MAB8292 Sigma-AldrichAnti-Parvovirus B19 Antibody, clone 521-5D

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo-expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G(2)/M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G(2)/M significantly, during B19V infection.

Fluorescence correlation spectroscopy (FCS) was used in monitoring human parvovirus B19 virus-like particle (VLP) antibody complexes from acute phase and past-immunity serum samples. The Oregon Green 488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2 s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the fluorescent VLPs with an acute phase serum sample, the mobility information obtained from the fluorescence intensity fluctuation by autocorrelation analysis showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1), corresponding to an average radius of 157 nm. In contrast, incubation of the fluorescent VLPs with a past-immunity serum sample gave an average diffusion coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7 x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed with acute phase or past-immunity sera. Thus, VLP-antibody complexes with different diffusion coefficients could be identified for the acute phase and past-immunity sera. FCS measurement of VLP-immune complexes could be useful in distinguishing between antibodies present in acute phase or past-immunity sera as well as in titration of the VLPs.

Erythroid lineage cells are target cells for human parvovirus B19, and a natural infection often results in transient anemia. To determine whether recombinant B19 capsid proteins (VP1/VP2) also inhibit human hematopoietic progenitor growth, a model system was set up. The B19 capsids were inoculated into primary cultures of hematopoietic stem cells derived from human fetal liver, resulting in a 70-95% reduction of BFU-E (burst-forming unit erythroid cells) as compared with the medium control. A similar effect was seen in human hematopoietic stem cell cultures derived from cord blood and adult bone marrow. Preincubation of the B19 capsids with either a monoclonal antibody to the virus or with B19 IgG positive human sera reduced the inhibitory effect. Furthermore, the inhibitory effect could be reduced by preincubating the target cells with a monoclonal antibody to the cellular receptor for the virus, the P antigen. These findings thus show that the inhibition of colony formation of human hematopoietic stem cells can occur in the absence of parvovirus B19 nonstructural proteins. We speculate that B19 capsid could provide a possible strategy to downregulate indigenous hematopoiesis in fetal stem cell transplantations.

Human parvovirus B19 can cause congenital infection with variable morbidity and mortality in the fetus and neonate. Although much information exists on the B19-specific antibody response in pregnant women, little information is available describing the cell-mediated immune (CMI) response at the maternal-fetal interface. The focus of this study was to characterize the CMI response within placentas from women who seroconverted to B19 during their pregnancies and compare it to controls. Immunohistochemical techniques were used to identify the various immune cells and the inflammatory cytokine present within placental tissue sections. Group 1 consisted of placentas from 25 women whose pregnancies were complicated by B19 infection; 6 women with good outcome (near-term or term delivery), and 19 with poor outcome (spontaneous abortion, nonimmune hydrops fetalis, or fetal death). Group 2 consisted of placentas from 20 women whose pregnancies were complicated with nonimmune hydrops fetalis of known, noninfectious etiology. Group 3 consisted of placentas from eight women whose pregnancies ended in either term delivery or elective abortion. The results of the study revealed a statistically significant increase in the number of CD3-positive T cells present within placentas from group 1 compared to group 2 or 3 (13.3 versus 2 and 1, respectively) (P less than 0.001). In addition, the inflammatory cytokine interleukin 2 was detected in every placenta within group 1 but was absent from all placentas evaluated from groups 2 and 3. Together, these findings demonstrate evidence for an inflammation-mediated cellular immune response within placentas from women whose pregnancies are complicated with B19 infection.

We previously reported detection of human parvovirus B19 DNA in livers from patients requiring transplantation for acute fulminant liver failure. In this study, we used immune adherence PCR (IA-PCR) to bind B19 virions in recipient native liver onto solid phase with specific monoclonal antibodies followed by PCR amplification of virion DNA. IA-PCR had sensitivity and specificity similar to conventional PCR. We examined liver tissue from 16 patients with non-A, non-B, non-C, non-E (NA-E) acute fulminant liver failure (AFLF) (6 of unknown etiology associated with aplastic anemia (AA), 4 of unknown etiology without AA; and 6 patients with AFLF of known etiology). IA-PCR detected B19 virions in 5 of 6 (83%) of livers from patients with idiopathic NA-E AFLF associated with AA and in 2 of 3 (75%) without AA, compared to 1 of 6 (17%) of livers from patients with AFLF of known etiology and to 6 of 34 (18%) of 34 control patients with chronic or neoplastic liver disease. Viral mRNA encoding the structural protein was detected in the liver tissue from three B19 IA-PCR positive patients with AFLF. Detection of B19 virions and mRNA for capsid proteins provided strong evidence for B19 infection during the course of NA-E AFLF and argues for involvement of B19 virus in liver injury.

A patient with refractory anemia with an excess of blasts in transformation developed pancytopenia and a concurrent interstitial pneumonia 110 days after allogeneic bone marrow transplantation. Bone marrow examination showed 0.4% giant proerythroblasts and 86.2% granulocytes, some of them large with a bizarre configuration and the others of normal size. Serum folate level was found low, 0.6 ng/ml. Immunocytochemistry with a B19-specific monoclonal antibody MAB8292 revealed B19 capsid antigen only in erythroblasts and large, bizarre granulocytes, but not in granulocytes of normal size. In situ hybridization of bone marrow cells using digoxigenin-labeled DNA probes detecting parvovirus B19 also demonstrated positive signals in 8.5% of marrow cells. Parvovirus B19 DNA was isolated from the serum and the bronchoalveolar lavage fluid of this patient by the polymerase chain reaction. These findings suggest that neutropenia may be caused by an involvement with parvovirus B19 though a deficiency of folic acid may have in part contributed to the genesis of neutropenia in the patient. The relevance of parvovirus B19 to the interstitial pneumonia remains unclear.