The authors have declared that no competing interests exist.
Conceived and designed the experiments: NP SE. Performed the experiments: AP AZ AS. Analyzed the data: FB. Contributed reagents/materials/analysis tools: SBi GU AS SBo EF. Wrote the paper: NP AP SE.
To evaluate the role of human bocavirus (hBoV) as a causative agent of respiratory disease, the importance of the viral load in respiratory disease type and severity and the pathogenicity of the different hBoV species, we studied all hBoV-positive nasopharyngeal samples collected from children who attended an emergency room for a respiratory tract infection during three winters (2009–2010, 2011–2012, and 2013–2014). Human bocavirus was detected using the respiratory virus panel fast assay and real-time PCR. Of the 1,823 nasopharyngeal samples, 104 (5.7%) were positive for hBoV; a similar prevalence was observed in all three periods studied. Among hBoV-infected children, 53.8% were between 1–2 years old, and hBoV was detected alone in 57/104 (54.8%) cases. All of the detected hBoV strains belonged to genotype 1. The median hBoV load was significantly higher in samples containing strains with both the N546H and T590S mutations compared to other samples (p<0.05). Children with a single hBoV-1 infection more frequently had upper respiratory tract infections (URTIs) than those who were co-infected (37.0% vs 17.8%, respectively, p = 0.04). The duration of hospitalization was longer among children with high viral loads than that observed among children with low viral loads (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and the use of aerosol therapy was more frequent among children with high viral loads than among those with low viral loads (77.1% vs 55.7%, respectively, p = 0.04). This study shows that hBoV is a relatively uncommon but stable infectious agent in children and that hBoV1 seems to be the only strain detected in Italy in respiratory samples. From a clinical point of view, hBoV1 seems to have in the majority of healthy children relatively low clinical relevance. Moreover, the viral load influences only the duration of hospitalization and the use of aerosol therapy without any association with the site of the respiratory disease.
Human bocavirus (hBoV) is a recently identified viral agent that belongs to the family
Despite there are studies suggesting that hBoV is able to infect the lower airways causing severe infections in both children and adults, the role of hBoV as a causative agent of respiratory disease is frequently questioned due to its common detection with other potential pathogens [
To evaluate the circulation of the different hBoV types and the possible relationships between viral load, virus genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children attending the emergency room of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Italy, due to a respiratory tract infection arising between November 1 and March 31 during 3 winters (2009–2010, 2011–2012, and 2013–2014). The study was approved by the Ethics Committee of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. Written informed consent of a parent or legal guardian was required, and children ≥ 8 years of age were asked to give their written assent. Patients’ demographic characteristics and medical histories were retrieved from hospital charts and were systematically recorded before and after the first visit to the emergency room using standardized written questionnaires [
Viral nucleic acids were extracted from each swab by means of a Nuclisens EasyMAG automated extraction system (Biomeriéux, Craponne, France), and the extract was tested for respiratory viruses using the respiratory virus panel xTAG RVP FAST v2 (Luminex Molecular Diagnostics, Inc., Toronto, Canada), which simultaneously detects influenza A virus (subtypes H1 or H3); influenza B virus; respiratory syncytial virus (RSV) types A and B; human parainfluenza virus types 1–4 (hPiV1-4); adenovirus (AdV); human metapneumovirus (hMPV); coronaviruses (hCoV) 229E, NL63, OC43 and HKU1, enterovirus/rhinovirus (EV/hRV); and hBoV, in accordance with the manufacturer’s instructions [
Viral nucleic acid extracts previously testing positive for hBoV were re-tested for confirmation by two different singleplex real-time PCRs using TaqMan Universal Master Mix II (Applied Biosystems, California, USA). Amplification and detection of viral DNA were performed with a 7900HT real-time PCR System machine (Applied Biosystems, California, USA). Conserved regions for RT-PCR primers and probes were identified in the hBoV NS-1 and NP-1 genes from the nucleotide sequence alignments available from GenBank (for NS1, DQ206700-08, DQ000495-96, and DQ200648, and for NP-1, DQ000495-96, AB243566-72, DQ296618-35, DQ353695-99, DQ299885, DQ267760-75, DQ284856, DQ295844, and AM109958-66;
The viral load was obtained using real-time PCR with the NS1 primers and probe previously described and a DNA plasmid used as the standard calibrator. The amplified target fragment of the plasmid was verified by sequencing. Plasmid DNA concentrations were detected with an ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE, USA). Each run included plasmid and negative controls. Standard precautions were taken throughout the PCR process to avoid cross-contamination. Negative controls and serial dilutions of the positive controls were included in every PCR assay. Finally, quantitative results were reported as DNA copies/mL of respiratory samples. The viral load was defined as low for values ≤106 log (copies/mL) and as high for values >106 log (copies/mL).
For genotyping, the viral VP-1/2 gene was amplified using a conventional PCR assay. Briefly, 4 sets of forward and reverse primers (
All alignments were performed using ClustalX 2.1 and BioEdit (version 7.1.3.0) software (Ibis Biosciences, Carlsbad, CA). Phylogenetic trees of the VP-1/2 protein gene were generated using the neighbour-joining method and p-distance model of the Molecular Evolutionary Genetics Analyses (MEGA) software, version 5.05 [
Tests for positive selection were conducted on the Datamonkey server [
Descriptive statistics of the responses were generated. Continuous variables were presented as mean values and standard deviations (SDs) and categorical variables as numbers and percentages. For categorical data, comparisons between groups were performed using a contingency table analysis with the
During the three study periods, 1,823 nasopharyngeal samples were collected in the emergency room. Of these, 104 (5.7%) tested positive for hBoV (
No. (%) of positive samples | |||||
---|---|---|---|---|---|
Age group (years) | Overall | Without co-infection | With co-infection(s) | Low viral load | High viral load |
< 1 | 30 (28.8) | 12 (21.0) | 18 (38.3) | 22 (33.3) | 8 (21.0) |
1–2 | 56 (53.8) | 32 (56.1) | 24 (51.1) | 31 (47.0) | 25 (65.8) |
≥ 3 | 18 (17.3) | 13 (22.8) | 5 (10.6) | 13 (19.7) | 5 (13.2) |
104 | 57 | 47 | 66 | 38 |
One missing value for age.
Viral load was categorized in two groups, and was considered “low” for values ≤6 log (copies/mL) and “high” for values >6 log (copies/mL).
p-value = 0.03 for comparison between subgroups of presence of co-infection(s), according to age group (Cochran-Armitage trend test).
p-value = 0.67 for comparison between subgroups of viral load, according to age group (Cochran-Armitage trend test).
The phylogenetic tree constructed using the VP1/VP2 sequences showed that all of the Italian hBoV strains detected during the three study periods belonged to hBoV genotype 1 (
Sequences originating from this study are indicated with black and red circles. HBoV reference stains are in bold. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches.
A total of 61/672 (9.1%) amino acid positions were observed to have at least one change in the VP1/VP2 sequence alignments (
Dots indicate identity. Amino acids highlighted in bold are under positive selective pressure. The bar length corresponds to the viral load levels. The viral load levels of strains with the T590S change and an additional change in a codon under positive selection are marked with an asterisk.
The VP1U region includes the conserved phospholipase A2 (PLA2) motif (nt 21–63). The VP1U sequences of all hBoV isolates identified in this study revealed conserved YXGXG (nt 16–20) and HDXXY (nt 41–45) motifs in the catalytic site of secreted PLA2. In addition, the amino acid residues at positions 21, 41, 42 and 63 have been hypothesized to form the catalytic network for enzymatic activity. In our hBoV strains, all the sequences had amino acids associated with efficient enzymatic activity (P21, H41, D42, and D63).
Of note, two hBoV strains (MI-267-Jan2014 and MI-272-Jan2014) had a peculiar amino acid sequence in the 19-amino acid segment starting at amino acid 411 (KVPTRRVQPYIRQTNWKHR), which has not been previously reported in hBoV strains included in the GenBank database (in red,
A global analysis of selective pressure made using the SLAC model indicated an estimated overall dN/dS ratio of 0.18. Overall, the site-specific analyses identified three sites (411, 536, and 546) as under positive selection by at least two methods used (SLAC, FEL, FUBAR, and MEME). The IFEL model was used to determine the selection pressure acting on the VP1/VP2 codons along the internal branches of the tree. Two positively selected codons (411 and 546) were identified. The selected sites, highlighted with arrows in
Method | Positive | Negative |
---|---|---|
SLAC | None | 29 sites |
FEL | 34 sites | |
FUBAR | 17, 40, 225, 361, |
38 sites |
IFEL | 17 sites | |
MEME | 225, 361, 392, 396, 401, 409, 410, |
- |
FEL: fixed-effects likelihood; FUBAR: fast unconstrained Bayesian approximation methods; IFEL: internal branch fixed-effects likelihood; MEME: mixed effects model of evolution; SLAC: single-likelihood ancestor.
Regarding the viral load, a wide range of hBoV DNA levels from 3.5x102 to 7.5x109 copies/mL were found in the clinical samples. In
Groups | No. | Viral load (DNA copies/mL | ||
---|---|---|---|---|
Median | Range | |||
A. Strains with T590 | 36 | 4.6 x 104 | 3.5 x 102–5.9 x 108 | |
B. Strains with only T590S | 61 | 4.5 x 104 | 5.8 x 102–7.5 x 109 | |
C. Strains with only N546H | 5 | 2.2 x 103 | 3.5 x 102–5.9 x 108 | |
D. Strains with N546H+T590S | 8 | 1.1 x 109 | 1.6 x 104–2.8 x 109 | |
A |
0.31 | |||
A |
0.33 | |||
A |
||||
B |
0.17 | |||
B |
||||
C |
In
Characteristic | Without co-infection(s) N = 57 | With co-infection(s) N = 48 | P value for comparison |
---|---|---|---|
n/N (%) | n/N (%) | ||
Males (%) | 28/54 (51.8) | 30/48 (62.5) | 0.28 |
Mean age ± SD, yrs | 2.30 ± 2.24 | 1.63 ± 1.31 | 0.06 |
Presence of fever” (%) | 47/48 (97.9) | 43/45 (95.6) | 0.61 |
High-grade fever° (%) | 24/48 (50.0) | 25/45 (55.6) | 0.59 |
Respiratory rate, breaths/min | 34.7 ± 8.0 | 37.7 ± 10.3 | 0.66 |
SpO2 in room air, mean % ± SD | 98.0 ± 1.7 | 96.8 ± 2.4 | 0.06 |
Clinical findings | |||
Cough | 41/49 (83.7) | 39/47 (83.0) | 0.93 |
Rhonchi | 2/49 (4.1) | 4/47 (8.5) | 0.43 |
Rales | 15/49 (30.6) | 15/47 (31.9) | 0.89 |
Wheezes | 7/49 (14.3) | 9/47 (19.1) | 0.52 |
Upper respiratory tract infection | 17/46 (37.0) | 8/45 (17.8) | |
Lower respiratory tract infection | 25/55 (45.4) | 24/46 (52.2) | 0.50 |
Hospitalisation rate, no.(%) | 7/49 (14.3) | 10/47 (21.3) | 0.37 |
Duration of hospitalisation, mean days ± SD | 7.4 ± 2.6 | 5.9 ± 2.2 | 0.35 |
Drug use, no. (%) | |||
Antibiotics | 38/49 (77.5) | 40/47 (85.1) | 0.34 |
Antipyretics | 42/49 (85.7) | 39/47 (83.0) | 0.71 |
Aerosol therapy | 29/49 (59.2) | 32/47 (68.1) | 0.36 |
Absence from community, mean days ± SD | 7.9 ± 5.0 | 9.2 ± 7.0 | 0.84 |
Similar illness within the family | 13/49 (26.5) | 23/47 (48.9) | |
White blood cell count (cells/μL) | 15,319 ± 6,805 | 11,889 ± 3,680 | 0.27 |
Neutrophils, % | 49.2 ± 28.4 | 38.5 ± 24.7 | 0.42 |
Lymphocytes, % |
26.3 ± 17.4 | 31.6 ± 4.6 | 0.99 |
Monocytes, % |
9.4 ± 4.6 | 11.2 ± 0.4 | 0.46 |
Basophils, % |
0.4 ± 0.3 | 0.2 ± 0.0 | 0.65 |
Eosinophils, % |
0.7 ± 0.6 | 0.6 ± 0.8 | 0.88 |
CRP, μg/dL | 6.2 ± 15.4 | 1.4 ± 2.2 | 0.32 |
CRP: C reactive protein; SD: standard deviation; SpO2: peripheral oxygen saturation.”38.0°C or more any time during the illness (before or at enrolment, or during follow-up);
aData were extracted from datasets of different studies that collected different information, therefore the denominators vary across characteristics.
bInformation available for 9 subjects only (7 without co-infection and 2 with co-infection).
Characteristic | Low viral load (≤106 hBoV DNA copies/mL) N = 67 | High viral load (>106 hBoV DNA copies/mL) N = 38 | P value for comparison |
---|---|---|---|
n/N (%) | n/N (%) | ||
Males (%) | 40/64 (62.5) | 18/38 (47.4) | 0.14 |
Mean age ± SD, yrs | 2.00 ± 2.04 | 1.98 ± 1.66 | 0.55 |
Presence of fever” (%) | 58/58 (100.0) | 32/35 (91.4) | 0.05 |
High-grade fever° (%) | 31/58 (53.5) | 18/35 (51.4) | 0.85 |
Respiratory rate, breaths/min | 36.8 ± 8.6 | 36.6 ± 11.1 | 0.73 |
SpO2 in room air, mean % ± SD | 97.3 ± 2.3 | 97.0 ± 2.1 | 0.49 |
Clinical findings | |||
Cough | 48/61 (78.7) | 32/35 (91.4) | 0.11 |
Rhonchi | 4/61 (6.6) | 2/35 (5.7) | 0.99 |
Rales | 19/61 (31.1) | 11/35 (31.4) | 0.98 |
Wheezes | 11/61 (18.0) | 5/35 (14.3) | 0.64 |
Upper respiratory tract infection | 18/57 (31.6) | 7/34 (20.6) | 0.26 |
Lower respiratory tract infection | 32/64 (50.0) | 17/37 (46.0) | 0.69 |
Hospitalisation rate, no.(%) | 10/61 (16.4) | 7/35 (20.0) | 0.66 |
Duration of hospitalisation, mean days ± SD | 5.0 ± 1.5 | 8.0 ± 2.2 | |
Drug use, no. (%) | |||
Antibiotics | 51/61 (83.6) | 27/35 (77.1) | 0.43 |
Antipyretics | 52/61 (85.2) | 29/35 (82.9) | 0.76 |
Aerosol therapy | 34/61 (55.7) | 27/35 (77.1) | |
Absence from community, mean days ± SD | 8.6 ± 6.3 | 8.2 ± 5.5 | 0.88 |
Similar illness within the family | 22/61 (36.1) | 14/35 (40.0) | 0.70 |
White blood cell count (cells/μL) | 13,191 ± 6,847 | 14,727 ± 4,337 | 0.28 |
Neutrophils, % | 41.9 ± 24.2 | 50.2 ± 32.1 | 0.52 |
Lymphocytes, % |
31.7 ± 13.1 | 19.1 ± 19.0 | 0.39 |
Monocytes, % |
11.7 ± 2.7 | 5.9 ± 3.6 | 0.09 |
Basophils, % |
0.4 ± 0.3 | 0.3 ± 0.4 | 0.90 |
Eosinophils, % |
0.7 ± 0.7 | 0.6 ± 0.5 | 0.99 |
CRP, μg/dL | 5.5 ± 14.6 | 1.6 ± 1.8 | 0.94 |
CRP: C reactive protein; SD: standard deviation; SpO2: peripheral oxygen saturation.”38.0°C or more any time during the illness (before or at enrolment, or during follow-up);
aData were extracted from datasets of different studies that collected different information, therefore the denominators vary across characteristics
bInformation available for 9 subjects only (6 with low and 3 with high viral load).
This study shows that in Italy during the winter periods 2009–2010, 2011–2012, and 2013–2014, the incidence of hBoV infection among children with respiratory disease was relatively low, limited to approximately 5% of cases, and did not significantly vary from year to year.
The phylogenetic analysis showed that all of the strains detected in this study belonged to hBoV genotype 1 and were closely related to the prototype strain identified by Allander et al. [
Most of the patients in whom hBoV1 was identified were younger than 3 years of age, further highlighting that younger children are the individuals most frequently infected by this viral agent [
Evaluation of the viral load has been considered a possible method to define when this virus is the real cause of a respiratory disease and when it is only a secondary infection. Unfortunately, this approach has had no success because although some studies have shown evidence for a strict correlation between high viral load and severe LRTI in children with a single hBoV infection [
However, the evolution of virulence appears to involve a variety of mechanisms in different viral systems, including mutations in regulatory regions and viral adaptation for utilization of alternative or expanded repertoires of cellular receptors. An alternative hypothesis to evaluate the importance of hBoV1 concerns the correlation between viral load levels and the presence of specific mutations. However, mutations associated with increased or reduced replication are rarely reported for hBoV. Recently, Hao et al. have reported that few nucleotide changes were correlated with a lower viral load [
In agreement with others [
In the present study, the dN/dS ratios for all pairwise comparisons were <1, which is in line with previous results showing that positive selection was extremely limited in parvoviruses [
In conclusion, this study confirms that hBoV is less common than other respiratory viruses but that the frequency of its detection in children with respiratory disease is in time stable. It was detected with a prevalence of about 5% in several consecutive seasons and no unusual clustering was observed among identified strains, with strains circulating in 2009 being closely related to those circulating in 2014. Moreover, only a minority of virus sites were found to be under positive selective pressure, and all the strains detected in respiratory tract infections of this Italian study belonged to genotype 1. From a clinical point of view, this study highlights that in otherwise healthy children, hBoV1 seems to have relatively low clinical relevance, because patients infected with hBoV alone mainly suffered from an URTI. The viral load was not associated with clinical characteristics of the infection, and viral mutations, despite affecting viral replication, did not affect the conditions or severity of the clinical presentation. Further studies are needed to clarify the clinical relevance of hBoV in children, particularly in those at risk for severe chronic underlying disease, and to evaluate the role of viral modification in conditioning the degree of viral virulence and the specific immune response.