Genus Bocavirus Research Articles

He who seeks finds: surveillance of Enteroviruses and Human Bocaviruses in Northern Italy wastewaters

Abstract Background Wastewaters surveillance has proven to be a valuable tool in detecting the spread of SARS-CoV-2 and other pathogens. Enterovirus (EV) and Human bocavirus (HBoV) are viruses that can infect humans, causing mild to severe pathologies transmitted also through the fecal-oral route. EVs are ssRNA viruses, grouped among four taxonomic species EV-A to D. HBoV is ssDNA virus mainly affects infants. Genus Bocavirus includes 4 genotypes: HBoV1 to 4, detected mainly in feces. EVs and HBoVs infections are often not notified, causing loss of data on the actual incidence. The aim of this study was to investigate the presence and diffusion of EVs and HBoVs in wastewaters. Methods 200 wastewater samples were collected during 2022, at the inlet of wastewater treatment plants in three cities of the Lombardy Region (i.e.: Brescia, Bergamo, and Cremona). EVs and HBoVs were detected by RT Real-time PCR and Real-time PCR respectively using specific primers for target region of the viral genome. Positive samples were genotyped by Sanger sequencing. Prevalence was calculated with the Blaker's method. Results Overall EV and HBoV prevalences were 20.5% (CI95% 15.5-26.2%) and 37% (CI95%.30.6-43:9%), respectively. EV was mainly detected in the mild season with a prevalence of 68.3% (CI95% 53.0-80.4%). Sequence analysis reported that EV-B species was the most prevalent in this study (9 samples). HBoVs positive samples’ prevalence was higher in cold season (71.6% CI95% 60.5-80.6%), than mild season. HBoV2 showed higher prevalence (85.6% CI95% 75.6-92.0%) than HBoV3. Conclusions This study provided insight into the seasonal spread of EVs and HBoVs circulating and can contribute to understand the disease burden. Wastewaters surveillance provides a wide and more complete epidemiological data than the clinical one of a single person, rather providing information of a community of individuals making the data very valuable to the competent authorities. Key messages • Wastewaters surveillance is a useful tool for investigating the spread and circulation of viruses in population. • EVs and HBoVS are widespread in Northen Italy population.

ITRAQ-based high-throughput proteomics analysis reveals alterations of plasma proteins in patients infected with human bocavirus.

Human bocavirus (HBoV) is a member of the genus Bocavirus, family Parvoviridae, and subfamily Parvovirus and was first identified in nasopharyngeal aspirates of Swedish children with acute respiratory tract infection (ARTI) in 2005. It is the causative agent of nasopharyngeal aspirate disease and death in children. The HboV genomic structure is a linear single-stranded DNA (ssDNA). Its clinical pathogenic characteristics have been extensively studied, however, at present the molecular mechanism underlying the pathogenesis of HBoV infection is not completely clear. In this study, a total of 293 differentially expressed proteins (DEPs) between ARTI cases and healthy plasma samples were characterized using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled bioinformatics analysis, among which 148 were up-regulated and 135 were down-regulated. Gene Ontology (GO) and Cluster of Orthologous Groups of proteins (COG) annotated an enrichment of DEPs in complement activation and biological processes like immunity, inflammation, signal transduction, substance synthesis, and metabolism. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis enriched DEPs mainly in the Wnt signaling pathway (ko04310), PPAR signaling pathway (ko03320), intestinal immune network for IgA production (ko04672), complement and coagulation cascades (ko04610), Toll-like receptor signaling pathway (ko04620) and B cell receptor signaling pathway (ko04662). Further, expression levels of three candidate proteins (upregulated PPP2R1A and CUL1, and downregulated CETP) were validated using western blotting. Our investigation is the first analysis of the proteomic profile of HBoV-infected ARTI cases using the iTRAQ approach, providing a foundation for a better molecular understanding of the pathogenesis of ARTI in children.

Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing.

The NP1 protein of minute virus of canines (MVC) governs production of the viral capsid proteins via its role in pre-mRNA processing. NP1 suppresses polyadenylation and cleavage at its internal site, termed the proximal polyadenylation (pA)p site, to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent third intron, which is necessary to properly enter the capsid protein open reading frame. We find the (pA)p region to be complex. It contains redundant classical cis-acting signals necessary for the cleavage and polyadenylation reaction and splicing of the adjacent upstream third intron, as well as regions outside the classical motifs that are necessary for responding to NP1. NP1, but not processing mutants of NP1, bound to MVC RNA directly. The cellular RNA processing factor CPSF6 interacted with NP1 in transfected cells and participated with NP1 to modulate its effects. These experiments further characterize the role of NP1 in parvovirus gene expression.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Unlike other parvoviruses, the bocavirus genus controls expression of its capsid proteins via alternative RNA processing, by both suppressing polyadenylation at an internal site, termed the proximal polyadenylation (pA)p site, and by facilitating splicing of an upstream adjacent intron. This regulation is mediated by a small genus-specific protein, NP1. Understanding the cis-acting targets of NP1, as well as the cellular factors with which it interacts, is necessary to more clearly understand this unique mode of parvovirus gene expression.

The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.

Human bocavirus induces apoptosis and autophagy in human bronchial epithelial cells.

Human bocavirus (HBoV) is classified in the Bocavirus genus within the Parvoviridae family, first identified from children with respiratory diseases. Previous studies have investigated the stimulating effect of HBoV on cell apoptosis and autophagy. In the present study, human bronchial epithelial cells (HBECs) were utilized to examine the mechanism of HBoV recombination expressing vector (pWHL-1) on the promotion of cell apoptosis and autophagy. The results from the present study indicated that pWHL-1 inhibited the proliferation of HBECs in a time-dependent manner. Additionally, pWHL-1induced apoptosis, as substantiated by an increased apoptotic rate and presence of autophagosomes. Following pWHL-1 transfection, proliferating cell nuclear antigen, caspase-3 and B cell lymphoma 2 (Bcl-2) protein expression levels were decreased, with the exception of Bcl-2 associated × (Bax) protein, which increased. mRNA and protein expression levels of microtubule-associated protein 1A/1B-light chain 3 (LC3) II and autophagy protein 5 were increased in pWHL-1-transfected HBECs, whereas, the mRNA and protein levels of LC3I and sequestosome 1 were decreased. Notably, pWHL-1 also enhanced the activation of p53 and inhibited AKT activation in HBECs. Results from the present study suggest that pWHL-1 induces apoptosis and autophagy, thus providing a novel insight into the effect of HBoV and its uses in respiratory diseases.

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

Porcine bocaviruses (PBoVs), new members of the Bocavirus genus, have been identified in swine worldwide. However, the antigenicity and epidemiology of PBoVs are still unclear. Here we used a recombinant baculovirus expression system to express the main capsid protein VP2 of Japan strain JY31b in insect Tn5 cells, and successfully produced the virus-like particles of PBoV (PBoV-LPs). The diameter and densities of the PBoV-LPs were estimated to be 30nm and 1.300g/cm3, respectively, which were similar to the values for the native virion of PBoV. Antigenic analysis demonstrated that the PBoV-LPs were not cross-reactive with porcine circovirus 2, but were cross-reactive with human bocavirus 1, 2, 3 and 4. An ELISA for detection of anti-PBoV IgG antibodies was established using PBoV-LPs as antigen, which proved to be useful for monitoring PBoV infection in both swine and wild boars. The preliminary epidemiology research showed that 90.7% of pigs and 59.5% of wild boars were positive for the anti-PBoV-IgG, suggesting that both species were also widely infected with PBoV. The seven PBoV strains detected in wild boars separated into four subgroups, demonstrating the genetic diversity of PBoV.

Yeast-generated virus-like particles as antigens for detection of human bocavirus 1-4 specific antibodies in human serum.

Human bocaviruses (HBoV) are non-enveloped, single-stranded DNA viruses, classified into the genus Bocavirus in the family Parvoviridae. Self-assembled virus-like particles (VLPs) composed of the major capsid protein VP2 of HBoV1-4 and mosaic VLPs composed of both VP2 and VP1 capsid proteins of HBoV1 were generated in yeast Saccharomyces cerevisiae and used to detect HBoV-specific IgG in human serum. Recombinant HBoV VLPs were similar to native HBoV particles in size and morphology. The prevalence of HBoV infection in a group of Lithuanian patients with clinical symptoms of respiratory tract infection was studied using purified yeast-generated VLPs as antigens in a competitive enzyme immunoassay (EIA). After depletion of cross-reactive antibodies, the seroprevalence of HBoV1 was 44.2% and the seroprevalence of HBoV2-4 was 35.7%. Mosaic VLPs consisting of HBoV1 VP1 and VP2 proteins showed a stronger reactivity with HBoV1 IgG-positive human serum specimens, and two equivocal serum specimens were reinterpreted as positive. Thus, mosaic VLPs offer a more sensitive tool for HBoV1 serology than currently available serodiagnostics tests based on VP2 VLPs. In conclusion, yeast S. cerevisiae represents an efficient expression system for generating recombinant HBoV1-4 VLPs of diagnostic relevance.

Differential contributions of porcine bocavirus NP1 protein N- and C-terminal regions to its nuclear localization and immune regulation.

Porcine bocavirus (PBoV), a newly identified parvovirus in the family Parvoviridae, has been reported worldwide in swine with post-weaning multisystemic wasting syndrome, respiratory disease or diarrhoea and in asymptomatic swine. NP1 is a protein unique to the genus Bocavirus and its function is not fully understood. In this study, we show that the N-terminal region of PBoV NP1 contains two classical nuclear localization signals (cNLSs) and a non-classical NLS. The N-terminal region also inhibits the promoter activity of IFN-β and IFN-stimulated response element activity the same as full-length NP1 protein, but the PBoV NP1 C-terminal region does not. PBoV NP1 also induces NFκB activation by increasing the phosphorylation of p65, and we demonstrate that the C-terminal region (aa 168-218) is responsible for the induction of NFκB, although the cNLS region of NP1 enhances this activation. The data suggest that PBoV NP1 contains two functionally independent domains in its N- and C-terminal regions. Thus, the N-terminal region of PBoV NP1 is critical for its nuclear localization and IFN-related promoter inhibition, and the C-terminal region is critical for its induction of NFκB.

Porcine bocavirus: achievements in the past five years.

Porcine bocavirus is a recently discovered virus that infects pigs and is classified within the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae). The viral genome constitutes linear single-stranded DNA and has three open reading frames that encode four proteins: NS1, NP1, VP1, and VP2. There have been more than seven genotypes discovered to date. These genotypes have been classified into three groups based on VP1 sequence. Porcine bocavirus is much more prevalent in piglets that are co-infected with other pathogens than in healthy piglets. The virus can be detected using PCR, loop-mediated isothermal amplification, cell cultures, indirect immunofluorescence, and other molecular virology techniques. Porcine bocavirus has been detected in various samples, including stool, serum, lymph nodes, and tonsils. Because this virus was discovered only five years ago, there are still many unanswered questions that require further research. This review summarizes the current state of knowledge and primary research achievements regarding porcine bocavirus.

Characterization of human bocavirus-like particles generated by recombinant baculoviruses

Human bocavirus (HBoV) is a nonenveloped, single-stranded DNA virus, classified recently into the genus Bocavirus in the family Parvoviridae. A recombinant baculovirus expression system was used to express the major capsid protein VP2 of HBoV1, HBoV2, HBoV3 and HBoV4 in insect cells. A large amount of the 61-kDa VP2 capsid protein (p61) of HBoVs was generated and efficiently released into the supernatant. The capsid protein was self-assembled into 22-nm-dia. virus-like particles (VLPs) with a buoyant density of 1.30g/cm3. The morphology of HBoVs-LPs was similar to that of the native HBoV particles, and immunogenic studies demonstrated the cross-reactivity among HBoV1, HBoV2, HBoV3 and HBoV4. When VP1 and VP2 protein of HBoV1 were co-expressed in insect cells, both proteins were detected in the same fraction after CsCl gradient centrifugation, suggesting that the VP1 protein is a minor structural protein of HBoVs. We developed an ELISA using purified VLPs as the antigen and used it to detect antibodies against HBoV1, HBoV2, HBoV3 and HBoV4. A high prevalence of antibodies against HBoVs was found in a general population of healthy Japanese, indicating that HBoVs have spread throughout Japan.

ABSTRACT 694

Background and aims: Human Bocavirus (HBoV), belongs to the genus Bocavirus of the Parvoviridae family. 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. Here we report a child who developed a life-threatening HBoV infection. Methods: Case: Previously healthy two years old girl with acute respiratory distress was admitted to the emergency department. On admission, there was a respiratory rate 62 breaths/minute with an oxygen saturation of %89. She had roncuses and bilateral breath sounds were decreased with prolonged expiration. Parenteral antibiotics, steroid and inhalation of short acting beta-2 agonist were initiated. Chest radiography revealed bilateral infiltrates. She developed severe respiratory failure despite the treatment and managed in PICU with mechanically ventilation. Cultures of blood and bronchoalveolar lavage were negative for bacteria on the day of admission. A nasopharyngeal aspirate was only positive for HBoV by PCR. The girl was extubated on the sixth day and compeletly recovered on the tenth day of hospitalization. Conclusions: Infections with HBoV is typically characterized with mild upper respiratory tract disease. It may also causes lower respiratory tract infections. For a few patients who develop severe symptoms with HBoV, the prognosis may range from fair to poor, rarely leading to life-threatening conditions. We aimed to emphasize this virus may lead to respiratory failure and need for mechanically ventilation support.

Complete sequence and phylogenetic analysis of a porcine bocavirus strain swBoV CH437

Porcine bocavirus (PBoV), a member of genus Bocavirus, family Parvoviridae, was first identified in 2009 in Swedish swine herds suffering from postweaning multisystemic wasting syndrome. Up to date, the different species of PBoVs have been reported in different countries. Especially, the virus isolated in China was complicated. In this study, we detected a novel PBoV strain swBoV CH437 from clinical samples collected in Gansu Province, Northwest China. The complete genome of swBoV CH437 was 5,275 nucleotides (nt) in length and contains three ORFs: ORF1 encodes NS1 (2,004 nt, 667 aa), ORF3 encodes NP1 (681 nt, 226 aa), and ORF2 encodes VP1 (2,049 nt, 682 aa) and VP2 (1,641 nt, 546 aa). Sequence analysis demonstrated that the NS1 gene shared 24.2-88.6 % nucleotide sequence identity, the NP1 shared 21.3-89.9 %, less than 95 % nucleotide sequence identity with other PBoV strains. Therefore, we propose that swBoV CH437 should be classified as a novel PBoV species.

High prevalence and genetic diversity of porcine bocaviruses in pigs in the USA, and identification of multiple novel porcine bocaviruses

Viruses in the genus Bocavirus are associated with respiratory and enteric disease in dogs and cattle. In addition, novel porcine bocaviruses (PBoVs) have been identified in domestic and wild pigs in recent years, but are of unknown relevance to date. The objectives of this study were to determine the prevalence ra tes and genetic diversity of PBoVs in pigs in the USA. Using newly established multiplex real-time PCR assays, 385 lung, lymph node, serum and faecal samples from pigs with various disease conditions were investigated. A high PBoV prevalence rate ranging from 21.3 to 50.8 % was identified in the investigated samples and often two or more PBoV species were detected in the same sample. Cloning and sequencing analysis of the partial non-structural protein NS1 and the capsid proteins VP1 and VP2 of DNA samples positive for PBoV groups 1 (n = 6), 2 (n = 16) and 3 (n = 42), including subgroups 3A, 3B or 3C, revealed a high genetic diversity especially for the PBoV G3 VP2 gene, whereas the PBoV group 1 VP1 gene displayed a low nucleotide polymorphism. Using primer walking, 18 partial or nearly complete genomes of PBoVs were obtained and six of the 18 nearly complete genomes represented novel PBoV species. Recombination analysis using partial NS1, VP1 and VP2 genes and the nearly complete genomes indicated possible recombination events within and between PBoVs. Further studies will be required to reveal the possible pathogenic role of these diverse PBoVs.

The DNA replication, virogenesis and infection of canine minute virus in non-permissive and permissive cells

Canine minute virus (CnMV), a kind of autonomous parvovirus, is a member of genus bocavirus in parvovirdae family. In our previous study, we constructed and obtained infectious clones of CnMV, analyzed genome characteristics, RNA transcription profile, and revealed some molecular mechanisms of cytopathic effect of target cells. The purpose of this study was to investigate DNA replication, virogenesis and infectious tropism of CnMV in non-permissive and permissive cells. We demonstrated that the genomic DNA of CnMV, besides WRD cells, could replicate significantly in some non-permissive cells (CrFK, EBtR and COS-7) following transfection with infectious clone of CnMV, pI-MVC. Moreover, by using Western blotting and immunofluorescence, we found that the NS1 protein of CnMV was obviously expressed in both 293, CrFK, EBtR and COS-7 cells transfected with pI-MVC. Meanwhile, two-rounds of reinfection on WRD cells (blind passage) of the transfected cell lysates in CrFK, EBtR and COS-7cells tranfected with pI-MVC showed that pI-MVC could produce infectious virions in these types of non-permissive cells. Furthermore, it is confirmed that CnMV only infected WRD cells (permissive cells for CnMV), could not infect any non-permissive cells including CrFK, EBtR, COS-7, HK293, A549 and A9 cells. Taken together, for the first time, we have demonstrated that bocavirus CnMV DNA could replicate and form infectious progeny virus in some non-permissive cells. And what is more, unlike other parvoviruses, CnMV did not infect some non-permissive cells, although the DNA replication of CnMV occurred in these cells.

Human bocavirus 1 infects commercially available primary human airway epithelium cultures productively

Human bocavirus 1 (HBoV1), a human parvovirus, belongs to the genus Bocavirus of the Parvoviridae family. It causes wheezing in young children with acute respiratory tract infections. HBoV1 has been shown to infect polarized human airway epithelium (HAE) made in house, and induces airway epithelial damage. In this study, two commercially available HAE cultures, EpiAirway and MucilAir HAE, were examined for HBoV1 infection. Both HAE cultures support fully productive HBoV1 infection. Infected EpiAirway and MucilAir HAE cultures showed loss of cilia, disruption of the tight junction barrier, and a significant decrease in transepithelial electrical resistance. Notably, HBoV1 persistent infection was demonstrated by maintaining HBoV1-infected EpiAirway HAE for as long as 50 days. After 2 days post-infection, progeny virus was produced consistently daily at a level of over 2×108 viral genome copies per culture (0.6cm2). This study is the first to use commercial sources of HAE cultures for HBoV1 infection. The availability of these cultures will enable a wide range of laboratories to study HBoV1 infection.

Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7-50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14-28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7-50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

Genetic characterisation of a porcine bocavirus detected in domestic pigs in Uganda

Porcine bocaviruses (PoBoVs) are small linear ssDNA viruses belonging to the genus bocavirus in the family Parvoviridae. The genome encodes four proteins—the non-structural protein 1 (NS1), the NP1 protein (unknown function) and the two structural proteins VP1 and VP2. In recent years, a number of different highly divergent PoBoV species have been discovered. PoBoVs have been shown to be present in pig populations in Europe, Asia and in the United States of America. In this study, we present the first data of the presence of PoBoV in Africa, specifically in Uganda. A PCR targeting a PoBoV species that have previously been detected in both Sweden and China was used to screen 95 serum samples from domestic pigs in Uganda. Two pigs were found to be positive for this specific PoBoV and the complete coding region was amplified from one of these samples. The amino acid sequence comparison of all these proteins showed a high identity (98–99 %) to the published Chinese sequences (strains: H18 and SX) belonging to the same PoBoV species. The same was true for the Swedish sequences from the same species. To the other PoBoV species the divergence was higher and only a 28–43 % protein sequence identity was seen comparing the different proteins.

Host range of Canine minute virus in cell culture

Canine minute virus (CnMV) is a member of the Bocavirus genus, together with Bovine parvovirus (BPV), which emerged as a new pathogen of dogs in 1967. The genus Bocavirus gained more recognition after the recent identification of Human bocavirus in pooled specimens from the respiratory tract of children. The cell culture host range of CnMV appears to be restricted to the Walter Reed canine cell (WRCC) line, although there is a report that indicates susceptibility of the Madin-Darby canine kidney (MDCK) cell line. In order to study the susceptibility of different cell lines to CnMV, the replication in cell cultures of canine, bovine, and human origin and the interaction of the virus with freshly isolated canine peripheral blood mononuclear cells were evaluated. The in vitro host range was unexpectedly wide. As shown by indirect fluorescent antibody and polymerase chain reaction assays, CnMV replicates efficiently in the A72 and MDCK canine cell lines. Bovine and human cell lines support the replication of CnMV less efficiently. In contrast, canine mononuclear blood cells are permissive to replication of CnMV in vitro. The present study revealed the ability of CnMV to replicate in continuous cell lines of different origin and, partially, in canine mononuclear cells.

Seroepidemiology of Human Bocaviruses 1–4

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). 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). 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. 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.

Human bocavirus—the first 5 years

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.