B19 Proteins Research Articles

Human Parvovirus B19 NS1 Protein Modulates Inflammatory Signaling by Activation of STAT3/PIAS3 in Human Endothelial Cells

The pathogenic mechanism by which parvovirus B19 may induce inflammatory cardiomyopathy (iCMP) is complex but is known to involve inflammatory processes, possibly including activation of JAK/STAT signaling. The nonstructural B19 protein NS1 acts as a transactivator triggering signaling cascades that eventually lead to activation of interleukin 6 (IL-6). We examined the impact of NS1 on modulation of STAT signaling in human endothelial cells (HMEC-1). The NS1 sequences were identified from B19 DNA isolated from the myocardia of patients with fatal iCMP. B19 infection as well as NS1 overexpression in HMEC-1 cells produced a significant upregulation in the phosphorylation of both tyrosine(705) and serine(727) STAT3 (P < 0.05). The increased STAT3 phosphorylation was accompanied by dimerization, nuclear translocation, and DNA binding of pSTAT3. In contrast, NS1 expression did not result in increased STAT1 activation. Notably, the expression levels of the negative regulators of STAT activation, SOCS1 and SOCS3, were not altered by NS1. However, the level of PIAS3 was upregulated in NS1-expressing HMEC-1 cells. Analysis of the transcriptional activation of target genes revealed that NS1-induced STAT3 signaling was associated with upregulation of genes involved in immune response (e.g., the IFNAR1 and IL-2 genes) and downregulation of genes associated with viral defense (e.g., the OAS1 and TYK2 genes). Our results demonstrate that B19 NS1 modulates the STAT/PIAS pathway. The NS1-induced upregulation of STAT3/PIAS3 in the absence of STAT1 phosphorylation and the lack of SOCS1/SOCS3 activation may contribute to the mechanisms by which B19 evades the immune response and establishes persistent infection in human endothelial cells. Thus, NS1 may play a critical role in the mechanism of viral pathogenesis in B19-associated iCMP.

Generation of multifunctional murine monoclonal antibodies specifically directed to the VP1 unique region protein of human parvovirus B19

Little is known about the VP1 unique region (VP1u), a part of one major capsid protein of human parvovirus B19 (B19), concerning its involvement in viral replication and infection cycle. Showing a phospholipase A 2 (PLA 2)-like activity, which is discussed to be necessary for viral release from host cell, its precise function remains unclear. The purpose of this study was to generate multifunctional monoclonal antibodies (mabs) for different applications that may be useful in investigating VP1u's relevance. To establish antiVP1u antibodies, spleen cells from Balb/c mice immunized with purified recombinant viral protein were used for generating antibody-producing hybridoma cell lines. Usability of the antibodies was tested in enzyme-linked immunosorbent assay (ELISA), Western-blot analysis, immunofluorescence and an inhibition assay of enzymatic activity of PLA 2. Three hybridoma cell lines secreting mab's specifically directed against the VP1u protein of B19 could be generated and functioned in every screening method used in this study. These antibodies are helpful tools for investigations in B19 research and diagnosis. Furthermore, the antibodies could help in gaining a deeper understanding of VP1u's role in viral replication and infection especially in the importance of its constitutive PLA 2-like activity.

Detection of Parvovirus B19 Nucleic Acids and Expression of Viral VP1/VP2 Antigen in Human Colon Carcinoma

The aim of this study was to evaluate whether parvovirus B19, a common infectious pathogen in humans, also was involved in human colon carcinoma. A total of 119 paraffin-embedded specimens of colon polyps, adenocarcinomas, carcinoma-adjacent tissues, and normal controls were processed for nested polymerase chain reaction (PCR), in situ hybridization (ISH), immunohistochemistry (IHC), and laser capture micro dissection detection of B19 DNA and protein. The expression of cyclo-oxygenase-2 (COX-2) in the colon- cancer cells (Lovo) transfected by inducible vector for VP1u was determined by western-blot analysis. B19 DNA was detected in 94.6% (35/37) of colon adenocarcinomas, 67.6% (25/37) of adjacent noncancerous tissues, 85.6% (30/35) of polyps, and 60.0% (6/10) of normal controls by nested PCR, respectively. Analysis of the microdissected material confirmed the presence of viral DNA in colonic neoplastic epithelium. ISH detected B19 DNA in 81.1% (30/37) of colon adenocarcinomas, 43.2% (16/37) of adjacent noncancerous tissues, 74.3% (26/35) of polyps, and 50.0% (5/10) of normal controls, respectively. B19 protein VP1/VP2 was found in 78.4% (29/37), 32.4% (12/37), and 57.1% (20/35) of colon adenocarcinomas, tumor-adjacent tissues, and polyps, respectively, but not in normal colons (none of 10). There were significant differences in nested PCR, ISH, and IHC between adenocarcinoma and non-neoplastic adjacent tissues, and between adenocarcinoma and normal controls. Transfection of colon-cancer cells (Lovo) by inducible vector for VP1u resulted in marked upregulation of cyclo-oxygenase-2 proteins. Parvovirus B19 nucleic acids commonly exist in human colon tissues and VP1/VP2 antigen is preferentially located in colon polyps and adenocarcinomas lesions. B19 viral products VP1u may induce important oncogenic pathways in colon-cancer cells.

Molecular and Functional Analyses of a Human Parvovirus B19 Infectious Clone Demonstrates Essential Roles for NS1, VP1, and the 11-Kilodalton Protein in Virus Replication and Infectivity

In an attempt to experimentally define the roles of viral proteins encoded by the B19 genome in the viral life cycle, we utilized the B19 infectious clone constructed in our previous study to create two groups of B19 mutant genomes: (i) null mutants, in which either a translational initiation codon for each of these viral genes was substituted by a translational termination codon or a termination codon was inserted into the open reading frame by a frameshift; and (ii) a deletion mutant, in which half of the hairpin sequence was deleted at both the 5' and the 3' termini. The impact of these mutations on viral infectivity, DNA replication, capsid protein production, and distribution was systematically examined. Null mutants of the NS and VP1 proteins or deletion of the terminal hairpin sequence completely abolished the viral infectivity, whereas blocking expression of the 7.5-kDa protein or the putative protein X had no effect on infectivity in vitro. Blocking expression of the proline-rich 11-kDa protein significantly reduced B19 viral infectivity, and protein studies suggested that the expression of the 11-kDa protein was critical for VP2 capsid production and trafficking in infected cells. These findings suggest a previously unrecognized role for the 11-kDa protein, and together the results enhance our understanding of the key features of the B19 viral genome and proteins.

Development of an improved method of detection of infectious parvovirus B19

Background Parvovirus B19, the only known pathogenic human parvovirus is the aetiologic agent of erythema infectiosum, transient aplastic crisis, pure red cell aplasia, and hydrops fetalis. Transmission is either by respiratory secretions or, as it can be present at high titre in plasma, by blood and blood products. B19 is only cultured with difficulty in vitro, and there is no readily available assay for detecting B19 infectivity or neutralizing antibodies. Objectives In this study, we evaluated different methods to detect viral infection for the purpose of developing automated methods for large-scale testing of viral infectivity, development of neutralizing antibody and viral inactivation assays. Study design Different cell lines were evaluated for their ability to support B19 infection and assays tested for sensitivity and ease of performing. A high-throughput assay was validated by determining infectious virus in blood pools and for determining neutralizing antibody in sera. Results B19 protein production was detected by immunofluorescence (IF) staining and increased viral DNA production by dot blot hybridization and quantitative PCR. The detection of RNA transcripts by RT-PCR assay and quantitative RT-PCR (qRT-PCR) was used as an indirect marker for infection. Of the cell lines tested, the subclone UT7/Epo-S1 showed the greatest sensitivity to B19 infection, with detection of viral transcripts by qRT-PCR the preferred assay. The assays were validated by experiments to determine the infectious titre of sera from acutely infected humans, to evaluate the presence of infectious virus in human donor plasma pools and to measure neutralizing antibodies.

T-helper cell-mediated interferon-γ, interleukin-10 and proliferation responses to a candidate recombinant vaccine for human parvovirus B19

Recombinantly expressed virus-like particles of human parvovirus B19 containing the two structural proteins VP1 and VP2 (VP1/2 capsids) or VP2 alone (VP2 capsids) elicit vigorous antibody responses in animal models, whereas only VP1/2 capsids elicit neutralizing antibodies. VP1 is, therefore, essential for protective B-cell immunity. In this study, we determined the ability of VP1/2 capsids containing VP1 and VP2 in the ratio recommended for vaccine use, and of sole VP2 capsids to stimulate T-helper (Th) cells to proliferate and to secrete interferon gamma (IF-γ) and interleukin-10 (IL-10) in humans long after natural infection. Similar proliferation, IF-γ and IL-10 responses were found with the VP1/2 and VP2 capsids. We conclude that, whereas VP1 contains important B-cell epitopes, VP2, the major structural protein of human parvovirus B19, appears to provide the major target for B19-specific Th-cells years or decades after natural infection.

Detection of IgE anti-parvovirus B19 and increased CD23+ B cells in parvovirus B19 infection: relation to Th 2 cytokines

The immune profile of a parvovirus B19-infected patient (male, 8 years old) was studied on day 0 (initial presentation) and on days 14 and 210 post symptom presentation (psp). Before infection, the patient was skin test positive to various allergens, including ragweed and tree and grass pollens, and had a serum IgE level of 150 IU/mL. On day 0, the patient was diagnosed as parvovirus B19 infected, as judged by the presence of IgG anti-parvovirus Abs in serum (EIA) and presentation of “slap cheek” rash. The patient’s serum IgE level increased from 150 IU/mL before infection to 256 IU/mL on day 0, was 233 IU/mL on day 14, and returned to preinfection levels on day 210. In contrast, there was little change in the levels of serum IgM, IgG, or IgA (nephelometry). IgE anti-parvovirus B19 protein (VP-N) was detected in serum (Western blot) on days 0, 14, and 210, despite the decrease in total IgE on day 210. Although there was no increase in total numbers of blood CD23+ B cells on day 0, by day 14 the numbers of these cells increased dramatically (93%), remaining high on day 210. In contrast, there were virtually no changes in total numbers of CD4+ and CD8+ T cells or CD16/56+ NK precursor cells on days 0–210. On day 0, when IgG and IgE anti-parvovirus were detected in serum, patient’s peripheral blood mononuclear cells (PBMC) expressed mRNA for the Th2 cytokines IL-4 and IL-10, but not for the Th1 cytokines IFN-γ or IL-2. However, by day 14 psp, PBMC expressed mRNA for the Th1 cytokines IFN-γ and IL-2, as well as for IL-4 and IL-10. This is the first demonstration of the existence of IgE anti-parvovirus B19 Ab. The presence of IgE anti-parvovirus B19 Ab in serum on day 0 and its persistence in serum 7 months psp suggests that IgE anti-parvovirus may be useful in prognosis of parvovirus B19 infection. Our results reinforce the idea that IgE, in general, may play a major role in anti-viral immunity, perhaps in conjunction with CD23+ cells. The results further suggest that clearance of this infection is accompanied by a switch to Th1 cytokines.

NS1 Protein of Parvovirus B19 Interacts Directly with DNA Sequences of the p6 Promoter and with the Cellular Transcription Factors Sp1/Sp3

The nonstructural proteins of parvovirus exert a variety of disparate functions during viral infection ranging from promoter regulation, involvement in DNA replication, and induction of apoptosis. Our interest was focused on the possible mechanism by which the NS1 protein mediates its effects on the p6 promoter of parvovirus B19. It is known that the p6 promoter is highly active in different cell lines and interaction with the viral NS1 protein results in a further increase of the activity. The protein may function by binding directly to the viral DNA or via an indirect binding through interaction with cellular transcription factors bound to the promoter. We examined the interaction of the NS1 protein with cellular transcription factors which are involved in regulating the promoter activity. After purified baculovirus-expressed NS1 protein in gel retardation assays was added, an altered complex formation was observed, indicating that NS1 protein interacts with Sp1/Sp3 transcription factors. Enzyme-linked immunosorbent assays verified these findings. The direct interaction of NS1 protein with p6 promoter elements was analyzed by a coprecipitation assay whereby labeled oligonucleotides spanning the entire promoter region were incubated with NS1 protein followed by an immunoprecipitation with NS1-specific antibodies. An eight-nucleotide-long, almost palindromic sequence (AGGGCGGA) was found as potential NS1-binding motif. Footprint analysis with oligonucleotides containing this DNA motif confirmed this result. Thus, transcriptional regulation by the NS1 protein may involve both the interaction with Sp1/Sp3 that binds to the promoter region and direct binding of NS1 to the promoter DNA.

Chronic human parvovirus B19 infection in rheumatic disease of childhood and adolescence.

Parvovirus B19 causes erythema infectiosum in children, but the virus is associated with an increasing range of different diseases. About 20% of infections are associated with delayed virus elimination and viremia persisting over several months or years. These persistent B19-infections are characterised by the presence of IgG against the non-structural protein NS1. This study aimed to find further evidence for an association of parvovirus B19 persistence with VP1/2- and NS1-specific IgG-antibodies in children suffering from rheumatic diseases of childhood. Forty-eight children and adolescents with joint complaints lasting longer than 1 year including patients with juvenile systemic sclerosis and juvenile dermatomyositis showed antibodies against the viral NS1-protein. Laboratory markers of inflammation, humoral immune response against parvovirus B19 proteins and the presence of viral genomes in patients' sera as well as in 124 healthy children were investigated. Almost 50% of the patients showed laboratory signs of chronic inflammation. B19-DNA was amplified in 31% of patients' sera and 7% of the controls (P<0.0001). VP2-specific IgM was detectable in 50% of the patients' and 6% of control sera. NS1-specific immune reactions were linked to persistent B19-infection as indicated by the presence of viral genomes in the peripheral blood and of VP2-specific IgM years after disease onset. To estimate the severity of the disease and the clinical course, the number of affected and functionally impaired joints were noted and compared with the records from patients' initial visit in the hospital. Disease related complications were registered. Impairment of activities of daily living was assessed by Childhood Health Assessment Questionnaire (CHAQ)- and Munich Quality of Life Questionnaire (KINDL)-tests. During observation the clinical state of four patients worsened, 27 improved, the others remained stable. Twenty-four children were restricted in their daily activities.

Immunoreactivity against linear epitopes of parvovirus B19 structural proteins. Immunodominance of the amino-terminal half of the unique region of VP1.

Three peptides corresponding to the 2-100 amino acids of VP1 unique sequence (VP1-F1), to the 99-227 amino acids of VP1 unique sequence (VP1-F2) and to the 237-781 amino acids of VP1 protein common to VP2 (VP1-F3 = VP2) were produced by prokaryotic expression. The three peptides, which span the entire VP1 structural protein of parvovirus B19 and also the entire VP2 protein, were used to evaluate the immunoreactivity against linear epitopes of these fragments in a large number of serum samples taken in different clinical situations with regards to B19 infection and in some commercial preparations of aspecific immunoglobulins. The data demonstrated that the specific VP1-F1 fragment, corresponding to the amino-terminal half of the VP1 unique region, is immunodominant and can elicit a long lasting immune response in comparison with VP1-F2 and VP1-F3 = VP2. Data regarding the presence of specific IgG to the three fragments in commercial preparations of immunoglobulins demonstrated that the dominant immune response was also against VP1-F1 linear epitopes while IgG against VP1-F2 and IgG against VP1-F3 = VP2 could be found only in high concentrations of Ig preparations. The reported data can be useful as a basis for the development of a B19 recombinant vaccine.

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Screening of Blood Donors for Human Parvovirus B19 and Characterization of the Results

Background and Objectives: Human parvovirus B19 (B19 virus) can be transmitted through blood transfusion and plasma–derived products. In a previous report, we utilized the simple hemagglutination method based on the interaction between the B19 virus and P antigen on human erythrocytes in order to screen the blood donors. We called this method receptor–mediated hemagglutination (RHA) [Lancet 1995;346:1237–1238]. In this paper, we report on a large–scale screening of the B19 virus by RHA and discuss the results. Materials and Methods: Donor sera from September 1995 to March 1997 and seroconversion panels were enrolled. Donor sera were examined by RHA for large–scale screening. The positive sera in the first screening were then further investigated by the RHA inhibition test, countercurrent immunoelectrophoresis (CIE), an enzyme–linked immunosorbent assay, and polymerase chain reaction (PCR). We also evaluated the infectivity and neutralizing activity of various kinds of sera by the erythroid colony forming unit (CFU–e) assay. To examine the detection limits of the B19 virus by RHA, B19–viremic sera were purified by sucrose gradient ultracentrifugation. Results: Among 257,710 sera specimens, 293 sera (0.11%) gave a positive reaction in the first screening using RHA. Out of these 293 sera specimens, 31 were positive for PCR, of which 28 were also RHA inhibition–positive, and 25 of the 28 CIE–positive. In the CFU–e injury assay, all the RHA inhibition (+) sera showed a decrease in the number of erythroid colonies. The RHA inhibition (–) PCR (+) B19 antibody (+) sera did not affect the erythroid colony formation and protected CFU–e from injury by the B19 virus. By measuring the amount of purified B19 protein and its RHA titer, the detection limit of the B19 virus by RHA was calculated to the 0.37±0.03 ng/ml. Conclusion: These results suggest that the RHA(+) RHA inhibition (+) sera were infectious in vitro. The combination of RHA and the RHA inhibition test is considered to be useful for the large–scale screening of infectious B19 virus in blood donors with high specificity.

Screening of Blood Donors for Human Parvovirus B19 and Characterization of the Results

Abstract Background and Objectives: Human parvovirus B19 (B19 virus) can be transmitted through blood transfusion and plasma‐derived products. In a previous report, we utilized the simple hemagglutination method based on the interaction between the B19 virus and P antigen on human erythrocytes in order to screen the blood donors. We called this method receptor‐mediated hemagglutination (RHA) [Lancet 1995;346:1237–1238]. In this paper, we report on a large‐scale screening of the B19 virus by RHA and discuss the results. Materials and Methods: Donor sera from September 1995 to March 1997 and seroconversion panels were enrolled. Donor sera were examined by RHA for large‐scale screening. The positive sera in the first screening were then further investigated by the RHA inhibition test, countercurrent immunoelectrophoresis (CIE), and enzyme‐linked immunosorbent assay, and polymerase chain reaction (PCR). We also evaluated the infectivity and neutralizing activity of various kinds of sera by the erythroid colony forming unit (CFU‐e) assay. To examine the detection limits of the B19 virus by RHA, B19‐viremic sera were purified by sucrose gradient ultracentrifugation. Results: Among 257,710 sera specimens, 293 sera (0.11%) gave a positive reaction in the first screening using RHA. Out of these 293 sera specimens, 31 were positive for PCR, of which 28 were also RHA inhibition‐positive, and 25 of the 28 CIE‐positive. In the CFU‐e injury assay, all the RHA inhibition (+) sera showed a decrease in the number of erythroid colonies. The RHA inhibition (‐) PCR (+) B19 antibody (+) sera did not affect the erythroid colony formation and protected CFU‐e from injury by the B19 virus. By measuring the amount of purified B19 protein and its RHA titer, the detection limit of the B19 virus by RHA was calculated to the 0.37±0.03 ng/ml. Conclusion: These results suggest that the RHA (+) RHA inhibition (+) sera were infectious in vitro. The combination of RHA and the RHA inhibition test is considered to be useful for the large‐scale screening of infectious B19 virus in blood donors with high specificity.

Persistent parvovirus B19 infections in immunocompromised children.

Immunocompromised patients have been shown to suffer from prolonged viral infections often without detectable immune response. Here chronic infections with low virus levels can be frequently observed. In these patients viral DNA can be detected over long periods by polymerase chain reaction (PCR). In this study parvovirus B19 presence was assessed by PCR, immunoblot and enzyme-linked immunosorbent assay in sera from children with mainly oncological and hematological diseases. In 45% of sera B19 DNA was observed. Of the children 25% had IgG antibodies to viral protein 1 and 2 (VP1/2) and 15% to nonstructural protein 1 (NS1). In 6% of children IgM antibodies to VP1/2 were detected. These results indicate that the number of children with immune response to B19 proteins is distinctly lower than the number of children with B19 DNA. Transfusions of blood products might have been a possible route for B19 infection. Establishment and maintenance of a persistent parvovirus B19 infection with or without immune response are enhanced in the analyzed immunocompromised children in comparison with immunocompetent children. A persistence of B19 DNA was demonstrated up to 10 months in patients sera.

DETECTION OF PARVOVIRUS B19 INFECTION IN FIRST AND SECOND TRIMESTER FETAL LOSS

Fetal and placental tissues and maternal sera from a series of 273 cases of first and second trimester fetal loss were collected to detect the frequency of parvovirus B19 infection. In addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used for the detection of parvovirus B19 infection. Sera were tested for B19-specific immunoglobulin M (IgM) and/or IgG using an enzyme-linked immunosorbent assay technique. Based on serology, 149 cases not related to B19 infection were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM and IgG at the time of abortion, indicating a recent maternal parvovirus B19 infection. In our histological examination, 10 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either in fetal tissues (n = 2) or in placental tissue (n = 8). However, this vacuolization was considered a fixation artifact and not identical to parvovirus B19-specific nuclear inclusions described in previous reports. Only 1 of these 10 cases had parvovirus B19 DNA detectable in placental tissue by PCR analysis. Neither in this case nor in any of the other cases tested was parvovirus B19 DNA or protein detectable by ISH or IHC, respectively. In none of 41 cases in which fetal tissues were available were congenital anomalies found. In conclusion, the frequency of maternal parvovirus B19 infection in this series of fetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from parvovirus B19 infection and the usage of nuclear histology for the detection of fetal parvovirus B19 infection is considered a nonspecific parameter that requires confirmation by PCR.

Comparison of Seven Commercial Tests for the Detection of Parvovirus B19-Specific IgM

Several enzyme immunoassays (EIA) for the detection of parvovirus B19 IgM (anti-B19 IgM) are now commercially available. In this study, seven commercial EIAs (Biotrin, DAKO, Viramed, Viratech, R-Biopharm, Mast) were compared with an in-house EIA (MvP-EIA) using native viral B19 particles and the reference IgM radioimmunoassay (MACRIA). A total of 88 sera were tested. Results agreed in 39/88 (44.3%) sera, whereas 47/88 (53.4%) were discrepant and 2/88 (2.3%) gave an equivocal result. Assay sensitivity ranged from 70.3 to 100% and specificity, from 75.9 to 100%. The best results were obtained with two EIAs (Biotrin, DAKO) using baculovirus-expressed B19 proteins as antigen. This study has shown that baculovirus-expressed B19 antibody tests are suitable tools for detecting anti-B19 IgM.

Antibodies to the Nonstructural Protein of Parvovirus B19 in Persistently Infected Patients: Implications for Pathogenesis

Three patients with persistent parvovirus B19 infection, as documented by the prolonged presence of IgM directed to the viral capsid proteins and detection of viral DNA in serum by dot-blot hybridization or polymerase chain reaction (PCR), were investigated for the presence of antibodies to the nonstructural protein NS-1 of parvovirus B19. This was done by using an ELISA based on recombinant NS-1 protein. Whereas control sera displayed no reactivity, sera from persistently infected patients showed a strong specific antibody response to NS-1. Patients were followed for 3-18 months, during which IgM titers declined but IgG directed to the nonstructural protein remained detectable. The appearance of NS-1-specific antibodies might indicate an altered course of viral infection leading to the establishment of persistently active infection and subsequent destruction of cells of nonerythroid lineage.

Morphology and antigenicity of recombinant B19 parvovirus capsids expressed in transfected COS-7 cells.

COS-7 cells transfected with parvovirus B19-simian virus 40 (SV40) hybrid vectors have previously been shown to express B19 structural proteins. In this study the morphology and antigenicity of B19 proteins expressed in these cells were investigated. At 84 h after transfection, approximately 10% of the COS-7 cells expressed B19 antigen, and the yield was equivalent to 2 x 10(3) to 2 x 10(5) B19 particles/transfected cell. The B19 proteins self-assembled into capsids that were morphologically and antigenically similar to native B19 virions, and could substitute for native antigen in a B19 IgM assay. Recombinant capsids lacking the recently described 11 kDa protein also resembled native virions.

Development of an IgM capture assay for the diagnosis of B19 parvovirus infection using recombinant baculoviruses expressing VP1 or VP2 antigens

Background: The clinical manifestations of human parvovirus B19 infection are often similar to those induced as the result of infection by other infectious agents such as rubella and some bacteria. Although diagnosis of B19 infection is feasible by detection of specific antibodies, the tests require viraemic serum as a source of antigen. This inevitably leads to problems of reproducibility and dependence upon appropriate high quality clinical material. Objectives: To develop a monoclonal antibody capture ELISA (MACEIA) for detecting anti-B19 IgM antibody in human sera, using recombinant baculoviruses expressing the B19 parvovirus VP1 and VP2 proteins and to compare this with MACEIA using a plasma derived B19 antigen. Study design: Sera from 85 patients with proven B19 infection and the paired convalescent sera from 26 anti-B19 IgM-positive acute samples were examined for B19-specific IgM antibody by a monoclonal antibody capture assay that utilised recombinant baculoviruses expressing B19 proteins in lieu of a plasma-derived B19 antigen. Control samples consisted of 24 anti-rubella IgM, 24 anti-EBV IgM and 102 negative sera from uninfected individuals. Results: Eighty-four of the 85 sera were anti-B19 IgM positive by MACEIA using recombinant baculovirus derived B19 antigen and by indirect immunofluorescence tests, whereas 79 were positive by MACEIA using plasma-derived antigen. Of the 26 convalescent samples which were positive as acute sera, 4 had become negative by 8 weeks post-infection. The expressed recombinant baculovirus antigens had identical molecular weights to the VP1 (84 kDa) and VP2 (58 kDa) proteins of virus purified from human plasma. Recombinant baculovirus-derived VP1 antigen was as effective as VP2 particles at detecting antibodies. Conclusions: Recombinant proteins VP1 and VP2, obtained from recombinant baculovirus-infected cell lysate, showed equal specificity to and higher sensitivity than, B19 virus purified from human plasma when used in MACEIA to detect B19-IgM antibody.

Human parvovirus B19 serology with recombinant VP1 and VP2 antigens: diagnosis of acute infections by detecting B19-specific IgM and IgA antibodies

Background: The availability of immunoassays for the laboratory diagnosis of human parvovirus B19 (B19) infection, which is commonly associated with erythema infectiosum in children and arthropathy and arthralgia in adults has been hampered by the lack of native B19 antigen. The production of abundant supplies of recombinant (r) B19 proteins, through the cloning of the B19 genome into expression vectors, has led to a proliferation of assays for detecting B19-specific antibodies. Objectives: This study was undertaken to evaluate serological assays for detecting B19-specific IgM and IgA antibodies using rVP1 and rVP2 B19 viral coat proteins. Their suitability in the diagnosis of acute B19 infections and the incidence of non-specific reactivity were determined. Study design: A panel of sera consisting of B19-specific IgM-positive and -negative samples was tested for B19-specific IgM and IgA antibodies in an indirect IFA using rVP1 antigen. These samples and a further panel collected from patients with other virus infections and samples containing rheumatoid factor were tested for B19-specific IgM in an antibody-capture ELISA and an indirect ELISA, both of which utilized rVP2 antigen. Results: Data from the two ELISAs using rVP2 antigen and the IFA with rVP1 antigen all showed significant correlation ( P≥0.001) with a reference RIA using native B19 antigen. Non-specific reactions were observed with Paul-Bunnell-positive and rubella virus-specific IgM antibody-positive sera in the ELISAs but not in the IFA. B19-specific IgA antibodies were detected in all sera containing B19-specific IgM antibodies but were also found in a small number of sera collected from healthy blood donors with no history of recent B19 infection. Conclusion: This study demonstrates the usefulness of assays employing rVP1 and rVP2 B19 antigens for detecting B19-specific antibodies. The use of IgM-specific ELISAs allows the processing of large numbers of samples and the absence of non-specific reactivity in the IFA may indicate a role for this assay as a confirmatory test.