Parvovirus B19 genome as a single, two-state replicative and transcriptional unit (original) (raw)
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Functional analysis and quantitative determination of the expression profile of human parvovirus B19
Virology, 2008
Comprehension of the pathogenetic potential of human parvovirus B19 requires the definition of the complete spectrum of cellular tropism and a functional analysis of the viral genome in infected cells. In this study, we carried out a systematic functional analysis of B19 virus genome in the course of infection of susceptible bone marrow mononuclear cells and myeloblastoid UT7/EpoS1 cells, in terms of dynamics of nucleic acid synthesis. A PCR array was designed and a comprehensive analysis was performed by quantitative PCR and RT-PCR, yielding extended information on the presence and abundance of the diverse classes of viral nucleic acids, on the temporal regulation of genome expression and on its relationship with the cell cycle. The analysis performed indicate that the synthesis of viral nucleic acids is correlated to the progression through the S phase of the cell cycle, that an extended pattern of transcriptional activity occurs throughout the course of infection, with a maximal rate of transcription preceding the onset of S-phase dependent replication of the viral genome, and that utilization of transcript processing signals is relatively constant throughout the course of infection. The information obtained led to the definition of a unified model of functional and expression profiling of parvovirus B19 genome.
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
Journal of Virological Methods, 1994
Modification of an in situ polymerase chain reaction (ISPCR) technique is described for the detection of B19 parvovirus infection. Specific amplification of B19 DNA inside fixed cells was followed by hybridisation with a digoxigenin-labelled probe and then visualised by immunochemical reaction. The assay had higher sensitivity compared to direct in situ hybridisation and still allowed cellular localisation and characterisation of infected cells. This assay can be used as a confirmatory method for PCR in tissues and will allow further identification of tissues permissive for B19 parvovirus infection.
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.
Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02
Journal of Virology
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 nonnal 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.
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.
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)
A new quantitative PCR for human parvovirus B19 genotypes
Journal of Virological Methods, 2015
Parvovirus B19 (B19V) is a minute ssDNA virus associated with a wide range of diseases from childhood erythema to fetal death. After primary infection, the viral genomes persist lifelong in solid tissues of most types. Quantification of the viral DNA is important in the timing of primary infection, assessment of tissue persistence and screening of blood donor plasma. In this study, we present a new PCR assay for detection and quantification as well as for differentiation of all three B19V genotypes. A new B19V qPCR was designed to target a 154-bp region of the NS1 area. Serum, plasma and solid tissue samples were suitable for testing in the assay. The WHO International Reference Panel for Parvovirus B19 Genotypes was utilized to validate the assay for detection of different genotypes of B19V in clinical material. Each panel member yielded, by the new qPCR, a quantity similar to the one reported by National Institute for Biological Standards and Control (NIBSC). The qPCR was specific for B19V and amplified and quantified all three genotypes with detection sensitivities of ≤10 copies/reaction. The differentiation of B19V genotypes was performed by Sanger sequencing of the amplified products.
1994
We performed a systematic study to identify cellular factors that bound to the terminal repeat region of B19 parvovirus. Using electrophoretic mobility shift assays, we detected one cellular factor which prominently bound to the repeat region. The factor was purified from K562 nuclear extract by specific DNA affinity column * Corresponding author. Mailing address: Hematology Branch, National Heart, Lung, and Blood Institute, Bldg. 10, Room 7C103, NIH, Bethesda, MD 20892. Fax: (301) 496-8396.