Enhanced inhibition of parvovirus B19 replication by cidofovir in extendedly exposed erythroid progenitor cells.

Human parvovirus B19 (B19V) commonly induces self-limiting infections but can also cause severe clinical manifestations in patients with underlying haematological disorders or with immune system deficits. Currently, therapeutic options for B19V entirely rely on symptomatic and supportive treatments since a specific antiviral therapy is not yet available. Recently a first step in the research for active compounds inhibiting B19V replication has allowed identifying the acyclic nucleoside phosphonate cidofovir (CDV). Herein, the effect of CDV against B19V replication was characterized in human erythroid progenitor cells (EPCs) cultured and infected following different experimental approaches to replicate in vitro the infection of an expanding erythroid cell population in the bone marrow. B19V replication was selectively inhibited both in infected EPCs extendedly exposed to CDV 500µM (viral inhibition 82%) and in serially infected EPCs cultures with passage of the virus progeny, constantly under drug exposure (viral inhibition 99%). In addition, a potent inhibitory effect against B19V (viral inhibition 92%) was assessed in a short-term infection of EPCs treated with CDV 500µM 1day before viral infection. In the evaluated experimental conditions, the enhanced effect of CDV against B19V might be ascribed both to the increased intracellular drug concentration achieved by extended exposure, and to a progressive reduction in efficiency of the replicative process within treated EPCs population.

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 24 hpi up to 48 hpi. The method, used together with quantitative PCR techniques, can be very useful to describe the kinetics of B19V infection within a heterogeneous cell population.

Single-cell chemiluminescence imaging of parvovirus B19 life cycle.

Human parvovirus B19 (B19V) is a single-stranded DNA virus. The genome encodes a multifunctional non-structural protein (NS), two capsid proteins (VP1, VP2) and other small non-structural proteins (7.5 kDa, 9 kDa, 11 kDa). Within sensitive cells, B19V can achieve a productive replicative cycle or, on the contrary, establish persistence; differences in its expression profile have been yet investigated following in vitro infections by methodologies enabling information on the entire infected cell population. Conversely, the present study reports quantitative data on the production of B19V analytes (DNA, RNAs, and proteins) at single cell-level, underlining cell-to-cell differences through the viral specific macromolecular synthesis process. Microscope imaging assays (in situ hybridization and immunocytochemical assays), exploiting chemiluminescence as principle detection and targeting viral nucleic acids and antigens, have been performed on a permissive cell line following in vitro infection. Chemiluminescence, involving the emission of photons deriving from a chemical reaction, provided the localization and quantitative detection of analytes down to a few molecules within infected cells. In our experimental conditions, B19V transcriptional activity, leading to the production of NS and VP RNAs, has been detected early in the viral cycle (from 12h post-infection, hpi) and before genome replication, starting at 24 hpi. The analysis of VP RNAs and related proteins suggested an inhibitory effect on capsid protein translation, as a post-transcriptional regulation events. Indeed, high levels of VP transcripts have been detected at early stages of infection while the proteins accumulated within cells only at 48-72 hpi.

miRNA and tropism of human parvovirus B19.

Parvovirus B19 has an extreme tropism for human erythroid progenitors. Here we propose the hypothesis explaining the tropism of human parvovirus B19. Our speculations are based on experimental results related to the capsid proteins VP1 and VP2. These proteins were not detectable in nonpermissive cells in course of these experiments, although the corresponding mRNAs were synthesized. Our interpretation of these results is an inhibition of translation in nonpermissive cells by human miRNAs. We bring support to our hypothesis and propose detailed experimental procedure to test it.

Chloroquine and its derivatives exacerbate B19V-associated anemia by promoting viral replication.

BACKGROUND: An unexpectedly high seroprevalence and pathogenic potential of human parvovirus B19 (B19V) have been observed in certain malaria-endemic countries in parallel with local use of chloroquine (CQ) as first-line treatment for malaria. The aims of this study were to assess the effect of CQ and other common antimalarial drugs on B19V infection in vitro and the possible epidemiological consequences for children from Papua New Guinea (PNG). METHODOLOGY/PRINCIPAL FINDINGS: Viral RNA, DNA and proteins were analyzed in different cell types following infection with B19V in the presence of a range of antimalarial drugs. Relationships between B19V infection status, prior 4-aminoquinoline use and anemia were assessed in 200 PNG children <10 years of age participating in a case-control study of severe infections. In CQ-treated cells, the synthesis of viral RNA, DNA and proteins was significantly higher and occurred earlier than in control cells. CQ facilitates B19V infection by minimizing intracellular degradation of incoming particles. Only amodiaquine amongst other antimalarial drugs had a similar effect. B19V IgM seropositivity was more frequent in 111 children with severe anemia (hemoglobin <50 g/L) than in 89 healthy controls (15.3% vs 3.4%; P = 0.008). In children who were either B19V IgM or PCR positive, 4-aminoquinoline use was associated with a significantly lower admission hemoglobin concentration. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that 4-aminoquinoline drugs and their metabolites exacerbate B19V-associated anemia by promoting B19V replication. Consideration should be given for choosing a non-4-aminoquinoline drug to partner artemisinin compounds in combination antimalarial therapy.

Placental endothelial cells can be productively infected by Parvovirus B19.

BACKGROUND: Parvovirus B19 vertical transmission occurs in about 30% of cases of maternal infection and may result in foetal hydrops or intrauterine foetal death. Details on the mechanism of transplacental transmission of B19 virus and subsequent foetal infection have not been elucidated. OBJECTIVE: To investigate the extent and distribution of B19 virus infection in placental tissues. STUDY DESIGN: Virological, histological, electron microscopy, immunohistochemical and immunofluorescence analysis of placental tissues obtained from a case of intrauterine foetal death caused by B19 virus. RESULTS: Real-time PCR analysis showed B19 virus DNA in placental samples. Histology, immunohistochemistry and electron microscopy demonstrated the concomitant infection of both foetal erythroid precursors and placental endothelial cells. In situ hybridisation for B19 virus nucleic acids, immunohistochemistry for B19 virus proteins and double labelling immunofluorescence confirmed that endothelial cells were productively infected by B19 virus. CONCLUSION: Foetal capillary endothelium in placental villi can be an additional target of productive B19 virus infection. Infection of placental endothelial cells may lead to a structural and functional damage critical both for altering maternal-foetal blood exchanges and for spreading the infection to the foetus, possibly concurring to the development of foetal hydrops and intrauterine foetal death.

Erythema infectiosum following generalized petechial eruption induced by human parvovirus B19.

Parvovirus B19 is a DNA virus responsible for a wide spectrum of clinical illnesses. Among dermatological manifestations, the most common is erythema infectiosum, also known as the fifth disease. In 1990 Harms et al first described a papular-purpuric gloves and socks syndrome (PPGSS) due to parvovirus B19. It is an acute acral dermatosis characterized by an eruption of petechiae and small purpuric papules affecting the hands and feet in a gloves-and-socks distribution. Recently it was observed that PPGSS may be associated with involvement of the peri-oral region and the chin (acropetechial syndrome) and other sites provoking unusual presentation of the rash. We describe a patient with an acral purpura with the features of the "acropetechial syndrome" involving the buttocks, genital and axillary regions who subsequently developed a maculopapular eruption with the characteristics of the fifth disease. Parvovirus B19 DNA was detected by polymerase chain reaction (PCR) both in skin vasculitic lesions and in the serum during the petechial eruption, before the onset of antibodies. The immune response coincided with the development of the exanthem, suggesting a direct role of parvovirus B19 in the pathogenesis of endothelial cell injury.

Human parvovirus B19 infection of monocytic cell line U937 and antibody-dependent enhancement.

Human parvovirus B19 (B19) infects human erythroid lineage cells. Accumulating evidence also shows that B19 is detectable in nonerythroid lineage cells in vivo, but the mechanism of infection is still not clear. In this study, we explored the mode of B19 infection of human monocytic cell line U937. An in vitro infection study demonstrated B19 binding of U937 and slow replication of B19-DNA with B19-NS1 mRNA transcription. B19-DNA replication in U937 was accompanied by undetectable level of B19-VP1 mRNA transcription, indicating that B19 infection of U937 cells may be abortive. Levels of B19-DNA and B19-NS1 mRNA transcription increased in the presence of anti-B19 IgG antibodies, but this effect decreased in the presence of anti-Fc receptor antibodies, showing antibody-dependent enhancement by B19 infection. Antibody-dependent enhancement also caused the increased production of TNFalpha in U937. This study is the first to suggest B19 infection of nonerythroid lineage cells with antibody-dependent enhancement.

Hypoxia enables B19 erythrovirus to yield abundant infectious progeny in a pluripotent erythroid cell line.

B19 may cause mild to severe clinical manifestations. Owing to the remarkable tropism of B19 for red blood cell progenitors, there is a lack of satisfactory cell lines fully permissive for B19. Because the local oxygen pressure may influence viral replication, we used hypoxia to improve the sensitivity of our infectivity assay in order to link B19 DNA detected by PCR to the presence of infectious B19 particles in plasma. Plasma samples and the WHO International Standard for B19 DNA detection by PCR were used to infect the pluripotent human erythroid cell line KU812F under different oxygen pressures. Specific human anti-B19 IgG was found to reduce infectivity. Low oxygen pressure led to higher yields of infectious B19 progeny and to a higher level of viral transcription than observed under normoxia. This sensitive infectivity assay is a promising model for studying B19 biology, identifying neutralising antibodies, and evaluating new virus inactivation methods.

An assay for parvovirus B19 neutralizing antibodies based on human hepatocarcinoma cell lines.

BACKGROUND: Although intravenous immune globulin (IVIG) is used widely for managing parvovirus B19 infections, IVIG products are not monitored routinely for the presence of parvovirus B19 neutralizing antibody. STUDY DESIGN AND METHODS: An assay has been developed to measure parvovirus B19 infectivity and neutralization activity based on two hepatocarcinoma cell lines (HepG2 and HuH7). The sources of parvovirus B19 were B19-DNA-containing plasma samples. Neosynthesized progeny in supernatants of infected cells were quantified by nested polymerase chain reaction. To validate the model, purified rabbit antibodies to different capsid protein sequences and IVIG preparations were tested. RESULTS: The number of parvovirus B19 infectious neovirions in supernatants of infected cells increased with infection time. Both rabbit antibodies and IVIG products inhibited parvovirus B19 infectivity when incubated overnight with virus. The efficacy of IVIG to neutralized parvovirus B19 was product-related. CONCLUSION: This assay for parvovirus B19 neutralization activity provides an improved and more specific method for selecting donors to produce IVIG with a high titer of parvovirus B19 neutralizing activity.

Removal of small non-enveloped viruses by nanofiltration.

BACKGROUND AND OBJECTIVES: Nanofiltration is one of the most effective virus reduction methods in the manufacturing process of plasma products. However, it is difficult to remove small viruses from high molecular weight protein preparations like immunoglobulin G or factor VIII complex by nanofiltration, because the size of the protein is similar to that of viruses. In order to separate the viruses from these proteins by nanofiltration, it is necessary to change the size of either one. In this study, we report that such non-enveloped viruses as human parvovirus B19 (B19), human encephalomyocarditis virus (EMC) or porcine parvovirus (PPV) aggregate in the presence of certain kinds of amino acids and could be easily removed by nanofiltration. MATERIALS AND METHODS: 0.3 M Glycine (or other amino acid) solution spiked with viruses was subjected to dead-end single filtration with a 35-nm pore-size filter. Virus removal by nanofiltration was either evaluated by PCR or by infectivity assay. RESULTS: B19 in a 0.3 M glycine solution was reduced to 1:10(7.5) (7.5-log) by nanofiltration with a 35-nm pore-sized filter, whereas in PBS it was not reduced. Similarly, B19 was also reduced when suspended in other amino acids solutions. This effect was also confirmed with the other small non-enveloped viruses EMC or PPV. When 5% globulin or 5% albumin was added to a 0.3 M glycine solution, the removal rate was decreased. CONCLUSIONS: These data suggest that viruses in the presence of certain kinds of amino acids could be aggregated and effectively removed by a filter that has a pore size larger than the size of the viruses.

Inactivation of parvovirus B19 during pasteurization of human serum albumin.

BACKGROUND: It has been shown that HSA may be contaminated with parvovirus B19 (B19) DNA. However, the presence of B19 DNA does not necessarily indicate infectious virus. HSA is pasteurized at 60 degrees C for 10 hours and it remains unclear whether this procedure inactivates B19. Studies with animal parvoviruses indicate considerable heat resistance at 60 degrees C. However, due to the lack of a suitable cell culture system, the pasteurization process has not been investigated in the past. STUDY DESIGN AND METHODS: The recently described cell clone KU812Ep6 was used to establish a system for investigation of B19 inactivation during pasteurization. Virus-infected cells were detected by immunofluorescent staining of viral capsid antigen and by RT-PCR assay of virus-specific capsid mRNA. RESULTS: B19 was inactivated after 10 minutes at 60 degrees C for > or = 4 log. In contrast, porcine parvovirus was widely resistant at 60 degrees C. Inactivation of B19 was independent of the analyzed albumin products (5, 20, and 25% albumin from three manufacturers) and from the specific virus source used for the inactivation experiments. Degradation of B19 DNA by deoxyribonuclease I treatment after pasteurization indicated that the virus capsid is destroyed during heat treatment. CONCLUSION: Heat resistance of B19 markedly differs from heat resistance of animal parvoviruses. While animal parvoviruses widely withstand pasteurization of albumin, B19 was rapidly inactivated. These results confirm the safety of pasteurized albumin and are in line with its good clinical safety record with respect to B19 infection. However, conclusions regarding the safety of other blood-derived medicinal products should not be derived from B19 inactivation in albumin, because different processes or different composition of product intermediates may significantly influence B19 stability during heat treatment.

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.

Parvovirus B19 and erythroid cells.

Parvovirus B19 is a human erythrovirus, i.e. which induces the death of erythroid progenitors. In such cells, until now only ubiquitous transcription factors have been described to regulate promoter driven gene expression. Their possible interactions with erythroid specific transcription factors merit further investigations. Effectively, the high level of replication of B19 in erythroid cells is not well understood. In addition to apoptosis, necrosis or inhibition of cell growth, the death of B19 infected erythroid progenitors has been never clearly reported as the result of immunological attack: this mecanism will merit further investigations. The interactions with other cell types in vitro remain at present not well defined but many obstacles have been mentioned which counteract B19 expression.

Increase in B19 viral load prior to relapse of anaemia in an AIDS patient with persistent B19 infection.

Anaemia caused by persistent parvovirus B19 infection can be remitted by intravenous immunoglobulin (IVIG) treatment. However, maintenance IVIG therapy is required in some AIDS patients as well as patients with combined immuno-deficiency because of relapse of anaemia. We used a simple semi-quantitative polymerase chain reaction (PCR) method to trace plasma B19 viral load after administration of IVIG. We noted that the rebound of B19 viral load occurred 6 weeks prior to detection of anaemia. This method may be helpful in determining the interval of maintenance IVIG therapy.

Fulminant parvovirus infection following erythropoietin treatment in a patient with acquired immunodeficiency syndrome.

We report the case of a 41-year-old black man with acquired immunodeficiency syndrome who developed a severe chronic anemia due to parvovirus infection. Bone marrow biopsy revealed erythroid aplasia. The infectious nature of the anemia was not recognized, and the patient was treated with erythropoietin. The patient's reticulocyte response was inadequate, however, and he remained anemic. A second bone marrow biopsy showed erythroid hyperplasia and prominent intranuclear parvovirus inclusions within erythroid progenitors. Erythropoietin was discontinued and was followed by a course of intravenous immunoglobulin, which resulted in rapid correction of anemia. To our knowledge, this is the first reported case of fulminant human parvovirus infection exacerbated by erythropoietin administration and documented by sequential bone marrow histologic examination. This case illustrates the critical importance of considering parvovirus in the etiology of chronic anemia with erythroid aplasia in immunocompromised patients.

[Virological diagnosis of an outbreak of fever and rashes caused by parvovirus B19, Cuba, 1995]. / Diagnóstico virológico de un brote de fiebre y rash producido por parvovirus B19, Cuba, 1995.

The results obtained in the study of an a outbreak of fever and rash occurred in Havana City in March, 1995, are reported. Dengue, measles, rubella, herpes simplex, and Epstein Barr were discarded as causal agents of the outbreak in the samples of 35 patients. Parvovirus B19 was identified as the causing agent of the outbreak by the detection of IgM antibodies and the polymerase chain reaction technique (PCR). The infection produced by this agent was confirmed in 14/18 samples (77.7%) by some of the techniques used. This study makes reference to the first outbreak of Parvovirus B19 that was proved in Cuba.