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A recombinant virus-like particle system derived from parvovirus as an efficient antigen carrier to elicit a polarized Th1 immune response without adjuvant.

Hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus (PPV) VP2 capsid protein carrying a CD8+ or CD4+ T cell epitope. Immunization of mice with a single dose of these hybrid pseudo-particles, without adjuvant, induced strong cytotoxic T lymphocyte and T helper (Th) responses against the reporter epitope. The Th response was characterized by a Th1 phenotype. We also analyzed in vitro the uptake mechanism of these parvovirus-like particles and the processing requirements associated with presentation by MHC molecules. Although previously shown to be presented by MHC class I molecules, these particles also enter very efficiently the MHC class II endocytic pathway, and behave as conventional exogenous antigens. Indeed, the processing of chimeric PPV:VLP was performed in endosomal/lysosomal acidic vesicles and the presentation of the foreign epitope carried by these particles was sensitive to brefeldin A and cycloheximide, showing that the foreign peptide was loaded on nascent MHC class II molecules. These results give some indication of how PPV:VLP can be presented by MHC class I and class II molecules, and underscore the wide potency of such VLP system to deliver foreign antigens for vaccine design.

Adjuvants, Immunologic↗

Four putative subtypes of human parvovirus B19 based on amino acid polymorphism in the C-terminal region of non-structural protein.

The nucleotide sequence of 10 isolates of human parvovirus B19 (B19) were determined and compared throughout 96.3% of the open reading frames (4145 nucleotides from nt. 509-4653). In the 4145 nucleotides, 122 mutation sites were found, of which 24 were accompanied by amino acid displacement. Furthermore, the polymorphism of the amino acids was seen in about 110 bases near the carboxy terminal of the non-structural protein, ranging from nt. 2011 to 2123, where four amino acid mutation points were found to exist. Based on the amino acid polymorphism of these four mutation sites in this area, 10 isolates of the B19 parvovirus could be divided into 4 subtypes (subtypes A, B, C, and D). The frequency of isolation of the subtypes depended on the time and location of collection of the B19 viremic blood specimens.

Amino Acid Sequence↗

Pure red cell aplasia associated with parvovirus B19 infection occurring late after allogeneic bone marrow transplantation.

Differential diagnosis for anemia late after allogeneic stem cell transplantation is broad. In this report, we describe a case of severe anemia secondary to pure red cell aplasia associated with human parvovirus B19 infection over 8 years after allogeneic bone marrow transplantation. Characteristics of parvovirus B19 infection and the immunosuppressed state after allogeneic stem cell transplantation are reviewed.

Anemia↗

Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing.

The extremely poor prognosis of malignant gliomas requires the investigation of other than standard therapies, i.e., the application of oncolytic viruses. In our study, we evaluated the effects of the oncosuppressive parvovirus H-1 on different established glioblastoma cell lines of rat and human origin and on short-term/low-passage cultures of human glioblastoma cells. We observed an efficient and dose-dependent killing of all glioma cell cultures at low multiplicities of infectious particles (MOI) per cell. Southern blot analysis of viral DNA amplification, RT-PCR analysis of viral RNA expression and Western blot analysis of the expression of viral structural (VP-1/VP-2) and nonstructural (NS-1) proteins demonstrated the biosynthesis of these viral macromolecular components in all of the cultures. Moreover, all the glioma cells were proficient for the production of infectious H-1 virus particles. The amount of virus production differed between a several fold increase of the input virus titer in most of the short-term/low-passage cultures up to 1,000-fold in one short-term glioma and in the rat cells. Glioma cells lines and, more importantly, short-term/low-passage cultures of human glioblastomas were found to be highly susceptible target cells for H-1 virus mediated cytotoxicity. The formation of fully infectious progeny particles in infected glioma cells offers the chance for the induction of secondary rounds of infection resulting in an advanced cytotoxic effect. These advantageous characteristics of H-1 virus infection of glioma cells, combined with the known low toxicity of H-1 virus in nontransformed cells, make parvovirus H-1 a promising candidate for oncolytic glioma therapy.

Animals↗

Parvovirus B19 infection in Taiwanese patients with hematological disorders.

Human parvovirus B19 has a strong tissue tropism for erythroid progenitor cells and is a causative agent for anemia. However, it remains unclear whether patients with hematological disorders are at a higher risk of B19 infection. In the present study, we evaluated the prevalence of B19 infection in 278 patients with hematological disorders by indirect antibody capture enzyme-linked immunosorbent assay. Virus inoculation into cell culture (of TF-6, WRL-68, and HL-60) was carried out using serum from patients with positive IgM anti-B19 and was then examined by nested polymerase chain reaction, dot-blot analysis, and sequence analysis. Our data demonstrated that the total seropositive rates of either IgG or IgM were 71.9%. The seropositive rates increase significantly with age (P < 0.001). After adjustment for age, the seropositive rate was significantly higher in our patients than in the general population with standardized rate ratio of 1.56 (95% CI = 1.43-1.68). No significant difference was found among different disease subgroups (P = 0.311). Nine patients (3.2%) had active B19 infection with positive IgM antibody, with four diagnosed as having idiopathic thrombocytopenic purpura (ITP). Viremia of B19 virus could be detected in eight of nine patients, including three patients in serum only, three patients in bone marrow only, and two patients in both serum and bone marrow. We conclude that patients with hematological disease have higher seropositive rates for B19 than occur in normal controls and that study of occult parvovirus B19 infection is recommended in patients with hematological disease.

Adult↗

Replication and cytopathology of human parvovirus B19 in human umbilical cord blood erythroid progenitor cells.

Human parvovirus B19 productively infected erythroid progenitor (EP) cells from umbilical cord blood, in vitro as shown by an increase of viral DNA in supernatant fluid assayed by dot blot hybridization and liquid scintillation counting. Progeny virus was released into the supernatant fluid of CD34+ EP cells which had been purified by immunomagnetic separation. This supernatant fluid was infectious for bone marrow cells. Erythroid bursts infected with virus showed characteristic cytopathic effect by electron microscopy consisting of cytoplasmic vacuolization, marginated chromatin, and nuclear inclusions of lattice or crystalline arrays. Cultures of umbilical cord blood EP cells may be useful for the propagation of parvovirus B19 serological testing reagents and the study of virus-host cell interactions.

Base Sequence↗

Nested polymerase chain reaction assay for the detection of B19 parvovirus DNA in human immunodeficiency virus patients.

Persistent B19 parvovirus infection has been recognized in immunocompromised patients, often occurring with a low-titer viremia. In this study, nested polymerase chain reaction (PCR) for the detection of B19 parvovirus DNA was carried out on the sera of 49 human immunodeficiency virus (HIV)-1-seropositive patients, negative for the detection of B19 DNA at dot blot hybridization assay and with different values of serum anti-B19 IgM (27 patients proved positive and 22 negative). Of the 49 HIV-seropositive samples tested by nested PCR, seven were positive for the detection of B19 DNA. All seven belonged to the group of subjects seropositive for specific anti-B19 IgM. The study shows that, in the presence of specific B19 IgM, circulating virus may still be present but can be detected only by PCR. In that B19 infection can occur with low-titer viremia in immunocompromised patients, PCR may be the only method for virus detection.

Adult↗

Circulating cytokines and chemokines in acute symptomatic parvovirus B19 infection: negative association between levels of pro-inflammatory cytokines and development of B19-associated arthritis.

The aim of the study was to characterise the profile and clinical correlates (arthritis, rash, and fatigue) of cytokines, chemokines, and other mediators in symptomatic acute parvovirus B19 infection. Serum was examined from cases of acute B19 infection (as defined by serum anti-B19 IgM positivity) (n = 84), and in normal persons (n = 43) for B19 markers (serum B19 antibodies and DNA), rheumatoid factor (RF), and antinuclear antibody (ANA). A panel of cytokines/chemokines was measured in duplicate using the Bioplex Protein Array system (BioRad Hemel Hempstead, UK). These included interleukin-1 beta (IL-1 beta), IL-4, IL-5, IL-6, IL-8, IL-13, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), macrophage chemoattractant protein-1 (MCP-1), granulocyte-monocyte colony stimulating factor (GM-CSF), transforming growth factor-beta1 (TGF-beta 1), endothelin-1 (ET-1), and neopterin. Acute symptomatic infection was characterised by specific IgG positivity (83%), serum B19 DNA positivity (96%), and raised levels of IL-4, IL-6, IL-8, TNF-alpha, IFN-gamma, MCP-1, GM-CSF, TGF-beta 1, and ET-1. Patients with acute B19-associated arthritis were found to have lower levels of IL-6, TNF-alpha, and GM-CSF than patients without arthritis, while those with rash had lower levels of TGF-beta 1. It is concluded that cytokine levels following acute symptomatic infection with parvovirus B19 indicate a state of immune activation. The profile of circulating mediators may provide insights into the possible pathogenesis of particular clinical manifestations of this infection.

Adolescent↗

Ultrastructural features of fetal erythroid precursors infected with parvovirus B19 in vitro: evidence of cell death by apoptosis.

Human parvovirus B19 cannot be cultured in standard cell lines and relatively little is known about the intracellular life-cycle of the virus. In this study, ultrastructural features of B19 infection were examined using haemopoietic cell suspension cultures derived from human fetal liver. Erythroblasts from infected cultures frequently contained crystalline arrays of both full and empty virus-like particles. The number and size of these arrays increased with the duration of culture, and their location changed from exclusively nuclear at 24 h post-infection to both nuclear and cytoplasmic at 3 days post-infection. Arrays were occasionally found in cytoplasmic protuberances which appeared to be pinching off from the cell. The location of the arrays corresponded to the distribution of viral capsid protein determined by immunolabelling at the light microscope level. Cells containing viral crystalline arrays also exhibited nucleolar degeneration, extreme margination of the nuclear heterochromatin, and cytoplasmic vacuolation. These features are typical of cells undergoing individual programmed cell death or 'apoptosis'. The triggering of apoptosis in erythroid precursors by parvovirus B19 may help to explain the apparent lack of a strong inflammatory response to fetal B19 infection and may have implications for understanding the mechanisms of viral spread throughout the host.

Apoptosis↗

A fetus with a parvovirus B19 infection and congenital anomalies.

A fetus with multiple structural defects was seen at prenatal ultrasound examination. After termination of the pregnancy a bilateral cleft lip, alveolus, and palate; micrognathia; and webbed joints were seen. Fetal tissues showed indications of infection, intranuclear inclusion bodies, chronic stress, haemolysis, arterial wall damage, and profuse haemorrhage. Parvovirus B19 DNA was detected in fetal tissues by dot hybridization after polymerase chain reaction. The possibility of parvovirus B19 infection leading to congenital malformations is discussed.

Adult↗

Fetal morbidity and mortality after acute human parvovirus B19 infection in pregnancy: prospective evaluation of 1018 cases.

OBJECTIVE: To determine more precisely the incidence of fetal complications following maternal parvovirus B19 infection at various gestational ages. METHODS: An observational prospective study of 1018 pregnant women whose acute B19 infection was serologically confirmed in our laboratory. RESULTS: The observed rate of fetal death throughout pregnancy was 6.3% (64/1018) (95% confidence interval [CI]: 4.9, 8.0). The fetal death rate for those infected within the first 20 weeks of gestation (WG) was 64/579 (11.0%). Fetal death was only observed when maternal B19 infection occurred before the completed 20 WG. The observed stillbirth proportion was 0.6% (6/960). Three of six stillbirth cases presented with fetal hydrops. The overall risk of hydrops fetalis was 3.9% (40/1018) (95% CI: 2.8, 5.3). Three of 17 cases with non-severe hydrops and 13 of 23 cases with severe hydrops received intrauterine transfusion(s). The proportion of fetuses with severe hydrops that survived following fetal transfusions was 11/13 (84.6%). All of the non-transfused fetuses with severe hydrops died. CONCLUSION: Our data demonstrate a relevant B19-associated risk of fetal death, which is largely confined to maternal B19 infection in the first 20 WG. Timely intrauterine transfusion of fetuses with severe hydrops fetalis reduces the risk of fetal death. Parvovirus B19-associated stillbirth without hydropic presentation is not a common finding.

Blood Transfusion, Intrauterine↗

Precise quantitation of human parvovirus B19 DNA in biological samples by PCR.

An application of a quantitative PCR-based method was developed for the detection of human parvovirus B19 DNA. The procedure was characterised according to guidelines for the validation of analytical procedures. Furthermore, the reliability was demonstrated by the correct quantitation of samples of an international collaborative study. This application might be useful for studies focussed on removal and/or inactivation procedures of human parvovirus B19 as well as for general screening purposes of biological materials.

Base Sequence↗

The structure of porcine parvovirus: comparison with related viruses.

The structure of baculovirus-expressed porcine parvovirus (PPV) capsids was solved using X-ray crystallography and was found to be similar to the related canine parvovirus (CPV) and minute virus of mice (MVM). The PPV capsid protein has 57 % and 49 % amino acid sequence identity with CPV and MVM, respectively, but the degree of conservation of surface-exposed residues is lower than average. Consequently, most of the structural differences are on the surface and are the probable cause of the known variability in antigenicity and host range. The NADL-2 and Kresse strains of PPV have distinct tissue tropisms and pathogenicity, which are mediated by one or more of the amino acid residues 381, 386, and 436. These residues are on or near the surface of the virus capsid, where they are likely to be associated with virus-cell interactions.

Amino Acid Sequence↗

Parvovirus-like particles as vaccine vectors.

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project.

Animals↗

A novel protein encoded by small RNAs of parvovirus B19.

A second small ORF contained within the parvovirus B19 genome has been shown to direct synthesis of a small 7.5-kDa protein. These results extend our previous studies in which we identified a family of 11-kDa proteins (J. St. Amand, C. Beard, K. Humphries, and C.R. Astell, Virology 183, 133-142, 1991; J. St. Amand and C.R. Astell, Virology, 192, 121-131, 1993). The presumed ORF encoding the 7.5-kDa protein was discovered on the left side of the viral genome and was identified by expression of the protein and in vitro mutagenesis. Two small RNA transcripts of B19 virus were demonstrated to be able to direct the synthesis of this 7.5-kDa protein by in vitro translation using rabbit reticulocyte lysates. Expression of 7.5-kDa protein was also observed in COS-7 cells transfected with a plasmid containing the B19 genome as well as in human peripheral mononucleocytes infected with B19 virus. In both cases the 7.5-kDa protein was detected by immunoprecipitation of cell lysates with polyclonal antibodies raised against a synthetic peptide corresponding to a portion of the predicted protein sequence. These data partially reveal the function of the abundant small RNA transcripts produced by parvovirus B19.

Amino Acid Sequence↗

Genomic organization and mapping of transcription and translation products of the NADL-2 strain of porcine parvovirus.

The NADL-2 strain of PPV was cloned into pUC19 and independent infectious clones were sequenced. This permitted a correction of published sequences and to predict a cruciform structure as an alternative to the 5'-hairpin of the "-" strand. This 5'-end structural covariance is shared with other parvoviruses of the same group and two alternative sequences ("flip" and "flop") were present in the region of the cruciform. Transcript and translation product mapping allowed the prediction of the location of the different expression signals. The 5'-startpoints of the transcripts were located at nucleotides 225 and 2035, respectively, and the polyadenylation site at nucleotides 4829-4833. This indicated that the TATA boxes at 196-TATA and 2004-AATA and the 4813-AATAAA polyadenylation sequence would be functional. Alternative splicing of capsid gene (VP) transcripts (either 2280-AG/GT or 2313-AG/GT spliced with 2386-AG/GA), to maintain or remove the first AUG (at 2287) in the ORF, yielded two 2.9-kb mRNAs containing a nested set of protein-coding sequences (VP-1 and VP-2 with predicted molecular mass 80.9 and 64.3 kDa, respectively). Three nonstructural (NS) protein gene transcripts were identified. The 4.7-kb transcript was not spliced in the NS gene and was predicted to code for a 75.5-kDa protein (NS-1; published value of phosphorylated form 84 kDa). The splicing sites of two different 3.3-kb NS transcripts were analyzed. These transcripts were predicted to code for the NS-2 protein (18.1 kDa). Of the two NS-2 transcripts, one had also the VP-intron removed downstream of the NS-2 coding sequences. A 2.9-kb transcript would code for an NS-3 protein (12.4 kDa) although such a protein has not been described before. A flow chart of the information from the viral DNA to the viral proteins is presented and several differences, both for the NS and the VP genes, with closely related parvoviruses are noted.

Amino Acid Sequence↗

Most of the VP1 unique region of B19 parvovirus is on the capsid surface.

B19 parvovirus is pathogenic in man and a vaccine is desirable. In convalescence after acute infection, the dominant humoral immune response is directed to the minor capsid protein called VP1, which differs from the major capsid protein by an additional NH2-terminal 227 amino acids. We have previously shown that this unique region contains multiple linear neutralizing epitopes. We produced seven recombinant B19 capsids that contained progressively truncated VP1 unique region sequences, each fused to a Flag peptide (AspTyrLysAspAspAspAspLys) at the NH2-terminus. Capsids containing normal VP2 and truncated Flag-VP1 proteins and, in some cases, only truncated Flag-VP1 chimeric proteins, were analyzed by ELISA, affinity chromatography, and electron microscopy using anti-Flag monoclonal antibody. All regions examined showed binding to anti-Flag antibody in multiple assays, indicating that most of the VP1 unique region is external to the capsid and accessible to antibody binding. These results have implications for the design of a B19 parvovirus vaccine and the use of empty capsids for presentation of heterologous protein antigens.

Amino Acid Sequence↗

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.

Base Sequence↗