Structural Insights into Human Bocaparvoviruses.

Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocavirus 1 (HBoV1) causes severe respiratory infections and HBoV2 to HBoV4 cause gastrointestinal infections in young children. Recent reports of life-threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms highlight the need to study these pathogens on a molecular and structural level for the development of therapeutics. Toward this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to a resolution of 2.8 to 3.0 Å by cryo-electron microscopy and three-dimensional image reconstruction. The bocaparvovirus capsids, which display different tissue tropisms, have features in common with other parvoviruses, such as depressions at the icosahedral 2-fold symmetry axis and surrounding the 5-fold symmetry axis, protrusions surrounding the 3-fold symmetry axis, and a channel at the 5-fold symmetry axis. However, unlike other parvoviruses, densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of a genus-specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring capsid surface topologies different from those of other parvoviruses. Structural comparisons at the strain (HBoV) and genus (bovine parvovirus and HBoV) levels identified differences in surface loops that are functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses and likely play similar roles in these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of antiviral strategies to control human bocavirus infections.IMPORTANCE Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study obtained the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, and HBoV3 and HBoV4 infect the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. Based on the comparison to other existing parvovirus capsid structures, this study suggests capsid regions that likely control successful infection, including determinants of receptor attachment, host cell trafficking, and antigenic reactivity. Overall, these observations could impact efforts to design antiviral strategies and vaccines for HBoVs.

Mapping Antigenic Epitopes on the Human Bocavirus Capsid.

UNLABELLED: Human bocaviruses (HBoV1 to -4) are emerging pathogens associated with pneumonia and/or diarrhea in young children. Currently, there is no treatment or vaccination, so there is a need to study these pathogens to understand their disease mechanisms on a molecular and structural level for the development of control strategies. Here, we report the structures of six HBoV monoclonal antibody (MAb) fragment complexes, HBoV1-15C6, HBoV2-15C6, HBoV4-15C6, HBoV1-4C2, HBoV1-9G12, and HBoV1-12C1, determined by cryo-electron microscopy and three-dimensional image reconstruction to 18.0- to 8.5-Å resolution. Of these, the 15C6 MAb cross-reacted with HBoV1, HBoV2, and HBoV4, while the 4C2, 12C1, and 9G12 MAbs recognized only HBoV1. Pseudoatomic modeling mapped the 15C6 footprint to the capsid surface DE and HI loops, at the 5-fold axis and the depression surrounding it, respectively, which are conserved motifs in Parvoviridae The footprints for 4C2, 12C1, and 9G12 span the surface loops that assemble portions of the 2-/5-fold wall (a raised surface feature between the 2-fold and 5-fold axes of symmetry) and the shoulder of the 3-fold protrusions. The MAb footprints, cross reactive and strain specific, coincide with regions with high and low sequence/structural identities, respectively, on the capsid surfaces of the HBoVs and identify potential regions for the development of peptide vaccines for these viruses. IMPORTANCE: Human bocaviruses (HBoVs) may cause severe respiratory and gastrointestinal infections in young children. The nonenveloped parvovirus capsid carries determinants of host and tissue tropism, pathogenicity, genome packaging, assembly, and antigenicity important for virus infection. This information is currently unavailable for the HBoVs and other bocaparvoviruses. This study identifies three strain-specific antigenic epitopes on the HBoV1 capsid and a cross-reactive epitope on the HBoV1, HBoV2, and HBoV4 capsids using structures of capsid-antibody complexes determined using cryo-electron microscopy and image reconstruction. This is the first study to report the highly conserved parvovirus DE loop at the 5-fold axis as a determinant of antigenicity. Additionally, knowledge of the strain-specific and conserved antigenic epitopes of the bocaviruses can be instrumental in characterization of the virus life cycle, development of peptide vaccines, and generation of gene delivery vectors for cystic fibrosis given the strict tropism of HBoV1 for human airway epithelial cells.

Original antigenic sin with human bocaviruses 1-4.

Human bocavirus (HBoV) 1 is a widespread parvovirus causing acute respiratory disease in young children. In contrast, HBoV2 occurs in the gastrointestinal tract and is potentially associated with gastroenteritis, whilst HBoV3 and -4 infections are less frequent and have not yet been linked with human disease. Due to HBoV1 DNA persistence in the nasopharynx, serology has been advocated as a better alternative for diagnosing acute infections. In constitutionally healthy children, we previously noted that pre-existing HBoV2 immunity in a subsequent HBoV1 infection typically resulted in low or non-existent HBoV1-specific antibody responses. A phenomenon describing such immunological events among related viruses has been known since the 1950s as 'original antigenic sin' (OAS). The aim of this study was to characterize this putative OAS phenomenon in a more controlled setting. Follow-up sera of 10 rabbit pairs, inoculated twice with HBoV1-4 virus-like particles (VLPs) or control antigens, in various combinations, were analysed with HBoV1-4 IgG enzyme immunoassays with and without depletion of heterotypic HBoV antibodies. There were no significant IgG boosts after the second inoculation in either the heterologously or the homologously HBoV-inoculated rabbits, but a clear increase in cross-reactivity was seen with time. We could, however, distinguish a distinct OAS pattern from plain cross-reactivity: half of the heterologously inoculated rabbits showed IgG patterns representative of the OAS hypothesis, in line with our prior results with naturally infected children. HBoVs are the first parvoviruses to show the possible existence of OAS. Our findings provide new information on HBoV1-4 immunity and emphasize the complexity of human bocavirus diagnosis.

Identification of past and recent parvovirus B19 infection in immunocompetent individuals by quantitative PCR and enzyme immunoassays: a dual-laboratory study.

Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs.

Human bocaviruses are commonly found in stools of hospitalized children without causal association to acute gastroenteritis.

UNLABELLED: Human bocaviruses (HBoVs) may be grouped into respiratory (HBoV1) and enteric (HBoV2-4) types. We examined this association of HBoV types and clinical symptoms in 955 children who had acute gastroenteritis (AGE, n = 172), acute respiratory tract infection (ARTI, n = 545) or symptoms of both (n = 238). Both nasal swab and stool specimens were studied for such patients. HBoV1 DNA was detected in 6.2 % of patients with ARTI and 9.2 % of patients with symptoms of both ARTI and AGE, but in only 1.7 % of patients with AGE alone. In about one half of the cases, HBoV1 was detected concomitantly in nasal swab and stool samples. HBoV2 was found in stool samples of patients with AGE (5.8 %), ARTI (5.1 %) and symptoms of both (5.5 %) but only rarely in nasal swabs. HBoV3 was found in the stools, but not in nasal swabs, in 0.6, 1.1 and 0.8 % of patients with, respectively, AGE, ARTI and both. HBoV4 was not found. All but one HBoV-positive stool sample of AGE patients contained a known gastroenteritis virus (rotavirus, norovirus, sapovirus, astrovirus or enteric adenovirus) that was probably responsible for the symptoms of the respective case. Sera of 30 HBoV-positive patients were available, and IgM antibodies for HBoVs were found in ten cases and HBoV DNA in eight of these. CONCLUSIONS: HBoV2 and HBoV3 were more commonly found in stool than in nasal swab samples, but the findings could not be causally linked with AGE. HBoV1 was commonly found in stool samples during ARTI, with or without gastrointestinal symptoms.

Primary and secondary human bocavirus 1 infections in a family, Finland.

Human bocavirus 1 (HBoV1) was detected in a young child hospitalized for pneumonia and subsequently in his twin brother and other family members. The mother's nasopharyngeal samples intermittently showed HBoV1 DNA; the grandmother had HBoV1 reinfection. Findings in this family lead to consideration of HBoV virulence, latency, and reactivation.

Possible involvement of human bocavirus 1 in the death of a middle-aged immunosuppressed patient.

An immunosuppressed 61-year-old man went to the hospital with fever, nonproductive cough, and increasing shortness of breath. The subject died 8 days later of respiratory complications. PCR of respiratory samples as well as a blood sample revealed exceptionally high DNA levels of the emerging pathogen, human bocavirus 1 (HBoV1), a recently found pathogen associated with respiratory symptoms in young children. We describe the clinical progression of the case and discuss the potential role of HBoV1 in the outcome.

Human bocavirus infection diagnosed serologically among children admitted to hospital with community-acquired pneumonia in a tropical region.

Human bocavirus (HBoV) is a human virus associated with respiratory disease in children. Limited information is available on acute infection with HBoV among children admitted to hospital with community-acquired pneumonia in tropical regions and the current diagnosis is inadequate. The aims were to diagnose and describe acute HBoV infections among children hospitalized for community-acquired pneumonia. In Salvador, Brazil, 277 children with community-acquired pneumonia were prospectively enrolled. Paired serum samples were tested by IgG, IgM, and IgG-avidity enzyme immunoassays (EIAs) using recombinant HBoV VP2. HBoV DNA was detected in nasopharyngeal aspirates and serum by a quantitative polymerase-chain reaction (PCR). HBoV DNA was detected in nasopharyngeal aspirates of 62/268 (23%) children and 156/273 (57%) were seropositive. Acute primary HBoV infection was reliably diagnosed (bearing at least two acute markers: Positive IgM, a fourfold increase/conversion of IgG, low IgG avidity or viremia) in 21 (8%) of 273 patients, 90% of 20 had HBoV DNA in nasopharyngeal aspirates, 83% with a high DNA load. The median age of infection with HBoV was 16 months, range 5-36. Community-acquired pneumonia was confirmed radiographically in 85% of 20 patients with acute HBoV infection diagnosed serologically. HBoV DNA was found in nasopharyngeal aspirates of 42/246(17%) children without an acute primary HBoV infection and available nasopharyngeal aspirate. Four children with HBoV secondary immune responses were detected, lacking both IgM and viremia. HBoV infection was diagnosed accurately in children aged 5-36 months with community-acquired pneumonia confirmed radiographically. PCR of nasopharyngeal aspirates is not a reliable marker of acute HBoV infection.

Occurrence of human bocaviruses and parvovirus 4 in solid tissues.

Human bocaviruses 1-4 (HBoV1-4) and parvovirus 4 (PARV4) are recently discovered human parvoviruses. HBoV1 is associated with respiratory infections of young children, while HBoV2-4 are enteric viruses. The clinical manifestations of PARV4 remain unknown. The objective of this study was to determine whether the DNAs of HBoV1-4 and PARV4 persist in human tissues long after primary infection. Biopsies of tonsillar tissue, skin, and synovia were examined for HBoV1-4 DNA and PARV4 DNA by PCR. Serum samples from the tissue donors were assayed for HBoV1 and PARV4 IgG and IgM antibodies. To obtain species-specific seroprevalences for HBoV1 and for HBoV2/3 combined, the sera were analyzed after virus-like particle (VLP) competition. While HBoV1 DNA was detected exclusively in the tonsillar tissues of 16/438 individuals (3.7%), all of them ≤8 years of age. HBoV2-4 and PARV4 DNAs were absent from all tissue types. HBoV1 IgG seroprevalence was 94.9%. No subject had HBoV1 or PARV4 IgM, nor did they have PARV4 IgG. The results indicate that HBoV1 DNA occurred in a small proportion of tonsils of young children after recent primary HBoV1 infection, but did not persist long in the other tissue types studied, unlike parvovirus B19 DNA. The results obtained by the PARV4 assays are in line with previous results on PARV4 epidemiology.

Seroepidemiology of human bocaviruses 1-4.

BACKGROUND: Recently, 3 new members of the genus Bocavirus, human bocavirus 2 (HBoV2), human bocavirus 3 (HBoV3), and human bocavirus 4 (HBoV4), were discovered. HBoV2-4 occur mainly in the gastrointestinal tract but rarely in the respiratory tract, contrary to human bocavirus 1 (HBoV1). METHODS: To investigate HBoV1-4 seroepidemiology among 195 adults and 252 wheezing children, we conducted immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme immunoassays with recombinant viruslike particles (VLPs). The children's sera were also tested for HBoV1-4 DNA by quantitative polymerase chain reaction (qPCR). RESULTS: Both rabbit and human antibodies to HBoV1-4 VP2 VLPs were found to be cross-reactive. After depletion of HBoV1-reactive antibodies, the HBoV2-4 approximate seroprevalences in adults were 34%, 15%, and 2% and in children aged 1-2 years 25%, 10%, and 5%, respectively. After depletion of HBoV2-4-reactive antibodies, the HBoV1 seroprevalence among adults decreased from 96% to 59%. No cross-reactivity of human anti-HBoV IgG was observed with bovine parvovirus1, parvovirus B19 or PARV4. No child was HBoV2-4 viremic. CONCLUSIONS: HBoV2-4 infect humans less commonly and elicit weaker B-cell responses than HBoV1. In our study HBoV2-4 did not seem to have a major etiological role in wheezing. Cross-reactivity with HBoV2-4 IgG partially accounts for the high HBoV1 seroprevalences previously reported. Correction for cross-reactivity is a prerequisite for VLP-based HBoV seroepidemiology.

Widespread infection with homologues of human parvoviruses B19, PARV4, and human bocavirus of chimpanzees and gorillas in the wild.

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.

Real-time quantitative PCR detection of four human bocaviruses.

Human bocavirus (HBoV) was discovered in 2005 and is associated with respiratory tract symptoms in young children. Three additional members of the genus Bocavirus, HBoV2, -3, and -4, were discovered recently from fecal specimens, and early results indicate an association between HBoV2 and gastrointestinal disease. In this study, we present an undifferentiating multiplex real-time quantitative PCR assay for the detection of these novel viruses. Differentiation of the individual bocavirus species can be subsequently achieved with corresponding singleplex PCRs or by sequencing. Both multiplex and singleplex assays were consistently able to detect ≤10 copies of HBoV1 to -4 plasmid templates/reaction, with dynamic quantification ranges of 8 logs and 97% to 102% average reaction efficiencies. These new assays were used to screen stool samples from 250 Finnish patients (median age, 40 years) that had been sent for diagnosis of gastrointestinal infection. Four patients (1.6%; median age, 1.1 years) were reproducibly positive for HBoV2, and one patient (0.4%; 18 years of age) was reproducibly positive for HBoV3. The viral DNA loads varied from <10(3) to 10(9) copies/ml of stool extract. None of the stool samples harbored HBoV1 or HBoV4. The highly conserved sequence of the hydrolysis probe used in this assay may provide a flexible future platform for the quantification of additional, hitherto-unknown human bocaviruses that might later be discovered. Our results support earlier findings that HBoV2 is a relatively common pathogen in the stools of diarrheic young children, yet does not often occur in the stools of adults.

Newly discovered KI, WU, and Merkel cell polyomaviruses: no evidence of mother-to-fetus transmission.

BACKGROUND: Three* human polyomaviruses have been discovered recently, KIPyV, WUPyV and MCPyV. These viruses appear to circulate ubiquitously; however, their clinical significance beyond Merkel cell carcinoma is almost completely unknown. In particular, nothing is known about their preponderance in vertical transmission. The aim of this study was to investigate the frequency of fetal infections by these viruses. We sought the three by PCR, and MCPyV also by real-time quantitative PCR (qPCR), from 535 fetal autopsy samples (heart, liver, placenta) from intrauterine fetal deaths (IUFDs) (N = 169), miscarriages (120) or induced abortions (246). We also measured the MCPyV IgG antibodies in the corresponding maternal sera (N = 462) mostly from the first trimester. RESULTS: No sample showed KIPyV or WUPyV DNA. Interestingly, one placenta was reproducibly PCR positive for MCPyV. Among the 462 corresponding pregnant women, 212 (45.9%) were MCPyV IgG seropositive. CONCLUSIONS: Our data suggest that none of the three emerging polyomaviruses often cause miscarriages or IUFDs, nor are they transmitted to fetuses. Yet, more than half the expectant mothers were susceptible to infection by the MCPyV.

Nanoassembly of nanostructures by cutting, bending and soldering of carbon nanotubes with electron beam.

We report that a series of in situ nanofabrication techniques of nanostructures, including cutting, bending and soldering of carbon nanotubes (CNTs), inside a field emission scanning electron microscope (FE-SEM) used for nanoassembly of nanostructures. The CNTs can be cut with electron beam assisted with oxygen gas. The cutting was developed for the bending of CNT, if some conditions of the cutting technique are changed. These include the increase of the acceleration voltage and/or setting the oxygen gas nozzle farther from the sample, and/or reducing the irradiation time. Using the proposed bending method angles larger than 90 degrees can be formed and the location of the kink can be set accurately. It is also shown that tungsten can be deposited on a substrate by the electron-beam-induced deposition, if the oxygen of the proposed cutting technique is replaced by W(CO)6. In this paper, these three nanofabrication methods were employed in the creation of a two dimensional (2D) nanostructure, the letters N and U, and a three dimensional (3D) nanostructure, the letter N. The 2D letters were constructed from 6 CNTs assembled on a substrate while the 3D letter N was bended from a single CNT and fixed to stand on a substrate. Based on the high performance of the proposed techniques, it is suggested that the cutting, bending, and soldering techniques inside SEM will become widely utilized in the fabrication and assembly of nanodevices and in the characterization of nanomaterials.

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.

Correction: B-Cell Responses to Human Bocaviruses 1-4: New Insights from a Childhood Follow-Up Study.

[This corrects the article DOI: 10.1371/journal.pone.0139096.].

B-Cell Responses to Human Bocaviruses 1-4: New Insights from a Childhood Follow-Up Study.

Human bocaviruses (HBoVs) 1-4 are recently discovered, antigenically similar parvoviruses. We examined the hypothesis that the antigenic similarity of these viruses could give rise to clinically and diagnostically important immunological interactions. IgG and IgM EIAs as well as qPCR were used to study ~2000 sera collected from infancy to early adolescence at 3-6-month intervals from 109 children whose symptoms were recorded. We found that HBoV1-4-specific seroprevalences at age 6 years were 80%, 48%, 10%, and 0%, respectively. HBoV1 infections resulted in significantly weaker IgG responses among children who had pre-existing HBoV2 IgG, and vice versa. Furthermore, we documented a complete absence of virus type-specific immune responses in six viremic children who had pre-existing IgG for another bocavirus, indicating that not all HBoV infections can be diagnosed serologically. Our results strongly indicate that interactions between consecutive HBoV infections affect HBoV immunity via a phenomenon called "original antigenic sin", cross-protection, or both; however, without evident clinical consequences but with important ramifications for the serodiagnosis of HBoV infections. Serological data is likely to underestimate human exposure to these viruses.

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.

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.

Expression of all six human Torque teno virus (TTV) proteins in bacteria and in insect cells, and analysis of their IgG responses.

Torque teno virus (TTV) is a non-enveloped human virus with a circular ( approximately 3800 nt) ssDNA genome. TTV transcription results in three viral mRNAs and six proteins, the function or antigenicity of which are unknown. The six open reading frames of TTV genotype 6 were expressed in bacteria and insect cells. Expression of the ORF1/1-encoded protein was inefficient, while expression of the others was successful, with ORF1 and ORF1/2 as arginine-rich region depleted. All six recombinant TTV proteins were antigenic. Of healthy adults, 11/25 (44%) showed strong IgG reactivity with one or more proteins. Four subjects, two of whom were genotype-6-DNA positive, were followed. One of the latter showed concurrently a strong IgG response against the ORF1 protein. The other showed appearance of IgG against the ORF2 protein concomitantly with resolution of the genotype-6 viremia. The genotype-6 sequences remained unaltered for years, suggesting that some mechanisms other than amino acid substitutions play a role in TTV immune evasion.