Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen).

BACKGROUND: The presence of a specific cellular receptor is thought to be necessary for susceptibility to viral infection. The erythrocyte P antigen is the cellular receptor for parvovirus B19. We hypothesized that the rare persons with the p phenotype, whose erythrocytes do not have this receptor, would be naturally resistant to B19 infection, which causes erythema infectiosum. METHODS: Blood samples were collected from two populations in cross-sectional studies. We determined the P antigen phenotype of the red cells and tested plasma for anti-B19-specific antibodies. Bone marrow from donors of known P antigen phenotype was inoculated with parvovirus B19. Infectivity was measured by assays of erythroid progenitor cells, dot blot analysis, and in situ hybridization for B19 DNA, and an immunofluorescence assay for viral-capsid proteins. RESULTS: Of the 17 subjects with the p red-cell phenotype, who did not have P antigen on their erythrocytes, none (0 of 11 and 0 of 6) had serologic evidence of previous parvovirus B19 infection. In contrast, the seropositivity rates in the two control groups were 71 percent (53 of 75, P < 0.001) and 47 percent (32 of 68, P = 0.03). In vitro, bone marrow from donors with the p phenotype maintained normal erythropoiesis despite very high concentrations of virus, with no evidence of infection of erythroid progenitor cells by parvovirus B19. CONCLUSIONS: People who do not have P antigen, which is the cellular receptor for parvovirus B19, are naturally resistant to infection with this pathogen.

Erythrocyte P antigen: cellular receptor for B19 parvovirus.

The pathogenic human parvovirus B19 replicates only in erythroid progenitor cells. This virus was shown to bind to blood-group P antigen, as measured by hemagglutination. Erythrocytes lacking P antigen were not agglutinated with B19. Purified P antigen (globoside) blocked the binding of the virus to erythroid cells and the infectivity of the virus in a hematopoietic colony assay. Target cells were protected from infection by preincubation with monoclonal antibody to globoside. Knowledge of a parvovirus receptor has implications for understanding the pathogenesis of parvovirus infections and for the use of parvoviruses in gene therapy.

Generation of neutralizing anti-B19 parvovirus human monoclonal antibodies from patients infected with human immunodeficiency virus.

The prevalence of IgG antibodies to human B19 parvovirus (anti-B19) is elevated in individuals infected with human immunodeficiency virus (HIV), especially during the later stages of HIV infection. In subjects with high titers of IgG anti-B19, 86% (19 of 22) had circulating B cells producing anti-B19. Immortalization of these cells with Epstein-Barr virus and generation of heterohybridomas by fusion with a mouse X human heteromyeloma resulted in the production of two cell lines producing IgG1 kappa monoclonal antibodies (MAbs). Both of these MAbs were specific for conformational epitopes on the VP2 capsid protein of B19 parvovirus and both were capable of neutralizing 50% of the viral infectivity in a human erythroid colony-forming unit assay at < or = 1 micrograms of MAb/mL. These human MAbs are potentially useful in the treatment of acute B19 parvovirus infection.

Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes.

Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infected erythroid cells embedded in Lowicryl K4M and LR White acrylic resins were established at the electron microscope level. The protocol was initially optimized using a positive control probe for whole human DNA which produced signal over the heterochromatin of all nucleated cells. In cultures harvested 2 days postinfection, B19 nucleic acid was detected mainly within the centrinuclear region of erythroid cells exhibiting characteristic margination of the chromatin. The B19 hybridization signal was largely unaffected by denaturation and was resistant to RNase digestion but sensitive to DNase digestion, indicating that it was mainly single-stranded B19 DNA. Relatively few gold particles were found over crystalline arrays of viral capsids, consistent with the observation that they are composed of mainly 'empty' capsids. B19 nucleic acid was detected in apparent transit from nucleus to cytoplasm through pores in the nuclear membrane. While the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section, it is a rapid and specific means of localizing viral nucleic acids with a high degree of resolution.

Granulocyte colony-stimulating factor treatment of mice modulates differently the sensitivity of blood and bone marrow hematopoietic progenitors to retroviral vector infection.

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) administration to mice on the retroviral-mediated transfer of the selectable marker gene neo to hematopoietic cells was studied. After G-CSF treatment, blood mononuclear cells and bone marrow cells were infected with the retrovirus, and the efficiency of gene transfer into myeloid progenitors was increased in peripheral blood and decreased in bone marrow cells. Bone marrow four to six months after transplantation from G-CSF treated mice revealed the presence of the transferred neo gene in 4 out of 104 mice, which is an eightfold lower proportion than in the control group. These data suggest that G-CSF in vivo treatment decreases bone marrow sensitivity and increases the sensitivity of peripheral blood cells to retroviral infection.

Induction of pre-B lymphoid leukemia following reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow progenitor cells.

We have previously shown that v-erb-B contained within a recombinant murine retroviral vector is capable of transforming pre-B lymphocytes (M. Miller, A. K. Kennewell, and G. Symonds, Leukemia, 6: 18-28, 1992) and early erythroid precursor cells [blast-forming units (erythroid) (M. Miller, A. Kennewell, Y. Takayama, A. Bruskin, J. M. Bishop, G. Johnson, and G. Symonds, Oncogene, 5: 1125-1131, 1990)] in vitro. To determine the sites and nature of v-erb-B-induced transformation in vivo, the hematopoietic systems of lethally irradiated mice were repopulated with v-erb-B-infected bone marrow. All mice became moribund within 4-12 weeks of reconstitution, with a median onset of disease at 6 weeks. Histopathological and flow cytometric evaluation of tissues from diseased mice, as well as morphological and phenotypic analysis (cytochemical as well as molecular) of the cell lines established from the mice, revealed that all but one of the mice examined at postmortem had developed a pre-B lymphoid leukemia or lymphoma. Abnormally high levels of mast cells in the spleen and bone marrow of the remaining mouse indicated a mast cell disease. The development of pre-B lymphoid malignancy in the majority of the reconstituted mice indicates a marked predisposition of v-erb-B to transform cells of the pre-B lymphoid lineage. The reconstitution of lethally irradiated mice with v-erb-B virus-infected bone marrow provides a model system for the analysis of events involved in the initiation and maintenance of acute lymphoid leukemia.

In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe.

An in situ hybridization assay using a digoxigenin-labelled probe was developed to detect B19 DNA in bone marrow erythroid elements of immunodeficient patients with hypoplastic anaemia. A 700 bp Bam HI-Hin dIII fragment of B19 DNA was used to construct the probe by incorporating deoxyuridine triphosphate labelled with digoxigenin. The in situ hybridized B19 DNA probe was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. Dark blue coloured inclusions at the enzyme site were detected in the nuclei of B19 infected erythroid cells at different stages of cell differentiation. Six out of the nine patients studied showed a positive reaction by in situ hybridization assay. The assay we developed proved highly specific and sensitive and it appears to be a suitable diagnostic test for investigating the possible role of B19 infection as a cause of haematopoietic disorders in immunocompromised hosts.

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.

Differential effect in vitro of tumor necrosis factor-alpha (TNF) on normal and virus-infected erythroid progenitors from Friend virus (FVA)-infected mice.

In vivo administration of tumor necrosis factor-alpha (TNF) suppresses both normal and Friend virus (FVA)-infected erythroid progenitor cells (CFU-E). To examine the mechanism of erythroid suppression by TNF, we examined TNF's direct effect on normal and virus-infected cells in vitro. Productively infected fibroblast cell lines, fresh acute virus-infected spleen cells, and virus-infected CFU-E were sensitive, whereas uninfected CFU-E were resistant to TNF cytotoxicity in vitro. When FVA-infected erythroblasts were depleted from the spleen cell population in vitro with antivirus antibodies, TNF suppression of the remaining (uninfected) cells was abrogated. In contrast, both normal and virus-infected macrophage progenitor cells and immature erythroid progenitor cells were equally sensitive to TNF cytotoxicity in vitro. Normal erythroblasts had significantly fewer TNF receptors than FVA-infected erythroblasts, which also were morphologically less mature. These results suggest that TNF can differentially suppress late-stage virus-infected erythroid progenitors in vitro.

Immunophenotyping of fetal haemopoietic cells permissive for human parvovirus B19 replication in vitro.

Human parvovirus B19 is known to inhibit erythroid colony formation in vitro, but the precise stage of differentiation at which erythroid precursors become capable of supporting viral replication has not been accurately determined. In order to address this issue, haemopoietic cells derived from first trimester fetal liver were cultured in medium containing B19 antigen-positive serum. Infected cells were phenotyped by combining immunohistology for cell-type specific antigens with non-isotopic in situ hybridization for B19 nucleic acid. Strong nuclear hybridization signal was detected as early as 8 h after infection in erythroid precursors labelling with antibodies to glycophorin A, glycophorin C, CD43, CD36 and HLA-ABC (pronormoblast or normoblast phenotype). Giant erythroid precursors labelling with the same five antibodies were a pathognomonic feature of infected cultures, but contained relatively little B19 nucleic acid. Hybridization signal was not detected in progenitor cells of more primitive erythroid phenotype or in nuclei of cells of other lineages, though B19 DNA was occasionally localized within the cytoplasm of macrophages. Double-labelling with antibody Ki-67 confirmed that proliferating cells were targets for B19 infection. Co-detection of cell-type specific antigens and viral nucleic acid is a powerful tool for investigating host cell specificity, and suggests that proliferating late erythroid precursors are the only haemopoietic cells fully permissive for B19 infection.

Severe in vitro inhibition of erythropoiesis and transient stimulation of granulopoiesis after bone-marrow infection with eight different HIV-2 isolates.

OBJECTIVE: To correlate severe anaemia and frequent neutropenia in HIV-2-infected patients with an inhibitory effect on bone-marrow progenitors common to several HIV-2 isolates. DESIGN: The effects of eight HIV-2 isolates on early (BFU-E) and late (CFU-E) erythroid progenitors and on granulomonocytic (CFU-GM) progenitors, produced in long-term bone-marrow cultures (LTBMC), were studied. METHODS: Absolute numbers of BFU-E, CFU-E and CFU-GM per culture flask were calculated weekly for each HIV-2-infected LTBMC using semi-solid clonogenic assays, and compared with those obtained in mock-infected LTBMC. Levels of significance for comparisons were determined by an analysis of variance (ANOVA). RESULTS: Pooled data from 24 series of LTBMC (three series for each HIV-2 isolate) revealed 80 and 100% inhibition of BFU-E and CFU-E on days 6 and 12 of LTBMC, respectively, while transient stimulation of CFU-GM was observed between days 14 and 20 of LTBMC, followed by total inhibition on day 30. CONCLUSIONS: These results confirm a direct inhibitory effect of HIV-2 on in vitro haematopoiesis. The similar pattern of erythroid progenitor inhibition obtained from seven out of eight isolates suggests that the inhibitory effect on erythropoiesis is a feature common to a large number of HIV-2 isolates, which correlates with clinical findings.

Hematopoietic target cells of anemogenic subgroup C versus nonanemogenic subgroup A feline leukemia virus.

Feline leukemia viruses (FeLVs) belonging to interference subgroup C induce fatal anemia resembling human pure red cell aplasia (PRCA). Subgroup A FeLVs, although closely related genetically to FeLVs of subgroup C, do not induce PRCA. The determinants for PRCA induction by a molecularly cloned prototype subgroup C virus (FeLV-Sarma-C [FSC]) have been localized to the N-terminal 241 amino acids of the surface glycoprotein (SU) gp70. To investigate whether the anemogenic activity of FSC reflects a unique capacity to infect erythroid progenitor cells, we used correlative immunogold, immunofluorescence, and cytological staining to study prospectively the hemopoietic cell populations infected by either FSC or FeLV-FAIDS-61E-A (F6A), a prototype of subgroup A virus. The results demonstrated that although only FSC-infected animals developed erythrocyte aplasia, the env SU and the major core protein (p27) were expressed in a surprisingly large fraction of the lymphoid, erythroid, and myeloid lineage marrow cells in both FSC- and F6A-infected cats. Between days 8 and 17 postinoculation, gp70 and p27 were detected in 43 to 73% of erythroid, 25 to 75% of lymphoid, and 35 to 50% of myeloid lineage cells, regardless of whether the cats were infected with FSC or F6A. Thus, anemogenic subgroup C and nonanemogenic subgroup A FeLVs have similar hemopoietic cell tropism and infection kinetics, despite their divergent effects on erythroid progenitor cell function. Acute anemia induction by subgroup C FeLV, therefore, does not reflect a unique tropism for marrow erythroid cells but rather indicates a unique cytopathic effect of the SU on erythroid progenitor cells.

Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice.

Nucleotide changes at both codons 317 and 321 in the VP2 capsid gene of the immunosuppressive strain of the murine parvovirus minute virus of mice, MVM(i), are required to create a virus capable of growing in A9 fibroblasts. This double mutant virus, ILB1, has growth characteristics very similar to those of the prototype fibrotropic strain MVM(p) in both single- and multiple-round infections of fibroblasts and is about 100-fold better at infecting fibroblasts than MVM(i). When only one nucleotide position is changed, either in codon 317 (as in ILB2) or in codon 321 (as in ILB3), the resulting viruses are less than twice as efficient as their parent MVM(i) at infecting fibroblasts. In the restrictive infection of A9 cells by the single mutants and MVM(i), gene expression and DNA replication were markedly reduced compared with ILB1 infection of the same cells or compared with infections of permissive hybrid cells by each of the viruses. This suggests that restriction acts predominantly at an early step in the infection. Since the phenotypes of ILB2 and ILB3 are essentially indistinguishable in restrictive infections, it is most likely that the individual loci affect the same step in the viral life cycle. The dramatic increase in fibroblast infectivity shown by ILB1 indicates a synergistic interaction between these two amino acid residues in the same rate-limiting process in fibroblast infection.

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.

Sequences present in a small region of the AKV virus envelope gene determine the efficiency with which pseudotyped spleen focus-forming virus infects erythroid target cells.

Induction of erythroleukemia in mice by the replication-defective spleen focus-forming virus (SFFV) relies on the presence of a helper virus to deliver the SFFV genome to erythroid target cells. Pseudotyping studies with different ecotropic murine leukemia viruses (MuLV) have shown that SFFV pseudotyped with Akv, the endogenous ecotropic virus of AKR mice, inefficiently gives rise to virus-induced erythroid bursts (vBFU-E) in vitro and fails to cause erythroleukemia in mice when compared to SFFV pseudotyped with Friend or Moloney MuLV. In order to locate the region(s) of the Akv genome responsible for its inability to act as a helper for SFFV, six different Moloney MuLV chimeras containing Akv envelope sequence substitutions were constructed. Virions with the chimeric envelopes were used to pseudotype SFFV and the complexes were analyzed for their ability to induce vBFU-E in vitro and erythroleukemia in mice. SFFV preparations pseudotyped with three of the constructs containing chimeric envelope genes efficiently gave rise to vBFU-E as did SFFV pseudotyped with Moloney MuLV. SFFV pseudotypes generated from the other three constructs, which all share a common 304-bp region located near the center of the Akv gp70 coding region, and Akv gave rise to very few vBFU-E. However, all SFFV preparations, with the exception of SFFV pseudotyped with Akv, induced erythroleukemia in mice. The results suggest that specific sequences present in the envelope gene of Akv are responsible for the inefficiency of the virus to infect erythroid target cells for SFFV, but additional Akv sequences outside those used in this study affect the ability of the Akv/SFFV virus complex to cause erythroleukemia in mice.

Modulation of normal human erythropoietic progenitor cells in long-term liquid cultures after HIV-1 infection.

Long-term bone marrow cultures (LTBMC) have been infected by two isolates of human immunodeficiency virus type 1 (HIV-1) (HIV-1 LAV and HIV-1 NDK) at multiplicities of infection ranging from 10(2) to 2.10(6) tissue culture infectious units (TCIU) per 10(6) bone marrow mononuclear cells (BMMNC). These infected cells are nonproducer cells and the viruses can be rescued by coculture with peripheral blood lymphocytes, cord blood lymphocytes, or BMMNC and not by the CEM cell line. HIV-1 clearly is not cytopathic for these cells. Following production and growth of erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E) for at least 6 weeks after infection with HIV-1 NDK, colony assays displayed a 50% inhibition of BFU-E production during 3 weeks of LTBMC. This was followed by a stimulation phase. On the contrary, HIV-1 LAV induces a 150% stimulation of BFU-E production, followed by 50% inhibition. Production of CFU-E was inhibited by 80-100% with the two isolates of HIV-1 after four weeks of LTBMC. Stimulatory and inhibitory activities were recovered from supernatants of infected LTBMC and lymphoid CEM cell lines, suggesting that HIV-1 induces release of a humoral factor responsible for disruption of hemopoietic progenitor cell production in vitro and consequently for hematologic abnormalities in AIDS patients.

Human B19 parvovirus infection: an example of multiple pathogenic effects determined by differences in host susceptibility.

B19 infection offers some general lessons about human viruses and their possible effects on the human host, as follows: (1) Ubiquitous apparently benign viruses may have severe effects on a compromised host. The virus may be invariable but the host can have diverse susceptibilities. (2) B19 and some other human viruses (though for none is the evidence so clear as for B19) have narrowly targetted effects. The host cell of B19 is a specialised progenitor of mature red cells: impairment of the function of this cell by B19 may cause profound anaemia. (3) The 'normal' host response to B19 may also cause disease, though this is self limiting. (4) The effects of malfunction of the virus' target cell are exacerbated when the immune response is impaired by congenital or acquired immunodeficiency, immunosuppressive therapy or, in the case of the fetus, developmental immaturity that allows the virus to persist.

Detection of burst-promoting activity in spleens of myeloproliferative sarcoma virus-infected mice using serum-free cultures.

Myeloproliferative syndrome was induced in adult DBA/2 mice by inoculation with myeloproliferative sarcoma virus (MPSV) and Friend murine leukemia virus (F-MuLV) as a helper virus. On day 26 after infection, the spleen weighed a maximum of 2.0 g (about 30 times the control weight). Assay of multipotent stem cells in vitro showed that the more enlarged spleens contained an increased number and concentration of mixed colony-forming units (CFU-mix) (at maximum, 11 times higher than the control). When the supernatant of cultured spleen cells was added to a serum-free bone marrow cell culture with or without erythropoietin (Epo) for detection of burst-promoting activity (BPA), it enhanced erythroid mixed colony (E-mix) formation only in the presence of Epo (p less than 0.05). Even when addition of Epo was delayed, it still induced a significant number of E-mix (p less than 0.05). These findings rule out a mimic effect of Epo resembling BPA and indicate the presence of BPA in the spleen. The culture supernatant also supported the proliferation of interleukin 3 (IL-3)-dependent 32Dcl cells. Therefore, although purification of the BPA substance has not yet been accomplished, BPA in the supernatant seems to depend on the presence of IL-3, which is known to be one of the factors stimulating multipotent hemopoietic stem cells. The presence of BPA- or CFU-mix-stimulating activity in the spleen after infection might be responsible for the development of panmyelosis, which is a characteristic of MPSV-induced myeloproliferative syndrome.

Ontogeny of the v-erbA oncoprotein from the thyroid hormone receptor: an alteration in the DNA binding domain plays a role crucial for v-erbA function.

The avian erythroblastosis virus v-erbA oncogene is imprecisely derived from a cellular gene (c-erbA) encoding a thyroid hormone receptor: the v-erbA protein has sustained both small terminal deletions and internal amino acid sequence changes relative to c-erbA. We report here that one of these missense differences between v- and c-erbA proteins, located in a zinc finger DNA binding domain, has dramatic effects on the biological activities of the encoded protein. Back mutation of the viral coding sequence to resemble c-erbA at this site severely impairs erythroid transformation and produces subtle changes in DNA binding by the encoded protein, suggesting that differences in DNA binding by the viral and cellular proteins may be involved in the activation of v-erbA as an oncogene.

Developmental potential of hematopoietic stem cells determined using retrovirally marked allophenic marrow.

Genetic markers of two general types have been used to assess the number of simultaneously productive stem cells in vivo, retrovirus markers and enzyme or hemoglobin variants. Use of the two techniques has led to different conclusions regarding stem-cell population organization, kinetics, and usage. To better understand this discrepancy, we have combined the two methods by retrovirally marking and transplanting stem cell populations of allophenic mice in which all tissues, including the hematopoietic system, are chimeric. Hematopoietic and lymphoid tissues of engrafted recipients were analyzed by Southern blotting to determine the number and extent of participation of individually marked stem cells. Genotypic chimerism of the same tissues was determined by quantitating electrophoretic variants of glucose phosphate isomerase. This procedure permitted the genotypic identification of individual stem-cell clones. The results demonstrate the participation of few pluripotent stem cells in the repopulation and maintenance of engrafted hematopoietic and lymphoid tissues. Furthermore, stem cells used during the period of early engraftment tended to be of one genotype (DBA/2), whereas stem cells used for long-term maintenance tended to be of the other, coexistent genotype (C57BL/6). We propose that this genotypic specificity reflects functional differences in stem-cell subpopulations and their relative prevalence in different mouse strains suggests a genetic component in the organization and usage of stem cells.