 15, 54). Allanderat al. reported a 42% incidence of acute otitis media in solely

TABLE 3. Diagnosis at discharge from hospital (%)

Study (reference)

% Patients from indicated study with indicated diagnosis upon dischargea:

UpperRTI Croup Bronchitis Bronchiolitis Pneumonia Asthma

exacerbationGastrointestinal

symptomsFebrileseizures

Allander et al., 2005 (2) NA NA NA NA NA NA NA NAAllander et al., 2007 (1) 0b 0 25 67 75 8 0 0Arnold et al., 2006 (6) 24 4 NA 26 24 24 16 NABastien et al., 2006 (7) 22 NA NA 11 17 NA NA NAChoi et al., 2006 (14) 5 8 NA 25 56 11 NA NAChung et al., 2006 (15) 24 0 76 0 0 0 12 NAFoulongne et al., 2006 (24) 15 NA NA 46 11 27 NA NAKaplan et al., 2006 (34) NA NA NA NA NA NA NA NAKesebir et al., 2006 (35) NA NA NA NA NA NA 25 NAMa et al., 2006 (46) —c NA 44 11 33 5 NA NAMaggi et al., 2007 (48) 0 0 0 55 45 0 11 0Manning et al., 2006 (49) 29 NA NA NA NA NA NA NANaghipour et al., 2007 (55) 0 0 14 NA 46 10 NA NAQu et al., 2007 (59) 0 0 29 NA 62 NA 9 5d

Volz et al., 2007 (74) 9 9 45 3 64 NA 9 9Weissbrich et al., 2006 (77) 40 NA 16 3 17 NA NA 9

a Data are percentages for all investigated patients unless otherwise indicated. NA, data not available.b Allander et al. (1) reported the diagnosis of acute otitis media for 42% of HBoV-positive children with bronchial obstruction without viral coinfection.c Study included only patients with lower RTIs.d Qu et al. reported one patient (10 months of age) with an acute life-threatening event.



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HBoV-positive patients (2). Except for this report, there arefew data on bacterial coinfections.

The possibility that HBoV, like the closely related bovineand canine bocaviruses (18, 19), could cause gastroenteritiswas raised in the first report on HBoV (2). Gastrointestinalsymptoms have been described for up to 25% of all patients (6,35, 54). Maggi et al. (48) detected HBoV DNA in stools of a6-month-old boy followed for neurological problems who hadpresented with diarrhea and bronchopneumonia. Both respi-ratory and stool specimens were positive for HBoV and anti-gen negative for rotaviruses, AdVs, astroviruses, and calicivirus1 and 2. Vicente at al. investigated the presence of HBoVDNA in 527 stool samples from ambulatory patients with gas-troenteritis (�36 months of age) with or without additionalrespiratory symptoms (73). Of these, 48 (9.1%) were positivefor HBoV DNA. Other enteric pathogens were found in 58%of all HBoV-positive fecal samples (Table 2). In contrast, Leeet al. detected HBoV DNA in only 0.8% of 942 hospitalizedchildren with gastroenteritis (40). Neske and coworkers re-ported a high frequency of HBoV DNA in stool samples de-rived from children who were also positive for HBoV DNA inNPAs (56).

Chest Radiography Findings

In the University of Bonn study, the majority of HBoV-positive patients (10/11) showed symptoms severe enoughfor physicians dealing with the patients to perform a chestradiograph, and 8 of 10 (80%) patients with a chest radio-graph had visible abnormalities (74). In these 11 patients nocoinfections were observed. This high percentage of pathol-ogy is in accordance with the results of other clinical re-search groups, which found similar pathology in 43% to 83%of cases (2, 24, 25).

The most common diagnosis in this study was (central) bron-chopneumonia; in 18% a segmental/lobar pneumonia was di-agnosed (74). Cases of HBoV-positive central pneumonia aswell as interstitial and lobar pneumonia, especially in newbornsand infants, have been described.

HBoV and Acute Wheezing

ARTIs have frequently been detected in infants with recur-rent airway obstruction (“wheezing”) and in older children andadults with asthma exacerbations (32, 63, 64, 80). In fact, thehighest frequency of laboratory confirmations are described bystudies of children with acute expiratory wheezing, usuallyattributed to viruses. Recently published clinical studies reportasthma exacerbation as a clinical entity in up to 27% of HBoV-positive patients (6, 24, 25), but several of the analyzed studiesdid not explicitly exclude relevant viral pathogens such as RSVand the HRVs. Half of the patients in the original study byAllander and coworkers presented with asthma as an underly-ing disease (2). Allander et al. subsequently reported that 49 of259 (19%) children hospitalized for acute wheezing in Finlandwere HBoV positive and speculated that this unusually highpercentage could imply that wheezing is the main manifesta-tion of HBoV infection. Fry et al. (27) found a statisticalassociation between HBoV detection and reporting wheezingamong patients with pneumonia in Thailand. Naghipour et al.

found that 5 HBoV-infected patients (24%) had a history ofasthma (55), while Maggi investigated respiratory specimensfrom 22 adult patients with acute asthma exacerbation and didnot detect HBoV (48). Chung et al. investigated nasopharyn-geal specimens from 231 children (1 month to 5 years of age)hospitalized with acute wheezing (16). Besides RSV (13.8%),HBoV was the most frequently detected virus (13.8%) in 5.6%without coinfection; HMPV and HCoV-NL63 were detected in7.8% and 1.3% of wheezing children, respectively.

HBoV in Immunocompromised Patients

Several clinical research groups have reported HBoV-posi-tive immunosuppressed/immunodeficient patients (6, 49, 69).Arnold and coworkers described two pediatric patients positivefor HBoV after organ transplantation (6). Smuts and cowork-ers reported HBoV detections for eight human immunodefi-ciency virus-infected pediatric patients (69), and Manning andcoworkers described two HBoV-positive immunosuppressedadult patients (49). Kupfer et al. have recently published theclinical case of a severe infection in a 28-year-old HBoV-positive female patient with malignant B-cell lymphoma (39).On admission, the patient had a pancytopenia, high fever, andclinical and radiological signs of pneumonia (reticulonodularinfiltrates in the computed tomography scan of the thorax).Despite the application of antibiotics, antifungals, and the an-tiviral ganciclovir, fever continued for 14 days. HBoV DNAwas detected retrospectively, suggesting HBoV as the sole po-tential pathogen in NPAs. Since unexplained pulmonary dis-ease is common in this group of patients, the role of HBoV incausing the symptoms is unclear. However, in studies to date,most symptomatic adults positive for HBoV DNA have falleninto a category of immunosuppression. On the other hand, thismay be the result of a selection bias, since these are the adultor elderly patients in which respiratory diagnostic specimensare taken in case of an infection. HBoV has not been identifiedin lymphoid tissue and in bone marrow and brain, respectively,from human immunodeficiency virus type 1-infected and un-infected adults upon autopsy (50).

Laboratory Results for HBoV-Infected Patients

Very few investigators have been able to document thecourse of markers of inflammation such as C-reactive protein(CRP) or white blood cell (WBC) count for HBoV-positivepatients. None of the first 11 HBoV-positive children treatedin our center (University of Bonn) in 2005/2006 fulfilled thelaboratory criteria of a suspected bacterial coinfection (WBC,�15 � 109/liter; CRP, �40 mg/liter) (67). The median WBCcount was 11.3 � 109/liter (range, 6.7 � 109 to 16.7 � 109) andthe median CRP concentration was 12.5 mg/liter (range, �0.03to 114) (74). Others reported median WBC and CRP concen-trations of similar magnitudes (46). In Allander’s recent reporton 12 children with HBoV infection (no viral coinfection) (1),9 displayed a radiologically confirmed pneumonia. The medianWBC was 9.1 (6.3 to 16.3) � 109/liter and the median CRP was18 (0 to 78) mg/liter.



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TREATMENT AND PREVENTION

The clinical impact of HBoV infection is uncertain, and todate there have been no studies on the benefits of particulartherapeutic approaches for HBoV-infected individuals and noproposals for novel therapeutics. Prospective controlled stud-ies evaluating specific treatment approaches for patients in-fected by HBoV will require analytically sensitive, specific, andclinically relevant diagnostic procedures. In our center (Uni-versity of Bonn), the high frequency of radiographicallyconfirmed pneumonia (70%) might explain the use of antimi-crobial chemotherapy for 82% of HBoV-positive patients.

A rapid HBoV testing method capable of identifying clini-cally relevant cases could reduce the unjustified and generallyineffective use of antibiotics in these patients.

If HBoV turns out to contribute significantly to the diseaseburden on children, possibilities for vaccine development willlikely be investigated, like it has been for RSV, HMPV, andparainfluenza viruses. To date, no such studies have been re-ported.

CONCLUSIONS AND FURTHER RESEARCH

Our current knowledge of HBoV infection suggests that thevirus is sometimes a passenger and sometimes a pathogen inacute respiratory tract disease. A better understanding of thenatural course of HBoV infection and an expanded arsenal ofdiagnostic tests capable of discriminating carriage from infec-tion will be necessary before any clinical questions can becomprehensively addressed. Detection of HBoV DNA inblood and serological assays have shown promising preliminaryresults. To date, retrospective studies report a peak of HBoVdetections during the first and second years of life. Some casereports have raised concerns about serious clinical outcomesamong immunosuppressed individuals. To date there is neithera method for virus culture nor an animal model of infection,but hopefully the introduction of serological detection of spe-cific antibodies will permit us some insight into the pathogen-esis and natural course of HBoV infection.

Many additional questions cannot yet be answered by thestudies that have been reported and should be addressed byfuture studies. How is HBoV transmitted, and is HBoV acausative agent of gastrointestinal diseases? Does the viruspersist in the human host? Could coinfection with HBoV in-crease the severity of concurrent viral infections? What is theimmune response to HBoV infection? Can HBoV cause exac-erbations of asthma and chronic obstructive pulmonary dis-ease?

HBoV might be one of the most recently identified respira-tory viruses, but its nature has attracted as much interest andraised as many questions as many of its better-characterizedrelatives. After decades of research, the most widespread andfrequent causes of human infections, the respiratory viruses,are still as confounding as ever.

ACKNOWLEDGMENTS

This work was partially supported by grants from the Else-Kroner-Fresenius-Stiftung (grant number A 01/05//F 00) and the EuropeanCommission (contract number LSHM-CT-2006-037276).

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