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Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus , respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10-30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.

Human bocavirus 1 (HBoV1), a nonenveloped single-stranded DNA parvovirus, causes mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children. HBoV1 often persists in nasopharyngeal secretions for months, hampering diagnosis. It has also been shown to persist in pediatric palatine and adenoid tonsils, which suggests that lymphoid organs are reservoirs for virus spread; however, the tissue site and host cells remain unknown. Our aim was to determine, in healthy nonviremic children with preexisting HBoV1 immunity, the adenotonsillar persistence site(s), host cell types, and virus activity. We discovered that HBoV1 DNA persists in lymphoid germinal centers (GCs), but not in the corresponding tonsillar epithelium, and that the cell types harboring the virus are mainly naive, activated, and memory B cells and monocytes. Both viral DNA strands and both sides of the genome were detected, as well as infrequent mRNA. Moreover, we showed, in B-cell and monocyte cultures and ex vivo tonsillar B cells, that the cellular uptake of HBoV1 occurs via the Fc receptor (FcγRII) through antibody-dependent enhancement (ADE). This resulted in viral mRNA transcription, known to occur exclusively from double-stranded DNA in the nucleus, however, with no detectable productive replication. Confocal imaging with fluorescent virus-like particles moreover disclosed endocytosis. To which extent the active HBoV1 GC persistence has a role in chronic inflammation or B-cell maturation disturbances, and whether the virus can be reactivated, will be interesting topics for forthcoming studies. IMPORTANCE Human bocavirus 1 (HBoV1), a common pediatric respiratory pathogen, can persist in airway secretions for months hampering diagnosis. It also persists in tonsils, providing potential reservoirs for airway shedding, with the exact location, host cell types, and virus activity unknown. Our study provides new insights into tonsillar HBoV1 persistence. We observed HBoV1 persistence exclusively in germinal centers where immune maturation occurs, and the main host cells were B cells and monocytes. In cultured cell lines and primary tonsillar B cells, we showed the virus uptake to be significantly enhanced by HBoV1-specific antibodies, mediated by the cellular IgG receptor, leading to viral mRNA synthesis, but without detectable productive replication. Possible implications of such active viral persistence could be tonsillar inflammation, disturbances in immune maturation, reactivation, or cell death with release of virus DNA, explaining the long-lasting HBoV1 airway shedding., (Copyright © 2021 Xu et al.)

Parvoviridae, a diverse family of small single-stranded DNA viruses was established in 1975. It was divided into two subfamilies, Parvovirinae and Densovirinae, in 1993 to accommodate parvoviruses that infect vertebrate and invertebrate animals, respectively. This relatively straightforward segregation, using host association as the prime criterion for subfamily-level classification, has recently been challenged by the discovery of divergent, vertebrate-infecting parvoviruses, dubbed "chapparvoviruses", which have proven to be more closely related to viruses in certain Densovirinae genera than to members of the Parvovirinae. Viruses belonging to these genera, namely Brevi-, Hepan- and Penstyldensovirus, are responsible for the unmatched heterogeneity of the subfamily Densovirinae when compared to the Parvovirinae in matters of genome organization, protein sequence homology, and phylogeny. Another genus of Densovirinae, Ambidensovirus, has challenged traditional parvovirus classification, as it includes all newly discovered densoviruses with an ambisense genome organization, which introduces genus-level paraphyly. Lastly, current taxon definition and virus inclusion criteria have significantly limited the classification of certain long-discovered parvoviruses and impedes the classification of some potential family members discovered using high-throughput sequencing methods. Here, we present a new and updated system for parvovirus classification, which includes the introduction of a third subfamily, Hamaparvovirinae, resolves the paraphyly within genus Ambidensovirus, and introduces new genera and species into the subfamily Parvovirinae. These proposals were accepted by the ICTV in 2020 March.

Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors. IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors., (Copyright © 2020 American Society for Microbiology.)

Background: Diagnosis of human bocavirus 1 (HBoV1) has been based on qualitative PCRs detecting HBoV1 DNA or detection of HBoV1 mRNA., Objective: This study aims to assess whether a rapid and automated HBoV1 antigen test is suitable for diagnosis of acute HBoV1 infection., Study Design: HBoV1 antigen detection has been compared with quantitative HBoV1 DNA PCR and HBoV1 mRNA RT-PCR., Results and Conclusion: We conclude that HBoV1 antigen detection has higher clinical specificity and positive predictive value than HBoV1 DNA qualitative PCRs, yet a lower sensitivity than HBoV1 mRNA detection. Additionally, HBoV1 antigen detection is beneficial in its rapidity and availability as a point-of-care test., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Parvoviruses are structurally simple viruses with linear single-stranded DNA genomes and nonenveloped icosahedral capsids. They infect a wide range of animals from insects to humans. Parvovirus B19 is a long-known human pathogen, whereas adeno-associated viruses are nonpathogenic. Since 2005, many parvoviruses have been discovered in human-derived samples: bocaviruses 1-4, parvovirus 4, bufavirus, tusavirus, and cutavirus. Some human parvoviruses have already been shown to cause disease during acute infection, some are associated with chronic diseases, and others still remain to be proven clinically relevant-or harmless commensals, a distinction not as apparent as it might seem. One initially human-labeled parvovirus might not even be a human virus, whereas another was originally overlooked due to inadequate diagnostics. The intention of this review is to follow the rocky road of emerging human parvoviruses from discovery of a DNA sequence to current and future clinical status, highlighting the perils along the way.

Background: Human bocavirus 1 is a commonly detected human parvovirus. Many studies have shown human bocavirus 1 as a pathogen in association with acute respiratory tract infections in children. However, because human bocavirus 1 persists in the upper airways for extensive time periods after acute infection, the definition and diagnostics of acute human bocavirus 1 infection is challenging. Until now, detection of human bocavirus 1 exclusively, high viral load in respiratory samples, and viremia have been associated with a clinical picture of acute respiratory illness. There are no studies showing detection of human bocavirus 1 messenger ribonucleic acid in the peripheral blood mononuclear cells as a diagnostic marker for acute lower respiratory tract infection., Case Presentation: We report the case of a 17-month-old Latvian boy who presented in intensive care unit with acute bilateral bronchiolitis, with a history of rhinorrhea and cough for 6 days and fever for the last 2 days prior to admission, followed by severe respiratory distress and tracheal intubation. Human bocavirus 1 was the only respiratory virus detected by a qualitative multiplex polymerase chain reaction panel. For the diagnosis of acute human bocavirus 1 infection, both molecular and serological approaches were used. Human bocavirus 1 deoxyribonucleic acid (DNA) was detected simultaneously in nasopharyngeal aspirate, stool, and blood, as well as in the corresponding cell-free blood plasma by qualitative and quantitative polymerase chain reaction, revealing high DNA-copy numbers in nasopharyngeal aspirate and stool. Despite a low-load viremia, human bocavirus 1 messenger ribonucleic acid was found in the peripheral blood mononuclear cells. For detection of human bocavirus 1-specific antibodies, non-competitive immunoglobulin M and competitive immunoglobulin G enzyme immunoassays were used. The plasma was positive for both human bocavirus 1-specific immunoglobulin M and immunoglobulin G antibodies., Conclusions: The presence of human bocavirus 1 genomic DNA in blood plasma and human bocavirus 1 messenger ribonucleic acid in peripheral blood mononuclear cells together with human bocavirus 1-specific immunoglobulin M are markers of acute human bocavirus 1 infection that may cause life-threatening acute bronchiolitis.

We report a case of lower respiratory tract infection with human bocavirus 1 (HboV1) in an immunodeficient 6-month-old boy leading to respiratory failure with fatal outcome. Polymerase chain reaction of serum/tracheal secretions revealed exceptionally high HboV1-DNA levels and immunoassays showed seroconversion indicating an acute primary HboV1 infection. All assays for other pathogens were negative, strongly suggesting that HboV1 was the causative agent in this case.

Human bocavirus 1 (HBoV1) can persist in nasopharynx and tonsils. Using HBoV1 serology, reverse-transcription polymerase chain reaction (PCR) for detecting messenger RNA (mRNA) and quantitative PCR for HBoV1 genome load count, we studied to what extent the HBoV1 DNA loads in nasopharynx correlate with acute infection markers. Tonsillar tissue, nasopharyngeal aspirate, and serum were obtained from 188 elective adeno-/tonsillectomy patients. Relatively high loads of HBoV1 DNA were detected in the nasopharynx of 14 (7%) primarily asymptomatic subjects with negative mRNA and/or serodiagnostic results. Quantitative HBoV1 DNA PCR may have lower specificity than HBoV1 mRNA detection for diagnosing symptomatic infection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Human bocavirus 1 (HBoV1), belonging to the Parvoviridae family, was discovered in 2005, in nasopharyngeal samples from children with respiratory tract infections. Three additional bocaviruses, HBoV2-4, were discovered in 2009-10. These viruses have mainly been found in faecal samples and their role in human diseases is still uncertain. HBoV1 causes a wide spectrum of respiratory diseases in children, including common cold, acute otitis media, pneumonia, bronchiolitis, and asthma exacerbations. HBoV1 DNA can persist in airway secretions for months after an acute infection. Consequently, acute HBoV1 infection cannot be diagnosed with standard DNA PCR; quantitative PCR and serology are better diagnostic approaches. Because of their high clinical specificity, diagnostic developments such as HBoV1 mRNA and antigen detection have shown promising results. This Review summarises the knowledge on human bocaviruses, with a special focus on HBoV1., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Members of the family Parvoviridae are small, resilient, non-enveloped viruses with linear, single-stranded DNA genomes of 4-6 kb. Viruses in two subfamilies, the Parvovirinae and Densovirinae, are distinguished primarily by their respective ability to infect vertebrates (including humans) versus invertebrates. Being genetically limited, most parvoviruses require actively dividing host cells and are host and/or tissue specific. Some cause diseases, which range from subclinical to lethal. A few require co-infection with helper viruses from other families. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Parvoviridae, which is available at www.ictv.global/report/parvoviridae.

Human bocaviruses (HBoVs) 1⁻4 belong to the Parvoviridae family, and they infect the respiratory or gastrointestinal tracts in children. We investigated the prevalence of HBoV1⁻4 DNAs in the blood and stool samples, and of HBoV1⁻4 IgG and IgM in the plasma samples, of children presenting with acute gastroenteritis (AGE). In addition, we identified HBoV co-infections with the five most frequent gastrointestinal pathogens. A total of 83 paired blood and stool samples were collected from children aged five years or less. Infection markers of HBoV1, 2, or 3 (viral DNA in blood and/or stool and/or antibodies) were detected in 61 out of 83 (73.5%) patients. HBoV1, 2, or 3 DNA as a monoinfection was revealed in 18.1%, 2.4%, and 1.2%, respectively, and 21.7% in total. In 56.1% of the HBoV DNA-positive patients, the presence in stool of another virus-most frequently norovirus or rotavirus-was observed. In conclusion, this study, for the first time, illustrates the prevalence and genetic diversity of HBoVs in Latvian children with gastroenteritis, and shows a widespread distribution of these viruses in the community. HBoV1 and 2 are commonly found as single infectious agents in children with AGE, suggesting that the viruses can be as pathogenic by themselves as other enteric agents are., Competing Interests: The authors declare no conflicts of interest.

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.

Aim: To assess the role of human bocavirus 1 (HBoV1) as a causative agent of non-severe community-acquired pneumonia (CAP) in children., Methods: Patients aged 2-59 months with non-severe CAP (respiratory complaints and radiographic pulmonary infiltrate/consolidation) attending a University Hospital in Salvador, Brazil were enrolled in a prospective cohort. From 820 recruited children in a clinical trial (ClinicalTrials.gov NCT01200706), nasopharyngeal aspirate (NPA), and acute and convalescent serum samples were obtained from 759 (92.6%) patients. NPAs were tested for 16 respiratory viruses by PCR. Acute HBoV1 infection was confirmed by measuring specific IgM and IgG responses in paired serum samples., Results: Respiratory viruses were detected in 693 (91.3%; 95%CI: 89.1-93.2) CAP cases by PCR. HBoV1-DNA was detected in 159 (20.9%; 95%CI: 18.2-24.0) cases. Of these 159 PCR positive cases, acute HBoV1 infection was confirmed serologically in 38 cases (23.9%; 95%CI: 17.8-31.0). Overall, acute HBoV1 infection was confirmed in 5.0% (38/759) of non-severe CAP patients. HBoV1 was detected in 151 cases with at least one other virus making 31.7% of all multiple virus (n = 477) detections. Among all 759 cases, 216 had one respiratory virus detected, and sole HBoV1 was detected in only 8 (3.7%). Acute HBoV1 infection was serologically diagnosed in 34 (22.5%) HBoV1-DNA-positive cases with another virus, compared to 4 (50.0%) cases with sole virus detection (p = 0.09)., Conclusion: HBoV1 was detected by PCR in one fifth of the children with non-severe CAP and acute HBoV1 infection was serologically confirmed in one quarter of these cases., (© 2017 Wiley Periodicals, Inc.)

Next-generation sequencing and metagenomics have revolutionized the discovery of novel viruses. In recent years, three novel protoparvoviruses have been discovered in fecal samples of humans: bufavirus (BuV) in 2012, tusavirus (TuV) in 2014, and cutavirus (CuV) in 2016. BuV has since been studied the most, disclosing three genotypes that also represent serotypes. Besides one nasal sample, BuV DNA has been found exclusively in diarrheal feces, but not in non-diarrheal feces, suggesting a causal relationship. According to both geno- and seroprevalences, BuV appears to be the most common of the three novel protoparvoviruses, whereas TuV DNA has been found in only a single fecal sample, with antibody detection being equally rare. Moreover, the TuV sequence is closer to those of non-human protoparvoviruses, and so the evidence of TuV being a human virus is thus far insufficient. Interestingly, besides in feces, CuV has also been detected in skin biopsies of patients with cutaneous T-cell lymphoma and a patient with melanoma, while all other skin samples have tested PCR negative. Even if preliminary disease associations exist, the full etiological roles of these viruses in human disease are yet to be resolved., Competing Interests: The authors declare no conflict of interest.

We report a case of human bocavirus 1 (HBoV1) bronchiolitis that led to life-threatening respiratory failure in a 9-month-old boy with no other pathogens detected. The virus-specific diagnosis was confirmed with the detection of HBoV1 DNA in respiratory samples and both DNA and IgM and IgG to HBoV1 in serum samples.

Background: Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection., Methods: Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection., Results: By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%)., Conclusions: A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Parvovirus B19 (B19V) DNA persists lifelong in human tissues, but the cell type harbouring it remains unclear. We here explore B19V DNA distribution in B, T and monocyte cell lineages of recently excised tonsillar tissues from 77 individuals with an age range of 2-69 years. We show that B19V DNA is most frequent and abundant among B cells, and within them we find a B19V genotype that vanished from circulation >40 years ago. Since re-infection or re-activation are unlikely with this virus type, this finding supports the maintenance of pathogen-specific humoral immune responses as a consequence of B-cell long-term survival rather than continuous replenishment of the memory pool. Moreover, we demonstrate the mechanism of B19V internalization to be antibody dependent in two B-cell lines as well as in ex vivo isolated tonsillar B cells. This study provides direct evidence for a cell type accountable for B19V DNA tissue persistence.

Parvovirus B19 (B19V) and human bocavirus 1 (HBoV1), members of the large Parvoviridae family, are human pathogens responsible for a variety of diseases. For B19V in particular, host features determine disease manifestations. These viruses are prevalent worldwide and are culturable in vitro, and serological and molecular assays are available but require careful interpretation of results. Additional human parvoviruses, including HBoV2 to -4, human parvovirus 4 (PARV4), and human bufavirus (BuV) are also reviewed. The full spectrum of parvovirus disease in humans has yet to be established. Candidate recombinant B19V vaccines have been developed but may not be commercially feasible. We review relevant features of the molecular and cellular biology of these viruses, and the human immune response that they elicit, which have allowed a deep understanding of pathophysiology., (Copyright © 2016 American Society for Microbiology.)

DNA in human skeletal remains represents an important historical source of host genomic information and potentially of infecting viruses. However, little is known about viral persistence in bone. We searched ca. 70-year-old long bones of putative Finnish casualties from World War II for parvovirus B19 (B19V) DNA, and found a remarkable prevalence of 45%. The viral sequences were exclusively of genotypes 2 (n = 41), which disappeared from circulation in 1970´s, or genotype 3 (n = 2), which has never been reported in Northern Europe. Based on mitochondrial and Y-chromosome profiling, the two individuals carrying B19V genotype 3 were likely from the Soviet Red Army. The most recent common ancestor for all genotypes was estimated at early 1800s. This work demonstrates the forms of B19V that circulated in the first half of the 20(th) century and provides the first evidence of the suitability of bone for exploration of DNA viruses.

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.

Recently a parvovirus called bufavirus (BuV) has been implicated as a causative agent of diarrhoea. To further reveal the epidemiology and genetic characteristics of BuV, this study was performed in Turkish children with diarrhoea. BuV was detected in 1.4% (8/583) of stool samples. All stool samples from healthy children (n = 148) were negative for BuV. Diarrhoea in BuV-positive patients was severe and occurred mainly during the colder months of the year. Complete genome sequences were generated from four BuVs. Only BuV3 was found, which was genetically and phylogenetically similar to Bhutanese BuV3, indicating that BuV3 is prevalent in Asian countries., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

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.

Background: Human parvovirus B19 (B19V) infection during early pregnancy increases the risk of miscarriage. Studies have inconsistently shown an elevated risk of infection among women with occupational contacts with children. Methodological differences, particularly in defining occupational exposure and in the type of reference group, may explain the conflicting findings., Methods: This cohort study compared B19V infections in pregnant day-care employees and healthcare professionals during a B19V epidemic in Finland. Women were identified from the files of nationwide trade unions and the National Supervisory Authority for Welfare and Health. Early-pregnancy maternal B19V IgG was analysed in 3710 women, and infections were defined as seroconversions after analysing in parallel the available umbilical cord blood samples of the 847 seronegative mothers. Independently of the serological status, the actual employment during pregnancy was assessed using registered information on employment history., Results: B19V infections were more common among day-care employees (22/331, 6.6%), than among those working in healthcare (12/326, 3.7%). The adjusted HRs of B19V infection, using proportional hazard regression, was 2.63 (95% CI 1.27 to 5.46) among all women and 5.59 (95% CI 1.40 to 22.4) among nulliparous women., Conclusions: Day-care employees are at an increased risk of B19V infection, which warrants preventive measures., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

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.

We identified a new genotype of bufavirus, BuV3, in fecal samples (0.8%) collected to determine the etiology of diarrhea in children in Bhutan. Norovirus GII.6 was detected in 1 sample; no other viral diarrheal pathogens were detected, suggesting BuV3 as a cause of diarrhea. This study investigates genetic diversity of circulating BuVs.

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 parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We identified human bocavirus (HBoV) DNA by PCR in cerebrospinal fluid from adults and children with encephalitis in Sri Lanka. HBoV types 1, 2, and 3 were identified among these cases. Phylogenetic analysis of HBoV1 strain sequences found no subclustering with strains previously identified among encephalitis cases in Bangladesh.

Among the immunocompetent, infections with parvovirus B19 (B19V) and human bocavirus (HBoV) 1 range clinically from asymptomatic to severe, while following allogeneic hematopoietic SCT (HSCT) B19V can cause a persistent severe illness. The epidemiology and clinical impact of HBoV1 and the other emerging parvovirus 4 (PARV4) among immunocompromised patients have not been established. To determine the occurrence and clinical spectrum of B19V, PARV4 and HBoV1 infections, we performed a longitudinal molecular surveillance among 53 allogeneic HSCT recipients for pre- and post-HSCT DNAemias of these parvoviruses. Quantitative real-time PCR showed B19V DNA in sera of 16 (30%) patients, at mean levels of 4.6 × 10(3), 9.9 × 10(7), 1.1 × 10(10) and 1.6 × 10(2) B19V DNA copies/mL pre-HSCT (9/53), and at 1 (6/53), 2 (4/53) and 3 months (1/25) post HSCT, respectively. However, no clinical manifestation correlated with the presence of B19V viremia. All B19V sequences were of genotype 1. None of the sera investigated contained PARV4 or HBoV1 DNAs. Our data demonstrate B19V viremia to be frequent among pediatric allogeneic HSCT recipients, yet without apparent clinical correlates. PARV4 or HBoV1 viremias were not seen in these immunocompromised patients.

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 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.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

We report 4 children with encephalitis associated with human bocavirus (HBoV) 1 or 2. All children were severely underweight, and 2 died; 1 of them had a matching HBoV2 nucleotide sequence isolated from serum and bocavirus like particles in the cerebrospinal fluid that were observed with electron microscopy. No further pathogens were detected in the cerebrospinal fluid of these patients.

Human bocavirus 1 (HBoV1) DNA is frequently detected in the upper airways of young children with respiratory symptoms. Because of its persistence and frequent co-detection with other viruses, however, its etiologic role has remained controversial. During 2009-2011, using HBoV1 IgM, IgG, and IgG-avidity enzyme immunoassays and quantitative PCR, we examined 1,952 serum samples collected consecutively at 3- to 6-month intervals from 109 constitutionally healthy children from infancy to early adolescence. Primary HBoV1 infection, as indicated by seroconversion, appeared in 102 (94%) of 109 children at a mean age of 2.3 years; the remaining 7 children were IgG antibody positive from birth. Subsequent secondary infections or IgG antibody increases were evident in 38 children and IgG reversions in 10. Comparison of the seroconversion interval with the next sampling interval for clinical events indicated that HBoV1 primary infection, but not secondary immune response, was significantly associated with acute otitis media and respiratory illness.

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., (Copyright © 2011 Wiley Periodicals, Inc.)

Objectives: Human bocavirus (HBoV) is frequently identified in children with respiratory tract infections, and its role in acute otitis media (AOM) has been suggested. The disease associations for the closely related bocaviruses HBoV2-4 remain unknown. Increasing evidence shows that probiotics may reduce the risk of AOM of viral origin. Objectives of the study was to examine the prevalence and persistence of bocaviruses in consecutive nasopharyngeal samples (NPS) of otitis-prone children, and whether an association exists between HBoV and the child's characteristics, respiratory symptoms, and AOM pathogens, and whether probiotics reduce the occurrence of HBoV., Methods: In a double-blind, placebo-controlled, randomized, 6-month intervention study, 269 otitis-prone children (aged 9 months to 5.6 years), consumed daily either one capsule of probiotics (Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Bifidobacterium breve 99 and Propionibacterium freudenreichii JS) or placebo. After a clinical examination and NPS collected at three-time points, the presence and persistence of HBoV1-4 DNA in NPS was determined by RT-qPCR at the baseline, after 3, and 6 months., Results: A high load (>10,000 copies/ml) of HBoV DNA was detected in 26 (17.1%) of 152 children, and 16 (10.5%) showed a prolonged presence of HBoV for at least 3 months. None had DNA of HBoV2-4. Higher number of siblings associated with increased HBoV prevalence (p=0.029). Prevalence or persistence of HBoV was not significantly associated with other characteristics, respiratory symptoms, or AOM pathogens. Probiotic intervention significantly reduced the number of HBoV DNA-positive samples (probiotic vs. placebo: 6.4% vs. 19.0%, OR=0.25, CI 95%=0.07-0.94, p=0.039)., Conclusions: HBoV, but not HBoV2-4, DNA occurs often in the nasopharynx of otitis-prone children, and may persist for 3-6 months. Probiotic treatment possibly reduced the presence of HBoV., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The disease spectrum associated with human bocavirus-1 infection remains to be fully defined. We report a case of bocavirus-1-associated bronchiolitis, leading to severe respiratory failure and extracorporeal membrane oxygenation in a 4-year-old child, and suggest blood testing for human bocavirus-1 in children with severe respiratory tract infection.

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.

Four species of human bocavirus (HBoV) have been recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae). Although detected both in respiratory and stool samples worldwide, HBoV1 is predominantly a respiratory pathogen, whereas HBoV2, HBoV3, and HBoV4 have been found mainly in stool. A variety of signs and symptoms have been described in patients with HBoV infection including rhinitis, pharyngitis, cough, dyspnea, wheezing, pneumonia, acute otitis media, fever, nausea, vomiting, and diarrhea. Many of these potential manifestations have not been systematically explored, and they have been questioned because of high HBoV co-infection rates in symptomatic subjects and high HBoV detection rates in asymptomatic subjects. However, evidence is mounting to show that HBoV1 is an important cause of lower respiratory tract illness. The best currently available diagnostic approaches are quantitative PCR and serology. This concise review summarizes the current clinical knowledge on HBoV species., (Copyright © 2011 John Wiley & Sons, Ltd.)

Human bocavirus (HBoV), discovered in 2005, can cause respiratory disease or no symptoms at all. We confirmed HBoV infection in an 8-month-old girl with hypoxia, respiratory distress, wheezing, cough, and fever. This case demonstrates that lower respiratory tract infection caused by HBoV can lead to severe and life-threatening disease.

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.

The efficacy of prednisolone in acute wheezing associated with human bocavirus infection was investigated in 232 hospitalized children (median age, 1.6 years). Clinical history, atopy status, and viral etiology were carefully studied. Outcomes included hospitalization time, duration of symptoms, and occurrence of relapses. No efficacy of prednisolone was found in children with serologically confirmed acute human bocavirus infection.

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.

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.