B cell immune response to human bocaviruses.

BACKGROUND: Human bocaviruses (HBoVs) have been demonstrated in respiratory and gastrointestinal infections; however, the immune response to them has not been studied in detail. In this study, we investigated the B cell immune responses to HBoV1 and HBoV2, representing two different species of bocaviruses in humans. METHODS: We analyzed the effects of stimulations with HBoV1 and 2 virus-like particles (VLPs) and of co-stimulation with HBoV1-rhinovirus (RV) on cells of the immune system by flow cytometry, transcriptomics, and luminometric immune assays. RESULTS: Human B cells, and particularly B regulatory cells (Breg cells), showed an increased immune response to HBoV1-VLPs stimulation. These immune responses were also supported by increased IL-1RA and PDL1 expressions in IL-10+ B cells from peripheral blood mononuclear cells (PBMCs) stimulated with HBoV1-VLPs. In addition, increased levels of IL-10 and IL-1RA were determined in the supernatants of PBMCs following HBoV1-VLPs stimulation. HBoV1-VLPs and RV co-stimulation increased the IL-10+ B cell population. Transcriptome analysis by next-generation RNA sequencing showed an increased expression of IL-10 signalling and Breg cell markers in PBMCs stimulated with HBoV1-VLPs. Furthermore, TGF-ß and chemoattractants MIP-1α, MIP-1ß and IP10 protein levels were high in the supernatants of PBMCs stimulated with HBoV1-VLPs. CONCLUSIONS: The findings demonstrate that in Breg cells, IL-10 signalling pathways, and anti-inflammatory activity are induced by HBoV1, which can explain the often mild nature of the disease. In addition, the immune regulatory response induced by HBoV1-VLPs may indicate a potential immunomodulatory role of HBoV1 on the immune system and may represent an immune regulatory strategy.

Cellular vimentin promotes the nuclear transport of the HBoV1 structural protein VP1u.

Human bocavirus 1 (HBoV1) is an important pathogen causing lower respiratory tract infection. The VP1 unique region (VP1u), consisting of 129 amino acids at the N-terminus of the HBoV1 structural protein VP1, is an important component of virus infection. Bioinformatics analysis predicted that HBoV1 VP1u exhibits two bipartite nuclear localization signals (NLS) and contains four basic regions (BRs). The two potential bipartite NLSs consist of BR2 and 3 and BR3 and 4, respectively. In this study, we inserted the truncated vp1u sequences into a double EGFP fusion expression vector and confirmed the vimentin (VIM)-HBoV1 VP1u interaction by mass spectrometry and immunoprecipitation. The results of our IFA analysis showed that all four VP1u BRs displayed strong nuclear transport functions. We further demonstrated that VP1u interacted with VIM and that they colocalized in the cytoplasm. VP1u expression in the cells enhanced the VIM expression, and the VP1u expression also increased upon VIM overexpression, although it was not affected by VIM knockdown. Upon VIM overexpression, VP1u nucleation was significantly enhanced, but was inhibited by VIM downregulation. These results indicate that the VP1u-VIM interaction could be involved in the nuclear transport of VP1u. VP1u nucleation might further affect HBoV1 replication and infection. This study could potentially help clarify the function of VP1u by further revealing the HBoV1 nuclear transport mechanism and provide a new approach for elucidating the molecular mechanism of HBoV1 replication.

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.

The 5' Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation.

The capsid mRNA transcripts of human bocavirus 1 (HBoV1) can be generated by alternative splicing from the mRNA precursor transcribed from the P5 promoter. However, the alternative translation regulation mechanism of capsid mRNA transcripts is largely unknown. Here we report that the polycistronic capsid mRNA transcripts encode VP1, VP2, and VP3 in vitro and in vivo The 5' untranslated regions (UTRs) of capsid mRNA transcripts, which consist of exons, affected not only the abundance of mRNA but also the translation pattern of capsid proteins. Further study showed that exons 2 and 3 were critical for the abundance of mRNA, while exon 4 regulated capsid translation. Alternative translation of capsid mRNA involved a leaky scan mechanism. Mutating the upstream ATGs (uATGs) located in exon 4 resulted in more mRNA transcripts polyadenylated at the proximal polyadenylation [(pA)p] site, leading to increased capsid mRNA transcripts. Moreover, uATG mutations induced more VP1 expression, while VP3 expression was decreased, which resulted in less progeny virus production. Our data show that the 5' UTR of HBoV1 plays a critical role in the modulation of mRNA abundance, alternative RNA processing, alternative translation, and progeny virus production.IMPORTANCE Alternative translation of HBoV1 capsid mRNAs is vital for the viral life cycle, as capsid proteins perform essential functions in genome packaging, assembly, and antigenicity. The 5' untranslated regions (UTRs) of capsid mRNAs are generated by alternative splicing, and they contain different exons. Our study shows that the 5' UTR not only modulates mRNA abundance but also regulates capsid expression. Two upstream ATGs (uATGs) that were upstream of the capsid translation initiation site in the 5' UTR were found to affect viral capsid mRNA polyadenylation, alternative translation, and progeny virus production. The results reveal that uATGs play an important role in the viral life cycle and represent a new layer to regulate HBoV1 RNA processing, which could be a target for gene therapy.

Nonstructural Protein NP1 of Human Bocavirus 1 suppresses the growth of A549 cell by promoting autophagy.

Human bocavirus 1 (HBoV1) refers to a human parvovirus causing acute respiratory tract infection in children. Bocaviruses encode an NP1 protein, which has 47% amino acid homology with NP1 of Minute Virus of Canines (MVC) and Bovine Parvovirus (BPV), but not with any protein of other parvoviruses. NP1 was found to induce apoptosis in Hela cells, which does not depend on viral replication and other protein expression. However, whether NP1 induces pulmonary cell death is unclear. In the present study, we investigate the impacts of NP1 on the autophagy and viability of A549 cells by expressing NP1. The plasmid containing NP1 gene was transfected into A549 cells. The apoptosis of A549 was evaluated by apoptosis detection kit and expression of caspase3. Cell viability and cell migration were detected by CCK8 kit and cell scratch test, respectively. The autophagy-related proteins and HMGB1 were detected by Western blot after NP1 expression in transfected cells. The real-time PCR was employed to detect HMGB1 mRNA. The secretory HMGB1 in supernatant of cell culture was measured by ELISA kit. The transient expression of NP1 did not induce apoptosis in A549 cells, but inhibited cell viability and migration. The expression of Beclin1 and LC3 II increased significantly and that of autophagy substrate P62 decreased dramatically upon transfection of NP1. The expression of NP1 reduced both levels of mRNA and protein HMGB1. The NP1 induced A549 autophagy was activated by STAT3 signaling pathway. HBoV1 NP1 induced autophagy in A549 cells by activating phosphorylation of STAT3 signaling pathway and inhibited A549 cell viability. This study provides insight into further elucidating the replication mechanism of HBoV1.

T84 Monolayer Cell Cultures Support Productive HBoV and HSV-1 Replication and Enable In Vitro Co-Infection Studies.

Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have been confirmed as human pathogens. While several cell lines support the growth of HSV-1, HBoV-1 was exclusively cultivated on air-liquid interface cultures, the latter being a rather complicated, slow, and low throughput system. One of the cell lines are T84 cells, which are derived from the lung metastasis of a colorectal tumor. In this study, we provide evidence that T84 also supports HBoV replication when cultivated as monolayers, while simultaneously being permissive for HSV-1. The cell culture model thus would enable co-infection studies of both viruses and is worth being optimized for high throughput studies with HBoV-1. Additionally, the study provides evidence for a supporting effect of HSV-1 on the replication and packaging of HBoV-1 progeny DNA into DNase-resistant viral particles.

HBoV-1: virus structure, genomic features, life cycle, pathogenesis, epidemiology, diagnosis and clinical manifestations.

The single-stranded DNA virus known as human bocavirus 1 (HBoV-1) is an icosahedral, linear member of the Parvoviridae family. In 2005, it was discovered in nasopharyngeal samples taken from kids who had respiratory tract illnesses. The HBoV genome is 4.7-5.7 kb in total length. The HBoV genome comprises three open-reading frames (ORF1, ORF2, and ORF3) that express structural proteins (VP1, VP2, and VP3), viral non-coding RNA, and non-structural proteins (NS1, NS1-70, NS2, NS3, and NP1) (BocaSR). The NS1 and NP1 are crucial for viral DNA replication and are substantially conserved proteins. Replication of the HBoV-1 genome in non-dividing, polarized airway epithelial cells. In vitro, HBoV-1 infects human airway epithelial cells that are strongly differentiated or polarized. Young children who have HBoV-1 are at risk for developing a wide range of respiratory illnesses, such as the common cold, acute otitis media, pneumonia, and bronchiolitis. The most common clinical symptoms are wheezing, coughing, dyspnea, and rhinorrhea. After infection, HBoV-1 DNA can continue to be present in airway secretions for months. The prevalence of coinfections is considerable, and the clinical symptoms can be more severe than those linked to mono-infections. HBoV-1 is frequently detected in combination with other pathogens in various reports. The fecal-oral and respiratory pathways are more likely to be used for HBoV-1 transmission. HBoV-1 is endemic; it tends to peak in the winter and spring. This Review summarizes the knowledge on HBoV-1.

Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System.

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)-insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.

Establishment of a Recombinant AAV2/HBoV1 Vector Production System in Insect Cells.

We have previously developed an rAAV2/HBoV1 vector in which a recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged into a human bocavirus 1 (HBoV1) capsid. Recently, the production of rAAV2/HBoV1 in human embryonic kidney (HEK) 293 cells has been greatly improved in the absence of any HBoV1 nonstructural proteins (NS). This NS-free production system yields over 16-fold more vectors than the original production system that necessitates NS expression. The production of rAAV with infection of baculovirus expression vector (BEV) in the suspension culture of Sf9 insect cells is highly efficient and scalable. Since the replication of the rAAV2 genome in the BEV system is well established, we aimed to develop a BEV system to produce the rAAV2/HBoV1 vector in Sf9 cells. We optimized the usage of translation initiation signals of the HBoV1 capsid proteins (Cap), and constructed a BEV Bac-AAV2Rep-HBoV1Cap, which expresses the AAV2 Rep78 and Rep52 as well as the HBoV1 VP1, VP2, and VP3 at the appropriate ratios. We found that it is sufficient as a trans helper to the production of rAAV2/HBoV1 in Sf9 cells that were co-infected with the transfer Bac-AAV2ITR-GFP-luc that carried a 5.4-kb oversized rAAV2 genome with dual reporters. Further study found that incorporation of an HBoV1 small NS, NP1, in the system maximized the viral DNA replication and thus the rAAV2/HBoV1 vector production at a level similar to that of the rAAV2 vector in Sf9 cells. However, the transduction potency of the rAAV2/HBoV1 vector produced from BEV-infected Sf9 cells was 5-7-fold lower in polarized human airway epithelia than that packaged in HEK293 cells. Transmission electron microscopy analysis found that the vector produced in Sf9 cells had a high percentage of empty capsids, suggesting the pseudopackage of the rAAV2 genome in HBoV1 capsid is not as efficient as in the capsids of AAV2. Nevertheless, our study demonstrated that the rAAV2/HBoV1 can be produced in insect cells with BEVs at a comparable yield to rAAV, and that the highly efficient expression of the HBoV1 capsid proteins warrants further optimization.

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes.

Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells. Despite a production yield of 1-2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%-70% of that produced using pAdHelper. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3-7 times higher than that using pAdHelper. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.

Detection of human bocavirus-1 in both nasal and stool specimens from children under 5 years old with influenza-like illnesses or diarrhea in Gabon.

OBJECTIVE: Human bocavirus (HBoV) is a viral pathogen which causes respiratory tract diseases and acute gastroenteritis worldwide. This virus mainly affected children under 5 years old. There is little information on HBoV in Gabon. Two first studies was conducted to determine the prevalence of respiratory and enteric viruses in children under 5 years old who visited health centers for influenza-like illness (ILI) or diarrhea in Gabon from March 2010 to June 2011. However, HBoV was not included in the screening. The aim of this retrospective study was to evaluate the prevalence and the HBoV genotype in children under 5 years old with ILI or diarrhea in Gabon. RESULTS: A total of 810 nasal swabs and 317 feces samples collected during the two first study were analyzed among which 32 (4.4%) and 7 (2.2%) were positive for HBoV respectively. While there were no significant differences in prevalence between age groups in children with ILI, all children with diarrhea were under 12 months of age. Moreover, 84.4 and 42.8% were diagnosed in co-infections with at least one other respiratory virus, or enteric viruses respectively. Finally, HBoV subtype 1 has been detected in both respiratory and gastrointestinal tracts with very low variability.

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes.

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

Complementation for an essential ancillary non-structural protein function across parvovirus genera.

Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.

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/cm(3). 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.

Effect of alum on immune response in mice induced by HBoV1 VP2 VLPs / 中国免疫学杂志

Objective:To study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs.Methods:BABL/c mice were randomly divided into VLPs experimental group, alum adjuvant experimental group, PBS control group and alum adjuvant control group,the experimental group mice were intramuscular immunization with HBoV1 VP2 VLPs and HBoV1 VP2 VLPs added alum,control group mice were immunization with alum or PBS buffer,then to study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs by cellular and humoral immune strength.Results: Alum adjuvant decreased cellular immune response induced by HBoV1 VP2 VLPs(P<0.001),enhance the HBoV1 VP2 VLPs immuned serum IgG titer(P<0.05)and IgG activity(P<0.01).Conclusion:Alum adjuvant can enhance humoral immune response induced by HBoV1 VP2 VLPs,but weaken cellular immune response induced by HBoV1 VP2 VLPs,when HBoV1 VP2 VLPs used as a prophylactic vaccine it should add alum adjuvant,when used as a therapeutic vaccine,it should not add alum adjuvant.