B19-infected Cells Research Articles

A flow-FISH assay for the quantitative analysis of parvovirus B19 infected cells

Human parvovirus B19 (B19V) replication is a process highly dependent on the cellular environment, therefore methodologies allowing for analysis at single cell level could represent effective tools to understand cell-to cell differences in the replication process and to investigate cell–virus interactions. Fluorescence in situ hybridization (FISH) can be combined with flow cytometry (flow-FISH) to enable the detection of target nucleic acid sequences in thousands of individual cells in a short amount of time. In the present study, a flow-FISH assay based on the use of a digoxigenin-labeled genomic probe has been developed to discriminate B19V infected cells following in vitro infection of UT7/EpoS1 cell line and EPCs (erythroid progenitor cells) generated from peripheral blood mononuclear cells. In B19V infected UT7/EpoS1 and EPCs, viral nucleic acids were detected by the flow-FISH assay starting from 24hpi up to 48hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population.

Development of chemiluminescent assays for the quantitative detection and imaging of 5-bromo-2′deoxyuridine-labeled DNA in parvovirus B19-infected cells

Incorporation of exogenous analogues is a widely used method to evaluate DNA synthesis in cultured cells exposed to exogenous factors such as infectious agents. Herein, two new quantitative methodologies exploiting ultrasensitive chemiluminescence (CL) detection of 5-bromo-2'-deoxyuridine (BrdU) have been developed: a CL microscope imaging assay to evaluate BrdU labelling at single-cell level and a CL dot-blot assay to measure the amounts of DNA produced in the course of an in vitro infection of proliferating cells. The assays have been optimized on UT7/EpoS1 cells cultured in presence of different concentrations of BrdU (from 3 to 100 μM) and used to monitor parvovirus B19 (B19) life cycle in infected cells. The CL microscope imaging assay provided a detailed localization of BrdU-labelled nuclei allowing to count positive cells and measure their related CL intensity signals. The CL dot-blot assay, coupled with a B19 capture procedure performed with a specific peptide nucleic acid probe, has been designed to discriminate and selectively quantify cellular and viral BrdU-labelled genomes. Quantitative evaluation of BrdU-labelled B19 DNA has been achieved by means of a CL calibration curve. The high detectability, down to 2 × 10(6) B19 genome copies, and the linear range extending up to 5 × 10(8) copies make the method suitable to evaluate the amounts of B19 DNA produced throughout a replicative viral cycle.

An Association Between Parvovirus B19 and Kikuchi–Fujimoto Disease

Although Kikuchi-Fujimoto disease (KFD) has a higher prevalence among Asian countries, it is a well-defined entity throughout the world. However, its etiology and pathogenesis remain undetermined. To study whether B19 infection is associated with idiopathic KFD (iKFD), we examined the presence of the viral genome and proteins in paraffin-embedded tissues of lymph nodes retrospectively from 33 iKFD patients and 16 age- and sex-matched control subjects by nested PCR (nPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). B19 was detected in 87.1, 69.7, and 57.6% of iKFD specimens by nPCR, ISH, and IHC, respectively, whereas the virus was positive in only 56.3, 31.3, and 25.0% of control tissues by the respective methods (nPCR: p = 0.029; ISH: p = 0.011; IHC: p = 0.032). The IHC-ISH double-staining assay demonstrated that B19-infected cells were mainly lymphocytes and a small number of histiocytes. These results showed for the first time a high frequency of localized persistence of B19 in lymph nodes from iKFD patients, suggesting that B19 might play an important role in the pathogenesis of iKFD.

Survival of parvovirus B19-infected cells by cellular autophagy

Human parvovirus B19 (B19) is a well-known pathogenic agent which causes apoptosis in erythrocyte lineage cells. Here, we provide the first evidence that mitochondrial autophagy is specifically found in the B19-infected cells. The protein expression ratio for LC3-II/LC3-I increased significantly in infected cells, indicating possible involvement of cellular autophagy in the infection process. Immunofluorescence confocal microscopy analyses revealed that B19 infection induced an intracellular autophagosome as judged by endogenous LC3 staining. Moreover, inhibition of autophagy by 3-MA significantly facilitated B19-infection-mediated cell death. These results suggest a novel mechanism by which B19-infected cells survive by cellular autophagy.

Parvovirus B19 and the pathogenesis of anaemia.

Human parvovirus B19 (B19) infection causes human bone marrow failure, by affecting erythroid-lineage cells which are well-known target cells for B19. The anaemia induced by B19 infection is of minor clinical significance in healthy children and adults, however, it becomes critical in those afflicted with haemolytic diseases. This condition is called transient aplastic crisis, and the pathogenesis is explained by the short life-span of red blood cells. Similarly, fetuses are thought to be severely affected by B19-intrauterine infection in the first and second trimester, as the half-life of red blood cells is apparently shorter than RBC at the bone marrow haematopoietic stage. On the other hand, B19 is also the causative agent of persistent anaemia in immunocompromised patients, transplant recipients and infants. The deficiencies of appropriate immune responses to B19 impair viral elimination in vivo, which results in enlargement of B19-infected erythroid-lineage cells. The B19-associated damage of erythroid lineage cells is due to cytotoxicity mediated by viral proteins. B19-infected erythroid-lineage cells show apoptotic features, which are thought to be induced by the non-structural protein, NS1, of B19. In addition, B19 infection induces cell cycle arrests at the G(1) and G(2) phases. The G(1) arrest is induced by NS1 expression prior to apoptosis induction in B19-infected cells, while the G(2) arrest is induced not only by infectious B19 but also by UV-inactivated B19, which lacks the ability to express NS1. In this review, we address the clinical manifestations and molecular mechanisms for B19-induced anaemia in humans and a mouse model, and of B19-induced cell cycle arrests in erythroid cells.

Identification and Characterization of a Family of 11-kDa Proteins Encoded by the Human Parvovirus B19

The human pathogenic parvovirus B19 directs the synthesis of two size classes of small abundant RNAs. It is shown that the smallest RNAs, of 500 and 600 nt, are translated into at least two 11-kDa proteins in B19-infected human leukemic bone marrow cells. A COS-7 cell expression system was used to demonstrate that the different forms of the protein result from translational initiation at multiple AUG codons in the same 94 aa ORF. The 11-kDa proteins were localized to the cytoplasm of transfected COS-7 cells using indirect immunofluorescence. However, their localization was at least partially nuclear in B19-infected cells. In COS-7 cells the expression of the major B19 structural and nonstructural proteins was not affected in the absence of the expression of the 11-kDa proteins.

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.