Human Bocavirus Capsid Messenger RNA Detection in Children With Pneumonia

Abstract

Background: The role of human bocavirus (HBoV) in respiratory illness is uncertain. HBoV genomic DNA is frequently detected in both ill and healthy children. We hypothesized that spliced viral capsid messenger RNA (mRNA) produced during active replication might be a better marker for acute infection.

Methods: As part of the Etiology of Pneumonia in the Community (EPIC) study, children aged <18 years who were hospitalized with community-acquired pneumonia (CAP) and children asymptomatic at the time of elective outpatient surgery (controls) were enrolled. Nasopharyngeal/oropharyngeal specimens were tested for HBoV mRNA and genomic DNA by quantitative polymerase chain reaction.

Results: HBoV DNA was detected in 10.4% of 1295 patients with CAP and 7.5% of 721 controls (odds ratio [OR], 1.4 [95% confidence interval {CI}, 1.0-2.0]); HBoV mRNA was detected in 2.1% and 0.4%, respectively (OR, 5.1 [95% CI, 1.6-26]). When adjusted for age, enrollment month, and detection of other respiratory viruses, HBoV mRNA detection (adjusted OR, 7.6 [95% CI, 1.5-38.4]) but not DNA (adjusted OR, 1.2 [95% CI, .6-2.4]) was associated with CAP. Among children with no other pathogens detected, HBoV mRNA (OR, 9.6 [95% CI, 1.9-82]) was strongly associated with CAP.

Conclusions: Detection of HBoV mRNA but not DNA was associated with CAP, supporting a pathogenic role for HBoV in CAP. HBoV mRNA could be a useful target for diagnostic testing.

Keywords: Human bocavirus; asymptomatic shedding; detection; mRNA; pneumonia.

Figure 1.

Detection of human bocavirus (HBoV) DNA and spliced capsid messenger RNA (mRNA). A, Assay design for detection of HBoV genomic DNA, viral capsid mRNA transcript R6 (assay 1), and capsid mRNA transcripts R3–5 (assay 2), modified from material published elsewhere [28]. The capsid mRNA detection assays span 2 different splice sites of alternate capsid mRNAs; nucleotide numbering from NC_007455. B, Five specimens with the highest viral DNA load at the Utah site were tested with (red) and without (blue) reverse transcriptase (RT), using the capsid mRNA detection assay. Only minimal cross-amplification was seen in the 3 specimens with the highest DNA load at levels that were 2.7 × 10⁴–1.6 × 10⁵-fold lower than the DNA loads in the corresponding specimens and 72–1300-fold lower than with the RT step.

Figure 2.

Sensitivity analysis of association of human bocavirus (HBoV) DNA and capsid messenger RNA (mRNA) detection with community-acquired pneumonia (CAP) in children enrolled at Primary Children’s Hospital (Utah) and Monroe Carell Jr Children’s Hospital at Vanderbilt (Tennessee). Monodetection denotes that no other respiratory pathogens were detected, per Etiology of Pneumonia in the Community Study results [27]. Asymptomatic and concurrent analysis is limited to asymptomatic controls and patients enrolled after January 2011 for Utah patients and after February 2011 for Tennessee patients. Concurrent analysis is limited to patients enrolled after January 2011. Asymptomatic analysis is limited to controls confirmed to remain symptom free for 14 days. Numbers indicate the size of each group, given the different stratifications. CI, confidence interval; OR, odds ratio.

Figure 3.

Human bocavirus (HBoV) capsid messenger RNA (mRNA) and DNA load in nasopharyngeal and oropharyngeal swab specimens from children with community-acquired pneumonia (CAP) and controls. A, At approximately 4 × 10⁴ copies/mL, the median HBoV capsid mRNA load in children with CAP was similar to that in controls. B, Median DNA load was approximately 2-fold higher in children with CAP (622 copies/mL) than in controls (375 copies/mL), but this difference was not statistically significant. However, a subset of children with CAP, as well as 2 controls, had HBoV DNA loads of 1 × 10⁶–1 × 10⁹ copies/mL. C, When stratifying HBoV DNA loads by HBoV capsid mRNA positivity, median HBoV DNA loads were 100000–1000000-fold higher in capsid mRNA–positive children with CAP (7.4 × 10⁷ copies/mL) and controls (4.6 × 10⁸ copies/mL) than in capsid mRNA–negative children with CAP (4.8 × 10² copies/mL) and controls (3.7 × 10² copies/mL). Conversely, HBoV DNA loads were similar in capsid mRNA– negative patients and controls and capsid mRNA–positive patients and controls. Children with no HBoV nucleic acid detected are not shown.