Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway / Escape inmunológico del parvovirus bovino mediante la inhibición de VP1 de la producción de IFN-β a través de la vía del receptor tipo RIG-I

Objective: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. Results: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.(AU)

Characterization of the GBoV1 Capsid and Its Antibody Interactions.

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties.

[Preparation and application of rabbit antiserum against structural protein VP2 of canine bocavirus].

OBJECTIVE: To prepare the rabbit polyclonal antibody against canine bocavirus (CBV) structural protein VP2 and identify its specificity. METHODS: The target sequence of gene fragment encoding VP2 C-terminal region (300 AA) was amplified from CBV infection clone. After the restriction enzyme digestion and nucleotide sequencing, the target gene was successfully inserted into pET32a(+) prokaryotic expression vector to form recombinant plasmid pET-32a(+)-VP2. Then pET-32a(+)-VP2 was transferred into E.coli (BL21), and VP2-his fusion protein was induced under the optimized induction of isopropyl ß-D-thiogalactopyranoside (IPTG). The products were identified and analyzed by SDS-PAGE. The purified fusion protein was inoculated into adult rabbits to develop polyclonal antibody. After the titer of the antiserum was detected by ELISA, Western blotting and immunofluorescence staining were performed to evaluate the features of the prepared antiserum. RESULTS: Restriction enzyme digestion and sequencing showed that prokaryotic expression vector pET-32a(+)-VP2 was successfully constructed. The soluble recombinant protein was highly expressed in E.coli BL21 as expected. After purification and inoculation into adult rabbits, the high-titer polyclonal antibody was prepared and the titer was 1:6 400 000. Western blotting and immunohistochemistry demonstrated that the specificity of the prepared polyclonal antibody was perfect. CONCLUSION: The polyclonal antibody against CBV structural protein VP2 has been successfully prepared.

Virus-like particles of porcine bocavirus generated by recombinant baculoviruses can be applied to sero-epidemic studies.

Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm(3), respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV.

Infecciones respiratorias agudas por bocavirus humanos en la población adulta ¿una rareza? / Acute respiratory infections by human bocavirus in the adult population. Really unfrequent?

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Optimación de estrategias en el manejo de la infección por el CMV en el trasplante / Optimization strategies in management of CMV infection in transplant patients

En la actualidad se emplean dos estrategias terapéuticas para prevenir el desarrollo de enfermedad orgánica por el CMV en el paciente trasplantado, la profilaxis universal y el tratamiento anticipado. Ambas son potencialmente optimizables. La primera, identificando con precisión a los pacientes con máximo riesgo de viremia con objeto de tratarlos selectivamente (profilaxis dirigida). En este sentido disponemos de marcadores genotípicos, biológicos en inmunológicos que podrían permitirlo. La segunda, a través de la monitorización conjunta de la carga viral del CMV en plasma y del nivel de LT CD8+ y CD4+ productores de IFN-g específicos frente al CMV (AU)

Currently, two therapeutic strategies are applied for preventing the development of CMV end-organ disease in transplant recipients: universal prophylaxis and preemptive antiviral therapy. Both are potentially optimable. As for the former strategy, precisely identifying patients at greatest risk of viremia would allow for a targeted prophylaxis. In this sense several genotypic, immunological and biological markers have been described that could be ancillary to that purpose. As for the latter strategy, combined monitoring of plasma CMV DNA load and peripheral levels of CMV-specific CD8 + and CD4 + IFN-γ producing T cells would permit a more rationale use of antivirals, thus avoiding overtreatment and derived toxicity (AU)

Human bocavirus NP1 inhibits IFN-ß production by blocking association of IFN regulatory factor 3 with IFNB promoter.

Human bocavirus (HBoV) mainly infects young children. Although many infected children suffer from respiratory or gastroenteric tract diseases, an association between HBoV and these diseases is not definite. Because modulation of type I IFN is crucial for viruses to establish efficient replication, in this study, we tested whether HBoV modulates type I IFN production. We observed that a nearly full-length HBoV clone significantly reduced both Sendai virus (SeV)- and poly(deoxyadenylic-thymidylic) acid-induced IFN-ß production. Further study showed that NP1 blocked IFN-ß activation in response to SeV, poly(deoxyadenylic-thymidylic) acid, and IFN-ß pathway inducers, including retinoic acid-inducible protein I, mitochondrial antiviral signaling protein, inhibitor of κB kinase ε, and TANK-binding kinase 1. In addition, NP1 interfered with IRF-3-responsive PRD(III-I) promoter activated by SeV and a constitutively active mutant of IRF-3 (IRF-3/5D). Although NP1 suppressed the IRF-3 pathway, it did not affect IRF-3 activation processes, including phosphorylation, dimerization, and nuclear translocation. Coimmunoprecipitation assays confirmed the interaction between NP1 and IRF-3. Additional deletion mutagenesis and coimmunoprecipitation assays revealed that NP1 bound to the DNA-binding domain of IRF-3, resulting in the interruption of an association between IRF-3 and IFNB promoter. Altogether, our results indicate that HBoV NP1 blocks IFN production through a unique mechanism. To our knowledge, this is the first study to investigate the modulation of innate immunity by HBoV. Our findings suggest a potential immune-evasion mechanism used by HBoV and provide a basis for better understanding HBoV pathogenesis.

Production, characterisation and applications of monoclonal antibodies to two novel porcine bocaviruses from swine in Northern Ireland.

The production, preliminary characterisation and applications of monoclonal antibodies (mAbs) against two novel swine bocaviruses isolated in cell culture from swine in Northern Ireland are described. Of the 17 stable final clones produced, four were characterised. All were of the IgG2a isotype and showed no cross-reactivity with either bocavirus strain. Partial neutralisation was observed with PBoV4 mAbs and homologous virus. The two mAbs selected for use in antigen-detecting ELISAs were successful in highlighting those fractions containing infectious virus within sucrose gradients. This is the first report of the production of specific reagents that will prove useful in the study of the biology of these viruses and swine bocavirus-associated diseases.

Comparison of Th-cell immunity against human bocavirus and parvovirus B19: proliferation and cytokine responses are similar in magnitude but more closely interrelated with human bocavirus.

Human parvovirus B19 (B19) has been, for decades, the only parvovirus known to be pathogenic in humans. Another pathogenic human parvovirus, human bocavirus (HBoV), was recently identified in respiratory samples from children with acute lower respiratory tract symptoms. Both B19 and HBoV are transmitted by the respiratory route. The vast majority of adults are IgG seropositive for HBoV, whereas the HBoV-specific Th-cell immunity has not much been studied. The aim of this study was to increase our knowledge on HBoV-specific Th-cell immunity by examining HBoV-specific T-cell proliferation, Interferon-gamma (IFN-γ), IL-10 and IL-13 responses in 36 asymptomatic adults. Recombinant HBoV VP2 virus-like particles (VLP) were used as antigen. HBoV-specific responses were compared with those elicited by B19 VP2 VLP. Proliferation, IFN-γ and IL-10 responses with HBoV and B19 antigens among B19-seropositive subjects were statistically similar in magnitude, but the cytokine and proliferation responses were much more closely correlated in HBoV than in B19. Therefore, at the collective level, B19-specific Th-cell immunity appears to be more divergent than the HBoV-specific one.

Evidence of prior exposure to human bocavirus as determined by a retrospective serological study of 404 serum samples from adults in the United States.

Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfate-polyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm(3) and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.

[Expression of recombinant VP2 gene in insect sf9 cells and screening of clinical specimens].

OBJECTIVE: To clone and express VP, gene from HBoV, and the expressed VP, protein was as the antigen in order to detect serum from children in Wenling area with lower respiratory tract infections. METHODS: The VP, gene was recombined with the genome of Baculovirus, which infected the insect cell. The fusion protein with HA tag was applied to confirm the specificity of expressed protein. Furthermore, the recombinant protein was observed using electron microscopy. The 176 serum from children in Wenling area with lower respiratory tract infections was screened using Western blot. RESULTS: The expressed VP2 protein was more than 60% in total proteins from insect cell, and MWt about 60 x 10(3). The virus-like particle (VLP) was observed using electron microscopy, and size about 20 nm. The 176 serum from children in wenling area with lower respiratory tract infections was screened using Western blot. The HBoV positive rate was 2.28% (4/176). CONCLUSION: The VP2 protein from human bocavirus was expressed in insect cell successfully. Through HA tag the VP2 protein was specific, and then the assay using SDS-PAGE with Western blot could detect and screen the antibody in serum from children with lower respiratory tract infections rapidly and accurately.

[Seroprevalence of antibody against human bocavirus in Beijing, China].

OBJECTIVE: To find out the importance of human bocavirus (HBoV) as an infectious agent for population in Beijing, China, seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen. METHODS: Serum specimens collected from infants and children who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics for health check up and adults visited the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for investigation. The major capsid protein VP2 from HBoV was expressed in E. coli strain BL21 (DE3) with the transformed PET30b vector inserted with full-length VP2 gene of HBoV and the specific antigenicity of this expressed protein was validated by previous study. Western blot was used to detect specific IgG antibody against HBoV in collected serum specimens diluted to 1:200. Mock expressed protein was E. coli cells strain BL21 (DE3) with the transformed PET30b vector without insert. Anti-His monoclonal antibody and rabbit anti-HBoV VP2 polypeptides hyper-immune serum were used as positive control for antibody detection. RESULTS: Out of 677 serum specimens tested, 400 (59.1%) were positive by Western blot. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were lower in the age groups of 1 and 2 months (41.4% and 31.3%, respectively) and were higher in the following ages from 6 months to 7 years (from 45.6% to 69.7%). The antibody positive rates were at a relatively constant level (about 70%) in the age groups from 7 years to 40 years and became lower (61.8% - 62.8%) in groups of age over 50 years. CONCLUSION: The high seroprevalence against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in Beijing, China as early as in 1996 and most of children had been exposed to HBoV by the age of 7 years. Infants under the age of 6 months were susceptible to infection with this virus.

CD4+ T helper cell responses against human bocavirus viral protein 2 viruslike particles in healthy adults.

BACKGROUND: Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae family, and its possible association with respiratory illness in infants has been discussed. To date, HBoV genomes have been detected worldwide in respiratory tract samples obtained from children with pulmonary diseases, whereas only limited data on virus-specific immunity are available, mainly because of the lack of recombinant viral antigens. METHODS: HBoV viruslike particles (VLPs) were produced in insect cells and characterized by electron microscopy and cesium chloride gradient centrifugation. HBoV viral protein 2 (VP2)-specific antibodies and CD4+ T helper cell responses were analyzed by enzyme-linked immunosorbent assay and enzyme-linked immunospot assay. RESULTS: VP2 capsid proteins of HBoV were produced in insect cells infected with a recombinant baculovirus, and the formation of icosahedral VLPs (diameter, 21-25 nm; sedimentation density, 1.33 g/cm(3)) was demonstrated. A significant increase in secretion of VP2-specific interferon-gamma was detected in cultures of peripheral blood mononuclear cells obtained from 69 healthy adults found to be positive for HBoV-specific immunoglobulin G antibodies, compared with control stimulations. In parallel, T cell responses against identically expressed parvovirus B19 VP2 VLPs were frequently observed in the individuals studied, without there being obvious cross-reactions between HBoV and parvovirus B19. CONCLUSIONS: Data suggest the presence of HBoV-specific immune responses in adults and strongly support a high prevalence of HBoV among humans.

Th1 and Th2 cytokine levels in nasopharyngeal aspirates from children with human bocavirus bronchiolitis.

BACKGROUND: Human bocavirus (hBoV) is regarded as one of the possible etiologic agents in lower respiratory tract infection and bronchial asthma exacerbation in children despite frequent co-detection with other respiratory viruses. The immunologic response in children with hBoV infection is still not clear. OBJECTIVES: To investigate the profiles of T helper-1 (Th1)/T helper-2 (Th2) cytokines in children with hBoV-associated bronchiolitis. STUDY DESIGN: This study utilized of 59 nasopharyngeal aspirates from 59 infants aged 24 months or younger, including 29 from children with hBoV-related bronchiolitis and 30 with respiratory syncytial virus (RSV)-related bronchiolitis. Eighteen infants hospitalized for elective surgeries were included as controls. Nasopharyngeal aspirates were tested simultaneously for cytokines interleukin (IL)-2, IL-4, IL-5, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha using the Cytometric Bead Array. RESULTS: Significantly higher concentrations of IFN-gamma (p=0.0001), IL-2 (0.006), and IL-4 (p=0.0002) were observed in hBoV positive specimens than in controls. The concentration of IL-10 (p=0.04) and TNF-alpha (p=0.006) in the RSV-positive group was significantly higher than in the hBoV-positive group, while there was no difference in other cytokines concentration between the two groups. CONCLUSIONS: These results showed that both of Th1 and Th2 cytokines were increased in children with hBoV-related bronchiolitis compared to normal controls, but Th2-polarized responses were not observed.

Human bocavirus--a novel parvovirus to infect humans.

For almost three decades parvovirus B19 has been described as the only member of the Parvoviridae to infect and cause illness in humans. This statement was correct until 2005 when a group of Swedish scientists identified a previously uncharacterized virus in pools of human nasopharyngeal aspirates obtained from individuals suffering from diseases of the respiratory tract. Comprehensive sequence and phylogenetic analysis allowed the identification of the new virus as a member of the Parvoviridae. Based on its close relation to the minute virus of canines and the bovine parvovirus, it was named human bocavirus (HBoV). Since the identification of HBoV, viral genomes have been frequently detected worldwide in nasopharyngeal swabs, serum and fecal samples almost exclusively derived from young children with various symptoms of the respiratory or the gastrointestinal tract. The detection of HBoV genomes tends to be associated with elevated rates of coinfections with further respiratory viruses, e.g. respiratory syncytial virus or metapneumovirus. First studies on virus-specific immune responses have described the presence of ubiquitous humoral and cellular immune reactions against HBoV in adults and adolescents, indicating a high seroprevalence of this new virus in humans.

Seroepidemiology of human bocavirus defined using recombinant virus-like particles.

BACKGROUND: Human bocavirus (HBoV) is a newly identified human parvovirus for which seroepidemiology and antigenic properties remain undefined. METHODS: The HBoV VP2 gene, expressed from a baculovirus vector, produced virus-like particles (VLPs), which were used to raise rabbit anti-HBoV antisera and to develop an enzyme-linked immunosorbent assay (ELISA). The VLP-based ELISA was used to screen for HBoV-specific immunoglobulin G antibodies in a convenience sample of 270 serum specimens, mostly from children, obtained at Yale-New Haven Hospital; 208 specimens were also screened for erythrovirus B19-specific antibodies by a B19 VLP-based ELISA. RESULTS: Immunofluorescence and ELISA showed that human parvoviruses HBoV and B19 are antigenically distinct. By the HBoV VLP-based ELISA, 91.8% and 63.6% of serum specimens from infants in the first and second months of life, respectively, were found to be seropositive, as were 45.4% from 3-month-old infants and 25.0% from 4-month-old infants. The percentages of HBoV-seropositive children increased to 40.7%-60.0% for children 5-47 months of age and to >85% for individuals >or=48 months old. However, the overall percentage of B19-seropositive individuals was <40.5% for all age groups screened. CONCLUSIONS: HBoV infection is common during childhood, but a minority of children and young adults screened have evidence of B19 infection.

Human bocavirus: passenger or pathogen in acute respiratory tract infections?

Human bocavirus (HBoV) is a newly identified virus tentatively assigned to the family Parvoviridae, subfamily Parvovirinae, genus Bocavirus. HBoV was first described in 2005 and has since been detected in respiratory tract secretions worldwide. Herein we review the literature on HBoV and discuss the biology and potential clinical impact of this virus. Most studies have been PCR based and performed on patients with acute respiratory symptoms, from whom HBoV was detected in 2 to 19% of the samples. HBoV-positive samples have been derived mainly from infants and young children. HBoV DNA has also been detected in the blood of patients with respiratory tract infection and in fecal samples of patients with diarrhea with or without concomitant respiratory symptoms. A characteristic feature of HBoV studies is the high frequency of coinciding detections, or codetections, with other viruses. Available data nevertheless indicate a statistical association between HBoV and acute respiratory tract disease. We present a model incorporating these somewhat contradictory findings and suggest that primary HBoV infection causes respiratory tract symptoms which can be followed by prolonged low-level virus shedding in the respiratory tract. Detection of the virus in this phase will be facilitated by other infections, either simply via increased sample cell count or via reactivation of HBoV, leading to an increased detection frequency of HBoV during other virus infections. We conclude that the majority of available HBoV studies are limited by the sole use of PCR diagnostics on respiratory tract secretions, addressing virus prevalence but not disease association. The ability to detect primary infection through the development of improved diagnostic methods will be of great importance for future studies seeking to assign a role for HBoV in causing respiratory illnesses.

ELISAs using human bocavirus VP2 virus-like particles for detection of antibodies against HBoV.

Human bocavirus (HBoV) has been identified worldwide in children with lower respiratory tract infections with an incidence of approximately 2-11%. The role of HBoV in pathogenesis, however, is largely unknown, and little is known about the epidemiology of the virus. To study the seroepidemiology of HBoV infection, the capsid protein was expressed in insect cells. Expression of the putative major capsid protein VP2 in insect cells led to the formation of virus-like particles exhibiting the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. The expressed particles were used to establish an enzyme-linked immunosorbent assay (ELISA) method, and serum samples from groups of children of various ages in China were tested for IgG antibodies against HBoV. HBoV antibodies were detected in as high as 36% of healthy children under 9 years. Of children hospitalized with lower respiratory tract infections, 31% were seropositive, and all age groups of these children showed a significantly higher level of HBoV IgG antibody than their healthy counterparts. When divided into age cohorts, results showed that more than 48% of healthy children had seroconverted by age of 4. Thus, HBoV appears to be a common infection in children. The potential pathogenesis of this virus, especially its role in lower respiratory tract infections in children warrants further investigation.

Serodiagnosis of human bocavirus infection.

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.