Structure of Parvovirus B19 Decorated by Fabs from a Human Antibody.

Parvovirus B19, one of the most common human pathogens, is a small DNA virus that belongs to the Parvoviridae As a result of previous infections, antibodies to B19 are present in most adults. B19 has a strong tropism to erythroid progenitor cells and is able to cause a series of medical conditions, including fifth disease, arthritis, myocarditis, hydrops fetalis, and aplastic crisis. No approved vaccine is currently available for B19, and there is a lack of structural characterization of any B19 epitopes. Here we present the first cryo-electron microscopy (cryo-EM) structure of a B19 virus-like particle (VLP) complexed with the antigen-binding fragment (Fab) of a human neutralizing antibody, 860-55D. A model was built into the 3.2-Å-resolution map, and the antigenic residues on the surface of the B19 capsid were identified. Antibody 860-55D bridges the capsid of B19 by binding to a quaternary structure epitope formed by residues from three neighboring VP2 capsid proteins.IMPORTANCE Parvovirus B19 is a common human pathogen and a particular threat to children, pregnant women, and patients with sickle cell disease or AIDS. Currently, neutralizing antibody is the most efficient treatment for acute B19 infections. Research on the antigenic properties of B19 will guide the usage of these antibodies and facilitate vaccine development. We have determined and report here the high-resolution structure of B19 virus-like particles (VLPs) complexed with the Fab of a human neutralizing antibody. The structure shows a quaternary structure epitope formed by three VP2 proteins and provides details on host recognition of human B19 virus.

Antibodies against human parvovirus B19 in pregnant women in Ogbomoso, Oyo State Nigeria.

Infection caused by Human parvovirus B19 (HPVB19) during pregnancy has been associated with adverse effects on fetus such as intrauterine fetal death but there is dearth information about the seroprevalence and risk factors among pregnant women. This study was carried out to determine the incidence of specific IgM antibodies to HPVB19 among pregnant women attending Bowen University Teaching Hospital, Ogbomoso, Oyo State. A total of 185 participants, in the age range 18-49 years, who consented voluntarily after thorough explanation of the purpose of the study were recruited for the study. Specific IgM antibodies were detected using commercially available third-generation ELISA kits. Data on socio-demographic characteristics and potential risk factors were collected using structured questionnaires. Chi-square (χ2) test was utilized to assess the association between the socio-demographic variables and HPVB19 status (P Ë‚ 0.05). Logistic regression was done to determine the strength of association between the potential risk factors and HPVB19 status (P Ë‚ 0.05). The overall incidence of IgM antibody recorded was 2.70% (5/185). Pregnant women in the age group 36-45 (7.7%) years had the highest record. None of the socio-demographic characteristics and risk factor considered were significantly associated with acquiring HPVB19. However, a one-fold risk was observed among those who had previous history of blood transfusion (95% CI = 0.949-0.997, OR = 1.0) The findings in this study confirmed the presence of antibodies to HPVB19 among pregnant women in Ogbomoso. Public awareness, enlightenment, and screening for the virus should be encouraged to prevent fetal complications during pregnancy.

Parvovirus B19 uptake is a highly selective process controlled by VP1u, a novel determinant of viral tropism.

The VP1 unique region (VP1u) of human parvovirus B19 (B19V) is the immunodominant part of the viral capsid. Originally inaccessible, the VP1u becomes exposed upon primary attachment to the globoside receptor. To study the function of the exposed VP1u in B19V uptake, we expressed this region as a recombinant protein. Here, we report that purified recombinant VP1u binds and is internalized in UT7/Epo cells. By means of truncations and specific antibodies, we identified the most N-terminal amino acid residues of VP1u as the essential region for binding and internalization. Furthermore, the recombinant VP1u was able to block B19V uptake, suggesting that the protein and the virus undertake the same internalization pathway. Assays with different erythroid and nonerythroid cell lines showed that the N-terminal VP1u binding was restricted to a few cell lines of the erythroid lineage, which were also the only cells that allowed B19V internalization and infection. These results together indicate that the N-terminal region of VP1u is responsible for the internalization of the virus and that the interacting receptor is restricted to B19V-susceptible cells. The highly selective uptake mechanism represents a novel determinant of the tropism and pathogenesis of B19V.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

INTRODUCTION: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. OBJECTIVES: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. METHODS: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. RESULTS: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. CONCLUSION: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.

The globoside receptor triggers structural changes in the B19 virus capsid that facilitate virus internalization.

Globoside (Gb4Cer), Ku80 autoantigen, and α5ß1 integrin have been identified as cell receptors/coreceptors for human parvovirus B19 (B19V), but their role and mechanism of interaction with the virus are largely unknown. In UT7/Epo cells, expression of Gb4Cer and CD49e (integrin alpha-5) was high, but expression of Ku80 was insignificant. B19V colocalized with Gb4Cer and, to a lesser extent, with CD49e. However, only anti-Gb4Cer antibodies could disturb virus attachment. Only a small proportion of cell-bound viruses were internalized, while the majority became detached from the receptor. When added to uninfected cells, the receptor-detached virus showed superior cell binding capacity and infectivity. Attachment of B19V to cells triggered conformational changes in the capsid leading to the accessibility of the N terminus of VP1 (VP1u) to antibodies, which was maintained in the receptor-detached virus. VP1u became similarly accessible to antibodies following incubation of B19V particles with increasing concentrations of purified Gb4Cer. The receptor-mediated exposure of VP1u is critical for virus internalization, since capsids lacking VP1 could bind to cells but were not internalized. Moreover, an antibody against the N terminus of VP1u disturbed virus internalization, but only when present during and not after virus attachment, indicating the involvement of this region in binding events required for internalization. These results suggest that Gb4Cer is not only the primary receptor for B19V attachment but also the mediator of capsid rearrangements required for subsequent interactions leading to virus internalization. The capacity of the virus to detach and reattach again would enhance the probability of productive infections.

Immunohistochemical detection of parvovirus B19 in "gloves and socks" papular purpuric syndrome: direct evidence for viral endothelial involvement. Report of three cases and review of the literature.

Papular-purpuric "gloves and socks" syndrome is a distinctive dermatosis featuring acral pruritus, edema, and petechiae. It has been attributed in most--but not all--reported cases to Parvovirus B19 infection, on the grounds of serological proof of recent infection or detection of viral DNA by polymerase chain reaction in patient serum or biopsies. We report the immunohistochemical detection of Parvovirus B19 VP2 structural protein in the endothelial lining of dermal blood vessels in 3 examples of Papular-purpuric "gloves and socks" syndrome and review previously described immunohistochemical investigations in cutaneous involvement by this infection.

The Effect of a Unique Region of Parvovirus B19 Capsid Protein VP1 on Endothelial Cells.

Parvovirus B19 (B19V) is a widespread human pathogen possessing a high tropism for erythroid precursor cells. However, the persistence or active replication of B19V in endothelial cells (EC) has been detected in diverse human pathologies. The VP1 unique region (VP1u) of the viral capsid has been reported to act as a major determinant of viral tropism for erythroid precursor cells. Nevertheless, the interaction of VP1u with EC has not been studied. We demonstrate that recombinant VP1u is efficiently internalized by rats' pulmonary trunk blood vessel-derived EC in vitro compared to the human umbilical vein EC line. The exposure to VP1u was not acutely cytotoxic to either human- or rat-derived ECs, but led to the upregulation of cellular stress signaling-related pathways. Our data suggest that high levels of circulating B19V during acute infection can cause endothelial damage, even without active replication or direct internalization into the cells.

Visualization of the externalized VP2 N termini of infectious human parvovirus B19.

The structures of infectious human parvovirus B19 and empty wild-type particles were determined by cryoelectron microscopy (cryoEM) to 7.5-A and 11.3-A resolution, respectively, assuming icosahedral symmetry. Both of these, DNA filled and empty, wild-type particles contain a few copies of the minor capsid protein VP1. Comparison of wild-type B19 with the crystal structure and cryoEM reconstruction of recombinant B19 particles consisting of only the major capsid protein VP2 showed structural differences in the vicinity of the icosahedral fivefold axes. Although the unique N-terminal region of VP1 could not be visualized in the icosahedrally averaged maps, the N terminus of VP2 was shown to be exposed on the viral surface adjacent to the fivefold beta-cylinder. The conserved glycine-rich region is positioned between two neighboring, fivefold-symmetrically related VP subunits and not in the fivefold channel as observed for other parvoviruses.

Decoration of virus-like particles with an enzymatic activity of biomedical interest.

The natural properties of virus-like particles (VLPs), like their nanometric size, polyvalence, monodispersity and biocompatibility, had called the attention of scientists from different fields. VLPs constitute an excellent platform for the development nanomaterials with a broad spectrum of applications, ranging from physics of soft matter to the development of vaccines and biological nanocarriers. To expand the repertoire of functions of VLPs, they can be decorated with different molecules. In this research, the α-glucosidase Ima1p of Saccharomyces cerevisiae was attached to the surface of in vitro assembled VLPs of parvovirus B19, by using the SpyTag/SpyCatcher system. The resulting particles were structurally characterized displaying a noticeable increase in size compared to the non-decorated VLPs. The study of the biochemical properties of the coupled enzyme indicate that it increased its Vmax by three-fold toward p-nitrophenyl-α-D-glucopyranoside (p-NPG) as substrate. In addition, the linked enzyme displayed a notorious 10 °C shift in its optimal temperature, from 35 °C for the non-attached enzyme, to 45 °C for the enzyme attached to VLPs. The decorated VLPs were also able to act on glycogen; therefore, these particles may be further developed as part of the therapy for treatment of lysosomal storage diseases derived from defects in the human acid α-glucosidase.

Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines.

Peptide presentation on primate erythroparvovirus 1 virus-like particles: In vitro assembly, stability and immunological properties.

Virus-like particles (VLPs) have demonstrated to be valuable scaffolds for the display of heterologous peptides for vaccine development and other specific interactions. VLPs of primate erythroparvovirus 1, generally referred as parvovirus B19 (B19V), have already been produced in-vivo and in-vitro from the recombinant VP2 protein of this virus. In this study, chimeric forms of B19V VP2 were constructed, and their ability to assemble into VLPs was evaluated. Chimeras were composed of the VP2 protein fused, at its N-terminus, with two peptides derived from the fusion glycoprotein (F) of the respiratory syncytial virus (RSV). The chimeric proteins self-assembled into VLPs morphologically similar to B19V virions. Stability of these VLPs was analyzed under denaturation conditions with guanidinium chloride (GdnHCl). Our results indicate that the presence of the heterologous fragments increased the stability of VLPs assembled by any of the VP2 chimeras. Specific proteolysis assays shown that a fraction of the N-termini of the chimeric proteins is located on the outer surface of the VLPs. Immunogenicity of VLPs against RSV was evaluated and the results indicate that the particles can elicit a humoral immune response, although these antibodies did not cross-react with RSV in ELISA tests. These results provide novel insights into the localization of the N-termini of B19V VP2 protein after in vitro assembly into VLPs, and point them to be attractive sites to display peptides or proteins without compromise the assembly or stability of VLPs.

In vitro encapsulation of heterologous dsDNA into human parvovirus B19 virus-like particles.

Virus-like particles (VLPs) have vast potential for applications in nanoscience and nanomedicine. These biological nanoparticles may be used for medical imaging, vaccination, or tissue-specific delivery of drugs or other bioactive molecules. VLPs of Human parvovirus B19 (B19 V) can be assembled in vitro from the recombinant VP2 protein. In this research, we describe a simple method for the encapsulation of heterologous linear dsDNA fragments of different sizes into B19 V-VP2 VLPs, in which the DNA and denatured VP2 protein are co-incubated and the assembly process is conducted by one dialysis step. Characterization of the particles by qPCR demonstrated the encapsulation of dsDNA, and indicates that the length of the dsDNA is critical for the encapsulation process. The strategy presented here opens the possibility to use this VLPs as a delivery system with future therapeutically applications.

Sensitive single-stage PCR using custom-synthesized internal controls.

A new approach for an internally controlled PCR was developed using a custom-synthesized oligonucleotide as the internal control. Three different PCR setups demonstrated the usefulness of this approach: (i) the addition of the respective internal control to samples containing ssDNA virus Parvo B19; (ii) the co-extraction of plasma samples and the respective internal control for the detection of the ssDNA virus TTV; and (iii) the addition of the respective internal control to a crude lysate of tail pieces for the genotyping of FVIII knockout mice, demonstrating that this approach is also applicable for dsDNA.

Persistent erythrovirus B19 urinary tract infection in an HIV-positive patient.

We report a urinary tract infection (UTI) with erythrovirus B19 in an HIV-1-positive homosexual man persisting for more than 7 months after the decline of viremia after a primary infection. During the course of the UTI, the patient complained of soreness in the kidney region and suffered from transient episodes of edema and hematuria. Proteinuria and elevated serum concentrations of creatinine further substantiated the hypothesis of a renal focus of a persistent erythrovirus B19 infection.

Strengths and limitations of the model virus concept.

New plasma- or cell culture-based pharmaceutical manufacturing processes must be validated for their ability to eliminate potentially contaminating pathogens. To evaluate the virus elimination potential of such a process, current guidelines propose the use of model viruses. This approach is discussed based on two examples. These examples show the strengths of this approach but also its limitations. The blood processing industry was recently challenged by the emergence of a West Nile Virus (WNV) epidemic in the United States. The susceptibility of WNV and a frequently used model virus to commonly used inactivation methods is compared. Current data show a good correlation. Due to its physico-chemical properties and the high viremic titers, B19 virus (B19V), a small (diameter 18-26 nm), robust, non-enveloped parvovirus, is a considerable challenge for the plasma processing industry. Mice minute virus (MMV), an animal parvovirus, is used as a model for B19V. Data show that B19V is considerably more susceptible to some physico-chemical inactivation methods than MMV. The examples of WNV and B19V show that the model virus concept is a practicable tool to evaluate the safety of plasma- or cell culture-derived pharmaceuticals regarding known and emerging viruses. It also underlines the need for investigational studies of relevant viruses if they can be handled in a normal virology laboratory, under moderate biosafety conditions.

The human parvovirus B19 non-structural protein 1 N-terminal domain specifically binds to the origin of replication in the viral DNA.

The non-structural protein 1 (NS1) of human parvovirus B19 plays a critical role in viral DNA replication. Previous studies identified the origin of replication in the viral DNA, which contains four DNA elements, namely NSBE1 to NSBE4, that are required for optimal viral replication (Guan et al., 2009). Here we have demonstrated in vitro that the NS1 N-terminal domain (NS1N) binds to the origin of replication in a sequence-specific, length-dependent manner that requires NSBE1 and NSBE2, while NSBE3 and NSBE4 are dispensable. Mutagenesis analysis has identified nucleotides in NSBE1 and NSBE2 that are critical for NS1N binding. These results suggest that NS1 binds to the NSBE1-NSBE2 region in the origin of replication, while NSBE3 and NSBE4 may provide binding sites for potential cellular factors. Such a specialized nucleoprotein complex may enable NS1 to nick the terminal resolution site and separate DNA strands during replication.

Effects of human Parvovirus B19 and Bocavirus VP1 unique region on tight junction of human airway epithelial A549 cells.

As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity.

Human parvovirus B19 virus-like particles: In vitro assembly and stability.

Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37°C, and a significant fraction of particles remain assembled after 30min at 80°C.