Serodiagnosis of human bocavirus infection

Abstract

Background: A new human-pathogenic parvovirus, human bocavirus (HBoV), has recently been discovered and associated with respiratory disease in small children. However, many patients have presented with low viral DNA loads, suggesting HBoV persistence and rendering polymerase chain reaction-based diagnosis problematic. Moreover, nothing is known of HBoV immunity. We examined HBoV-specific systemic B cell responses and assessed their diagnostic use in young children with respiratory disease.

Patients and methods: Paired serum samples from 117 children with acute wheezing, previously studied for 16 respiratory viruses, were tested by immunoblot assays using 2 recombinant HBoV capsid antigens: the unique part of virus protein 1 and virus protein 2.

Results: Virus protein 2 was superior to the unique part of virus protein 1 with respect to immunoreactivity. According to the virus protein 2 assay, 24 (49%) of 49 children who were positive for HBoV according to polymerase chain reaction had immunoglobulin (Ig) M antibodies, 36 (73%) had IgG antibodies, and 29 (59%) exhibited IgM antibodies and/or an increase in IgG antibody level. Of 22 patients with an increase in antibody levels, 20 (91%) had a high load of HBoV DNA in the nasopharynx, supporting the hypothesis that a high HBoV DNA load indicates acute primary infection, whereas a low load seems to be of less clinical significance. In a subgroup of patients who were previously determined to have acute HBoV infection (defined as a high virus load in the nasopharynx, viremia, and absence of other viral infections), 9 (100%) of 9 patients had serological evidence of primary infection. In the control group of 68 children with wheezing who had polymerase chain reaction results negative for HBoV in the nasopharynx, 9 (13%) had IgM antibodies, including 5 who displayed an increase in IgG antibody levels and were viremic. No cross-reactivity with human parvovirus B19 was detected.

Conclusions: Respiratory infections due to HBoV are systemic, elicit B cell immune responses, and can be diagnosed serologically. Serological diagnoses correlate with high virus loads in the nasopharynx and with viremia. Serological testing is an accurate tool for disclosing the association of HBoV infection with disease.

Table 1

Primer sequences used to amplify the capsid genes for cloning.

Figure 1

Coomassie blue-stained SDS PAGE showing the prokaryotically expressed virus protein 2 (VP2) (A) and the unique part of virus protein 1 (VP1u) (B). Arrows indicate VP1u and VP2. M, molecular weight standard; p, affinity purified protein; w, lysate with expressed VP gene; w/o, lysate without virus protein gene.

Figure 2

Representative results of human bocavirus (HBoV) IgG (A) and IgM (B) immunoblotting, using virus protein 1 (VP1u) or virus protein 2 (VP2) antigen-harboring membranes, as indicated. Membrane strips were treated with the first (I) and second (II) samples of serum pairs and show IgG conversions for both VP1u and VP2 (A) and the appearance of IgM for VP2 (B). Black and white arrows indicate the positions of the VP1u and VP2 antigens, respectively. M, molecular weight.

Table 2

Human bocavirus (HBoV) virus protein 2 (VP2) immunoblot results for 117 wheezing children, compared with nasopharyngeal aspirate (NPA) [17] and serum PCR results for HBoV.