Time-resolved FRET -based approach for antibody detection - a new serodiagnostic concept.

Förster resonance energy transfer (FRET) is a phenomenon widely utilized in biomedical research of macromolecular interactions. In FRET energy is transferred between two fluorophores, the donor and the acceptor. Herein we describe a novel approach utilizing time-resolved FRET (TR-FRET) for the detection of antibodies not only in a solution-phase homogenous assay but also in single- and two-step solid-phase assays. Our method is based on the principle that the Y-shaped immunoglobulin G molecule is able to simultaneously bind two identical antigen molecules. Hence, if a specific IgG is mixed with donor- and acceptor-labeled antigens, the binding of antigens can be measured by TR-FRET. Using donor- and acceptor-labeled streptavidins (SAs) in conjunction with a polyclonal and a monoclonal anti-SA antibody we demonstrate that this approach is fully functional. In addition we characterize the immune complexes responsible for the TR-FRET signal using density gradient ultracentrifugation and solid-phase immunoassays. The homogenous TR-FRET assay described provides a rapid and robust tool for antibody detection, with a wide potential in medical diagnostics.

Serodiagnosis of primary infections with human parvovirus 4, Finland.

To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle-based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus-infected injection drug users.

Expression and serological characterization of polyomavirus WUPyV and KIPyV structural proteins.

The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged < or = 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged < or = 2 y. This difference shows the importance of conformational VP1 antigenicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood.

Serologically verified human bocavirus pneumonia in children.

WORKING HYPOTHESIS: Human bocavirus (HBoV) is a newly identified parvovirus frequently found in children suffering from acute respiratory and intestinal infections. The aim of the present study was to evaluate, by using a newly developed antibody assay, the role of HBoV in pediatric community-acquired pneumonia (CAP) and the seropositivity rate to HBoV in a prospective study in North-Italian children. MATERIALS/PATIENTS AND METHODS: During a 15-month study period, 124 children were admitted due to presumptive pneumonia, and in 101 of them, pneumonia was radiologically confirmed. The etiology of CAP was studied by antibody assays to 16 microbes, including the newly developed enzyme immunoassay for HBoV. RESULTS: Serological evidence of acute HBoV infection was found in 12 (12%) children, being single in 7 and mixed in 5 cases (4 with other viruses and 3 with bacteria). IgM was positive in 11 cases. A diagnostic rise in IgG antibodies between paired sera was observed in six cases. HBoV was the second most common virus next to respiratory syncytial virus (17%). The seropositivity rate to HBoV increased with age, reaching nearly 100% before school age. CONCLUSIONS: The present results show that HBoV is able to induce significant antibody responses and suggest that HBoV may be a fairly common cause of pneumonia in children. Seroconversion to HBoV in most children takes place in early childhood.

Absence of human bocavirus from deceased fetuses and their mothers.

BACKGROUND: The human bocavirus (HBoV), a newly discovered parvovirus, is closely related to the bovine parvovirus and the canine minute virus, which are known to cause adverse pregnancy outcomes. Another human parvovirus, B19, can lead to fetal hydrops, miscarriage and intrauterine fetal death (IUFD). OBJECTIVES: To determine the prevalence of HBoV DNA in aborted fetuses and IUFDs. The HBoV serology of the mothers was also studied. STUDY DESIGN: We retrospectively studied all available fetuses (N=535) autopsied during 7/1992-12/1995, and 1/2003-12/2005 in Helsinki, Finland. All available formalin-fixed paraffin-embedded fetal tissues - placenta, heart and liver - of 120 miscarriages, 169 IUFDs, and 246 induced abortions were studied by quantitative PCR. We also measured the HBoV IgM and IgG antibodies in the corresponding maternal sera (N=462) mostly of the first trimester. The IgM-positive sera underwent HBoV PCR. RESULTS: None of the fetal tissues harbored HBoV DNA. A total of 97% (448/462) of the mothers were positive for IgG antibodies to HBoV, while only 0.9% (4/462) exhibited HBoV-specific IgM antibodies without viremia or respiratory symptoms. One IgM-positive mother had an unexplained fetal loss. CONCLUSIONS: We did not find HBoV DNA in any of the deceased fetuses. Almost all pregnant women were HBoV-IgG positive.

Clinical assessment and improved diagnosis of bocavirus-induced wheezing in children, Finland.

Human bocavirus (HBoV) is a widespread respiratory virus. To improve diagnostic methods, we conducted immunoglobulin (Ig) G and IgM enzyme immunoassays with recombinant virus-like particles of HBoV as antigen. Acute-phase and follow-up serum samples from 258 wheezing children and single serum samples from 115 healthy adults in Finland were examined. Our assays had a sensitivity of 97% and a specificity of 99.5%. Of adults, 96% had immunity; none had an acute infection. Of 48 children with serologically diagnosed acute HBoV infections, 45 were viremic and 35 had virus in nasopharyngeal aspirates (NPAs). Of 39 HBoV NPA PCR-positive children co-infected with another virus, 64% had a serologically verified HBoV infection. HBoV caused illness of longer duration than rhinovirus and of equal severity to that of respiratory syncytial virus. Among children with bronchiolitis, >25% had acute HBoV infections. Accurate HBoV diagnosis requires serologic analysis or PCR of serum; PCR of NPAs alone is insufficient.

Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples: implications for respiratory transmission and latency.

BACKGROUND: Merkel cell polyomavirus (MCPyV) was discovered recently. It is considered a potential causative agent of Merkel cell carcinoma, a life-threatening skin cancer. OBJECTIVES: To study the prevalence of MCPyV in a large number of clinical samples of various types. Most of the samples were examined also for the other newly found polyomaviruses KI (KIPyV) and WU (WUPyV). STUDY DESIGN: Altogether 1390 samples from immunocompetent or immunocompromised patients, including (i) tonsillar tissues and sera from tonsillectomy patients; (ii) nasopharyngeal aspirates (NPAs) and sera from wheezing children and (iii) nasal swabs, sera and stools from febrile leukemic children were studied for MCPyV. The tonsils, nasal swabs and stools were also studied for KIPyV and WUPyV. RESULTS: MCPyV DNA was detected in 14 samples altogether; 8 of 229 (3.5%) tonsillar tissues, 3 of 140 (2.1%) NPAs, 2 of 106 (1.9%) nasal swabs and 1 of 840 (0.1%) sera. WUPyV and KIPyV were detected in 5 (2.2%) and 0 tonsils, 1 (0.9%) and 4 (3.8%) nasal swabs and 0 and 2 (2.7%) fecal samples, respectively. The patients carrying in tonsils MCPyV were of significantly higher age (median 42years) than those carrying WUPyV (4years, p<0.001). CONCLUSIONS: MCPyV DNA occurs in tonsils more frequently in adults than in children. By contrast, WUPyV DNA is found preferentially in children. MCPyV occurs also in nasal swabs and NPAs, in a frequency similar to that of KIPyV and WUPyV. The tonsil may be an initial site of WUPyV infection and a site of MCPyV persistence.