Functional analysis and quantitative determination of the expression profile of human parvovirus B19 (original) (raw)
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Parvovirus B19 genome as a single, two-state replicative and transcriptional unit
Virology, 2006
The variation in the amount of parvovirus B19 DNA and different classes of RNA in permissive and non-permissive infected cells was analysed by means of quantitative real-time PCR and RT-PCR assays. In the permissive bone marrow mononuclear cells, UT7/Epo and KU812Ep6 cells, viral DNA usually increased within 48 hpi, rarely exceeding 2 Logs with respect to input DNA. Viral RNA was always present within 2-6 hpi, its increase paralleled that of viral DNA up to 36-48 hpi, and all the different classes of viral RNA were constantly represented in stable relative amounts throughout the infection cycle. In the non-permissive TF-1 cells, viral DNA did not increase and only one most represented single class of viral RNA was detected. Our data do not support the current model for B19 virus replication and transcription, consisting in different early and late expression patterns, but suggest an alternative model, indicating that the B19 virus genome should be considered a single, two-state replicative and transcriptional unit.
Journal of General Virology, 1994
Erythroid progenitor cells are the main target for B19 parvovirus infection. The UT7 cell line demonstrates a marked erythroid differentiation on induction by erythropoietin (EPO) (UT7/EPO cells) and therefore appears to be a potential target for B19 parvovirus. We aimed to evaluate the presence and localization of B19 nucleic acids in UT7/EPO cells by in situ hybridization. Three digoxigenin-labelled probes were used: two recognized specifically the non-structural region of the B19 genome and one probe was structural region-specific. In our experiment UT7/EPO cells were not permissive to B19 infection. Transcription led to nonstructural and structural gene transcripts without DNA replication or capsid protein synthesis.
Human parvovirus B19: A review
Summary. – Parvovirus B19 (B19V) is a small non-enveloped single-stranded DNA (ssDNA) virus of the family Parvoviridae, the subfamily Parvovirinae, the genus Erythrovirus and Human parvovirus B19 type spe- cies. It is a common community-acquired respiratory pathogen without ethnic, socioeconomic, gender, age or geographic boundaries. Moreover, the epidemiological and ecological relationships between human parvovirus B19, man and environment have aroused increasing interest in this virus. B19V infection is associated with a wide spectrum of clinical manifestations, some of which were well established and some are still controversial, however, it is also underestimated from a clinical perspective. B19V targets the erythroid progenitors in the bone marrow by binding to the glycosphingolipid globoside (Gb4), leading to large receptor-induced structural changes triggering cell death either by lysis or by apoptosis mediated by the nonstructural (NS)1 protein. The pattern of genetic evolution, its peculiar properties and functional profile, the characteristics of its narrow tro- pism and restricted replication, its complex relationship with the host and its ample pathogenetic potential are all topics that are far from a comprehensive understanding. The lack of efficient adaptation to in vitro cellular cultures and the absence of animal models have limited classical virological studies and made studies on B19V dependent on molecular biology. The present review looks at the nature of this virus with the view to provide more information about its biology, which may be useful to the present and future researchers. Keywords: human parvovirus B19; respiratory pathogen; biology; genome; fifth disease; transi
Transfusion Medicine and Hemotherapy, 2010
scribed parvoviruses and has primarily been associated with respiratory infections. Parvoviruses are non-enveloped, isometric viruses with a diameter of 18-26 nm. The particles consist of 60 copies of the capsid protein and contain single-stranded DNA of positive or negative polarity. The B19V genome has a length of 5,596 nucleotides. On the right and on the left, the encoding sequence of 4,830 nucleotides is flanked by inverted terminal repetitive sequences with a length of 383 nucleotides each. Out of these, 365 nucleotides possess the sequence of a palindrome, which leads to the formation of a hairpin like doublestranded structure at both end of the genome (terminal hairpins). DNA strands with positive or negative polarity are distributed in virions with equal frequency. Replication: At least nine overlapping mRNA transcripts are formed during replication. All transcripts initiate at a common promoter (p6) [10]. There are two groups of spliced mRNAs, which encode for virus structure proteins VP1 and VP2, as well as the two proteins with 11 kDa and 7.5 kDa: There is only one unspliced mRNA species encoding for the non-structure protein NS1 with a molecular weight of 77 kDa. Structure proteins: The two structure proteins VP1 and VP2 (capsid proteins) are encoded by the 3'-terminal half of the genome. The main structure protein VP2 (58 kDa) differs from VP1 (84 kDa) by a shorter reading frame (it is by 226 N terminal amino acids shorter). As in the case of all other parvoviruses, the surface of B19V consists of 60 copies of the capsid protein. Virus preparations contain 95-96% VP2 and 4-5% VP1. The structure of empty recombinant virus particles was analyzed in detail by X-ray structure analysis [11], and the infectious particles were characterized by cryo-electron microscopy [12]. Non-structure proteins (NS1): A high homology exists between the NS1 proteins of different parvoviruses. Conserved areas show a significant homology with the T-antigen of polyoma viruses and with the E1-protein of papilloma viruses. NS1 is located in the nucleus of B19V-infected cells and is involved 1 Current Knowledge about the Pathogen 1.1 Characteristics of Parvovirus B19
Characterization of the Early Steps of Human Parvovirus B19 Infection
Journal of Virology, 2012
The early steps of human parvovirus B19 (B19V) infection were investigated in UT7/Epo cells. B19V and its receptor globoside (Gb4Cer) associate with lipid rafts, predominantly of the noncaveolar type. Pharmacological disruption of the lipid rafts inhibited infection when the drug was added prior to virus attachment but not after virus uptake. B19V is internalized by clathrindependent endocytosis and spreads rapidly throughout the endocytic pathway, reaching the lysosomal compartment within minutes, where a substantial proportion is degraded. B19V did not permeabilize the endocytic vesicles, indicating a mechanism of endosomal escape without apparent membrane damage. Bafilomycin A 1 (BafA1) and NH 4 Cl, which raise endosomal pH, blocked the infection by preventing endosomal escape, resulting in a massive accumulation of capsids in the lysosomes. In contrast, in the presence of chloroquine (CQ), the transfer of incoming viruses from late endosomes to lysosomes was prevented; the viral DNA was not degraded; and the infection was boosted. In contrast to the findings for untreated or BafA1-treated cells, the viral DNA was progressively associated with the nucleus in CQ-treated cells, reaching a plateau by 3 h postinternalization, a time coinciding with the initiation of viral transcription. At this time, more than half of the total intracellular viral DNA was associated with the nucleus; however, the capsids remained extranuclear. Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear penetration.
Enhanced Cell-Based Detection of Parvovirus B19V Infectious Units According to Cell Cycle Status
Viruses, 2020
Human parvovirus B19 (B19V) causes various human diseases, ranging from childhood benign infection to arthropathies, severe anemia and fetal hydrops, depending on the health state and hematological status of the patient. To counteract B19V blood-borne contamination, evaluation of B19 DNA in plasma pools and viral inactivation/removal steps are performed, but nucleic acid testing does not correctly reflect B19V infectivity. There is currently no appropriate cellular model for detection of infectious units of B19V. We describe here an improved cell-based method for detecting B19V infectious units by evaluating its host transcription. We evaluated the ability of various cell lines to support B19V infection. Of all tested, UT7/Epo cell line, UT7/Epo-STI, showed the greatest sensitivity to B19 infection combined with ease of performance. We generated stable clones by limiting dilution on the UT7/Epo-STI cell line with graduated permissiveness for B19V and demonstrated a direct correlatio...
Biological and Immunological Relations among Human Parvovirus B19 Genotypes 1 to 3
Journal of Virology, 2007
The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6-and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ϳ10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1-or type 2-infected subjects (long-term immunity) was examined with homo-and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.
Journal of Virology, 1998
Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans -activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photin...
Persistence of human parvovirus B19 in human tissues
Pathologie Biologie, 2002
Human parvovirus B19 infection causes various clinical symptoms, such as rash, arthropathy, anemias and fetal death, but it can also remain asymptomatic. The arthropathies and anemias can become chronic for several years, not infrequently resembling autoimmune syndromes. B19 replicates only in red blood cell precursors of bone marrow or fetal liver, resulting in high-titred short-lived viremia, but viral DNA is detectable also in cells of several other types. Recently B19 DNA has been found, by very sensitive amplification tests, in certain tissues not only of symptomatic but also of healthy individuals for several years or decades after B19 infection. The mere presence of B19 DNA in these tissues of a symptomatic patient (e.g. joints in chronic arthritis or skin in dermatomyositis) thereby does not prove that the present disease is caused by B19. The diagnosis has to be verified by other innovative means. How and why viral DNA persists in the tissues of healthy individuals is under investigation. 2002 Éditions scientifiques et médicales Elsevier SAS chronic infection / disease association / DNA persistence / erythrovirus B19 / polymerase chain reaction (PCR)