Juha Lindner

Prevalence of Parvovirus B19 and Human Bocavirus DNA in the Heart of Patients with no Evidence of Dilated Cardiomyopathy or Myocarditis

BACKGROUND Although the DNA of parvovirus B19 (B19V) is frequently detected in patients with dilated cardiomyopathy or myocarditis, whether the parvovirus causes disease is questionable, since even in healthy individuals the virus persists in various tissues. The same question applies to human bocavirus (HBoV). We have determined the prevalence and quantity of B19V and HBoV DNA in heart tissue of patients who were not experiencing virus-related heart diseases and analyzed whether the seroprevalence corresponded to DNA prevalence in the heart. METHODS Samples of left-atrium heart tissue and serum were obtained from 100 patients who underwent open-heart surgery. Serum immunoglobulin (Ig) G and IgM against proteins encoded by B19V and HBoV were detected by enzyme-linked immunoabsorption assay and immunoblotting. B19V and HBoV DNA concentrations were determined by quantitative real-time polymerase chain reaction (PCR) in heart tissue and serum samples. Nested PCRs for VP1, K71, and GT3 identified the B19V genotypes. RESULTS The prevalences of serum IgG specific for B19V and HBoV were 85% and 96%, respectively. Of all the patients, 85% had B19V DNA detected in heart tissues, and 4% displayed low-level B19V viremia, whereas only 5% of heart tissue samples and none of the serum samples demonstrated HBoV DNA. The sensitivity of B19V serological testing for B19V DNA in heart samples was 0.96 (95% confidence interval, 0.92-1.0). Specificity was 0.8 (95% confidence interval, 0.6-1.0), and the positive predictive value was 0.96 (95% confidence interval, 0.92-1.0). B19V genotypes 1 and 2 were present in 11% and 89% of heart tissues samples, respectively. B19V genotype 3 was not detected in any of the samples. CONCLUSIONS Our data suggest that B19V but not HBoV demonstrates a lifelong persistence in the heart. The detection of B19V DNA in heart tissue showed no correlation with clinical symptoms. We strongly recommend that serological testing become a standardized procedure for future studies, to obtain representative data concerning the prevalence of B19V in the heart.

Humoral Immune Response Against Human Bocavirus VP2 Virus-Like Particles

Human bocavirus (HBoV) was recently detected in samples from children and infants with infections of the respiratory tract. Here we analyze the prevalence of IgG and IgM antibodies against HBoV virus-like VP2 particles in healthy adult blood donors and children using a newly established standardized enzyme-linked immunosorbent assay. Virus-specific IgG antibodies were frequently detected in infants with active viremia and respiratory illness (10/24, 42%) and in young children without detectable HBoV genomes in their blood (27/52, 52%). In sera obtained from healthy adults, ubiquitous VP2-specific antibodies were found in 280/299 (94%) cases. HBoV-specific IgM antibodies were detected in 10/24 (42%) of sera samples obtained from HBoV DNA-positive children, and in 6/24 (25%) the sera displayed equivocal responses. In contrast, VP2-specific IgM was not detectable in samples obtained from 52 children without detectable amounts of HBoV genomes in their blood. Only 2/299 sera samples from healthy adult blood donors were found to be IgM-positive (1%), and equivocal IgM responses were observed in 9/299 (3%) individuals. In conclusion, a high IgG seroprevalence of HBoV in the adult population was observed, whereas the presence of virus-specific IgM was associated with viremia. These data show that ELISA test systems for the detection of HBoV-specific antibodies are a valuable tool for serological diagnosis of this new emerging pathogen.

Prevalence and clinical aspects of human bocavirus infection in children

Human bocavirus (HBoV) was recently described as a new member of the Parvoviridae. In order to investigate the suggested association of HBoV with respiratory and gastric disease in infants and young children, sera of 357 paediatric patients hospitalized with infectious and non-infectious diseases were retrospectively analyzed for the presence of HBoV DNA and virus-specific antibodies using quantitative PCR and ELISA, respectively. HBoV seroprevalence was determined to range from 25% in infants younger than 1 year of age to 93% in children aged more than 3 years. Viral loads between 1 x 10(2) and 1.2 x 10(6) geq/mL were observed in 6.7% (20/297) of sera obtained preferentially from young children suffering from infectious diseases. HBoV genomes were furthermore detected in 5% (3/60) of sera collected from individuals with non-infectious illnesses. HBoV DNA was present most frequently in patients with respiratory disease (9.6%). Whereas only 5.2% of patients with upper respiratory tract disease were viraemic, HBoV DNA was found in 14.6% and 10.0% of patients with lower respiratory tract illness and pneumonia, respectively. Acute HBoV infections were also observed in 7.5% of patients with gastroenteritis and in one child with inflammatory bowel disease. None of 77 patients hospitalized for various other infectious diseases (e.g. rash, urinary tract infection, meningitis) displayed viraemia. In 60.9% and 47.8% of DNA-positive children, HBoV-specific IgM and IgG was observed, respectively. The present prospective study provides comprehensive data on the clinical association of acute HBoV infection with respiratory illness and on the seroprevalence of virus-specific antibodies in children.

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.

Clinical and epidemiological aspects of human bocavirus infection

Abstract Human bocavirus was recently described as a novel member of the Parvoviridae to infect humans. Based on accumulating clinical and epidemiological data the virus is currently being associated with respiratory infections in young children and infants and is furthermore discussed as causative agent of gastrointestinal illness.

CD4+ T Helper Cell Responses against Human Bocavirus Viral Protein 2 Viruslike Particles in Healthy Adults

Abstract 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 immunsorbent 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/cm3) was demonstrated. A significant increase in secretion of VP2-specific interferon-γ 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.

Adaptive immune responses against parvovirus B19 in patients with myocardial disease

BACKGROUND Parvovirus B19 (B19V)-DNA is frequently detected in endomyocardial biopsies (EMBs) from patients with acute myocarditis (AMC) and dilated cardiomyopathy (DCM), but also in various healthy tissues. The clinical relevance of this DNA-persistence is unclear. OBJECTIVES To investigate potential pathogenic influences of B19V-DNA in EMBs, we analyzed B19V-specific adaptive immune responses in AMC/DCM patients and healthy controls. STUDY DESIGN 15 AMC/DCM patients with detectable B19V-DNA in EMBs and 51 controls were analyzed for signs of acute B19V-infections and virus-specific immune responses by PCR, ELISA, Western line, and ELISpot-assays. RESULTS Productive B19V-infection was determined in three patients. Slightly lower levels of B19V-specific T-cells were observed in patients as compared to the controls, no differences were observed in virus-specific serology. Viral DNA-load in EMBs could not be correlated to the number of B19V-specific T-cells. No differences in T-cell response, viremia and/or serological markers indicative for viral pathogenesis were observed in patients with inflammatory cardiomyopathy. CONCLUSIONS Discrepancies in B19V-specific adaptive immunity were not observed in AMC/DCM patients as compared to controls. The data indicate that the exclusive detection of B19V-DNA in EMBs is not sufficient to associate B19V with AMC/DCM but should be complemented with additional virological and immunological parameters in further studies.

Urea-mediated cross-presentation of soluble Epstein-Barr virus BZLF1 protein.

Soluble extracellular proteins usually do not enter the endogenous human leukocyte antigen (HLA) I–dependent presentation pathway of antigen-presenting cells, strictly impeding their applicability for the re-stimulation of protein-specific CD8+ cytotoxic T lymphocytes (CTL). Here we present for the Epstein-Barr virus (EBV) BZLF1 a novel strategy that facilitates protein translocation into antigen-presenting cells by its solubilisation in high molar urea and subsequent pulsing of cells in presence of low molar urea. Stimulation of PBMC from HLA-matched EBV-seropositive individuals with urea-treated BZLF1 but not untreated BZLF1 induces an efficient reactivation of BZLF1-specific CTL. Urea-treated BZLF1 (uBZLF1) enters antigen-presenting cells in a temperature-dependent manner by clathrin-mediated endocytosis and is processed by the proteasome into peptides that are bound to nascent HLA I molecules. Dendritic cells and monocytes but also B cells can cross-present uBZLF1 in vitro. The strategy described here has potential for use in the development of improved technologies for the monitoring of protein-specific CTL.

No detection of human bocavirus in amniotic fluid samples from fetuses with hydrops or isolated effusions

BACKGROUND Human bocavirus (HBoV) is a recently identified parvovirus associated with respiratory disease in infants. Animal bocaviruses have been shown to cause intrauterine infection, fetal anasarca and abortion in late gestation. OBJECTIVES To investigate whether HBoV infection is associated with fetal hydrops, fetal anemia or isolated fetal effusions. STUDY DESIGN We determined the prevalence of HBoV and parvovirus B19 (B19) DNA in amniotic fluid samples from fetuses with hydrops, anemia or isolated effusions using different real-time PCR protocols, and the HBoV IgG and IgM positivity rate in pregnant women with fetal hydrops or normal ultrasound findings by a non-commercial virus-like particle-based enzyme immunoassay. RESULTS None of 87 amniotic fluid samples tested was HBoV DNA positive. Twelve of 60 fetuses with hydrops or anemia were found B19 DNA positive. Anti-HBoV IgG antibodies were detected in 100% (19/19) and 94% (47/50) of serum samples from pregnant women with fetal hydrops and normal ultrasound findings, respectively. All serum samples were found negative for anti-HBoV IgM. CONCLUSION We suggest that HBoV is not a common cause of fetal hydrops, anemia or isolated effusions. This has to be confirmed by further studies of proven gestational HBoV infection.

Seroepidemiology of human bocavirus in Apulia, Italy

A serological survey was performed to determine the prevalence of antibodies against human bocavirus in an Apulian population. Anti-hBoV IgG antibodies were analysed in 1206 inhabitants (age range, 1month-84years) using a standardized ELISA test based on the use of recombinant hBoV VP2 virus-like particles. In total, 1075 (89.1%) of 1206 participants (mean age 32±24.8years) displayed anti-hBoV-IgG. The seroprevalence increased significantly (p<0.0001) in children from 2-4years (64.2%) to 5-9years (96.4%). A similar trend was observed in both male and female subjects. In conclusion, our results show that hBoV infection is common in this population, especially in children.