Article

Detection of human Parvovirus B19 DNA by using the polymerase chain reaction

Department of Pediatrics, Children's Medical Center, Medical College of Virginia, Richmond 23298.
Journal of Clinical Microbiology (Impact Factor: 4.23). 02/1990; 28(1):65-9.
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ABSTRACT The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699 nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a 699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR. PCR was 10(4) times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 10(7) times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19 infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22 serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid, ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when testing only a single serum sample.

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Available from: William C Koch, Nov 12, 2014
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    • "PCR can detect fewer particles of CPV than ELISA and it is 10 to 100 folds more sensitive than electron microscopy (Schunck et al., 1995). PCR has successfully been applied to detect human parvovirus (Koch and Adler, 1990), porcine parvovirus (Molitor et al., 1991) and CPV (Mochizuki et al., 1993; Hirasawa et al., 1994; Uwatoko et al., 1995) in faecal samples. The time taken in sample preparation, important for removing the inhibitory substances present in clinical samples, limits PCR test "
    Indian Journal of Virology 01/2006; · 0.36 Impact Factor
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    • "Parvovirus IgM and IgG-specific antibodies were detected by immunoblotting as described [16]. Detection of parvovirus B19 DNA in the patient's serum was carried out by PCR amplification as described [17]. PCR reactions were performed on 3 Al serum samples run in duplicates, and positive and negative controls were run concurrently. "
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    ABSTRACT: Parvovirus B19-induced chronic anemia has been associated with failure to mount an effective neutralizing antibody response. We describe an adolescent male with a 13-year history of parvovirus B19-induced anemia as the primary manifestation of X-linked hyper IgM immunodeficiency (XHIM). This patient, whose serum IgG concentration was at the low end of the normal range and who mounted IgG antibody responses to T cell-dependent antigens, suffered from a nonsense mutation (R11 --> X) in the CD40 ligand (CD40L) gene. This resulted in low-level expression of a mutant CD40L predicted to lack the cytoplasmic domain. Intravenous immunoglobulin therapy alone or in combination with interferon gamma, given in the context of impaired Th1 cytokine production, suppressed but did not eradicate the infection. These results highlight the critical function of the CD40/CD40L pathway in parvovirus B19 infection and suggest that subtle defects in this pathway may underlie cases of chronic parvovirus B19 infection atypical of XHIM.
    Clinical Immunology 12/2005; 117(3):231-7. DOI:10.1016/j.clim.2005.08.005 · 3.99 Impact Factor
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    ABSTRACT: Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype. Parvorokkovirus eli B19 on hyvin yleinen ihmisiä infektoiva virus joka aiheuttaa ihmiselle monenlaisia sairauksia. Tavallisin on lapsilla ja nuorilla esiintyvä ihottumatauti, parvorokko. Raskauden aikana virus voi siirtyä äidistä sikiöön aiheuttaen jopa sikiön kuoleman. B19-virus lisääntyy luuytimessä, punasolujen esiasteissa, joten se voi tiettyjä veritauteja sairastaville henkilöille aiheuttaa rajun lyhytkestoisen anemian, joka hoitamattomana voi olla kohtalokas. Immuunipuutteiset henkilöt voivat sairastua vuosienkin mittaiseen anemiaan. Parvorokkoviruksen aiheuttama niveltulehdus on yleinen etenkin naisilla ja kestoltaan sekä vaikeusasteeltaan vaihteleva, vaikeimmat tapaukset jopa nivelreuman kaltaisia. Tässä työssä löysimme uuden, ennestään tuntemattoman parvovirusvariantin (LaLi), joka perimäainekseltaan eroaa sekä B19-prototyypistä että vuonna 1998 löydetystä V9-variantista. Nykyisin parvorokkovirukset jaetaankin kolmeen tyyppiin: perinteiset isolaatit kuuluvat tyyppiin 1, LaLi:n kaltaiset tyyppiin 2 ja V9:n kaltaiset tyyppiin 3. Herkkiä geenimonistusmenetelmiä käyttämällä parvorokkoviruksen DNA:n on osoitettu säilyvän perusterveiden, oireettomien ihmisten kudoksissa vieläpä vuosia infektion jälkeenkin. Näin ollen kudosperäistä B19-DNA-löydöstä sellaisenaan ei voida käyttää taudinmääritykseen. Osoitimme myös tyypin 2 DNA:n varastoituvan ihmisten kudoksiin, jopa prototyyppiä yleisemmin. Kudosten parvorokkovirus-DNA:n varastointimekanismia ei vielä tunneta, mutta osoitimme virusgeenien säilyvän pilkkoutumatta ja muuntumatta. Vielä ei tiedetä kykenevätkö nämä varastoidut genomit joissain tilanteissa tuottamaan esim. RNA:ta, proteiineja tai jopa infektiivistä virusta, ja siten aiheuttamaan ihmisille myöhäiskomplikaatioita. Parvorokkoviruksen on osoitettu leviävän myös verituotteiden välityksellä. Evaluoimme verituotteiden seulontaan tarjolla olevat kaupalliset geenimonistusmenetelmät uusien virustyyppien suhteen ja osoitimme, etteivät kaikki käytössä olevat testit löydä uusia parvorokkovirustyyppejä. Lisäksi tarkastelimme uusien virustyyppien esiintyvyyttä suomalaisten verenluovuttajien keskuudessa seulomalla n. 140 000 luovuttajan näytteet: tästä joukosta löytyi ainoastaan tyyppiä 1. Toisin sanoen, vaikka tyypin 2 DNA:ta löytyi yleisesti terveiden henkilöiden ihonäytteistä, tyypin 2 virusta ei suomalaisten verenluovuttajien keskuudessa havaittu. Vertaillessamme B19-prototyypin ja uusien B19-tyyppien biologisia ominaisuuksia sekä infektiokykyä soluviljelmissä osoitimme, että myös uudet parvorokkovirustyypit ovat biologisesti aktiivisia, infektiokykyisiä viruksia. Lisäksi vertailimme tyyppien 1 ja 2 immunologiaa ja vasta-ainediagnostiikkaa: osoitimme että kumpaa tahansa virustyyppiä kohtaan muodostuneet vasta-aineet tunnistavat myös toisen virustyypin, ja näyttäisivät suojaavan kummankin virustyypin uusintainfektioilta.
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