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The 1994 human parvovirus B19 epidemic in Denmark: diagnostic and epidemiological experience.

In 1994 the first human parvovirus B19 (B19) epidemic to be documented in Denmark was recorded from February 2 to September 30. In total, 10,333 serum samples were tested for specific B19 IgM and IgG antibodies, using IDEIA Parvovirus B19 IgM and IgG kits. The prevalence of B19 IgM positivity was 11% for the whole period and 29% at the peak of the epidemic in week 14, declining from week 39 and onwards to 1-3%. The prevalence of B19 IgG (IgM-negative samples) was 60%, indicating an earlier infection, and the same for men and women. The gender distribution of tested patients was the same at the beginning of the epidemic as at the end of the epidemic and a year after its peak, i.e. 86% of samples were from women and only 14% from men. Age distribution for women was the same for the three periods (median age 34 years). For men the median age was 32 years, 39 years and 31 years, respectively. Only a few samples from children were tested. No change in test pattern was observed during the three periods. Approximately 75% of all samples tested were from women of childbearing age (18-45 years old), suggesting a fear of fetal complications in an actual or future pregnancy, rather than a serological verification of clinical symptoms. From the sparse clinical information that accompanied the serum sample we were not able to demonstrate that women were more likely than men to have a symptomatic B19 infection. With reservations we estimate that 14% of adverse pregnancy outcome is correlated with a B19 infection.

Adolescent↗

[Human parvovirus B19-induced aplastic crisis in a case with spherocytosis].

A 43-year-old female with spherocytosis was admitted to the hospital because of fever, headache, and rapidly progressive anemia. On admission, she had severe anemia (Hb 3.9 g/dl) with marked decrease in reticulocytes (0.3%). Some premature leukocytes, such as promyerocyte, were observed in the peripheral blood. Bone marrow smear revealed normocellular marrow with normal presence of erythroid cells. Anti-human parvovirus B19 IgM and IgG antibody was positive in the serum on admission. The DNA of the virus was also detected in the peripheral blood by PCR assay. Thus, this case was diagnosed as acute aplastic crisis caused by human parvovirus B19 infection. Blood transfusion was performed on day 1 and day 2. The symptoms gradually disappeared and increase in reticulocyte (11.5%) was observed on day 5. Premature leukocytes in the peripheral blood disappeared on day 2. She recovered from anemia and was discharged on day 7.

Adult↗

Monoclonal antibodies that distinguish antigenic variants of canine parvovirus.

Canine parvovirus (CPV) is classified as a member of the feline parvovirus (FPV) subgroup. CPV isolates are divided into three antigenic types: CPV type 2 (CPV-2), CPV-2a, and CPV-2b. Recently, new antigenic types of CPV were isolated from Vietnamese leopard cats and designated CPV-2c(a) or CPV-2c(b). CPV-2c viruses were distinguished from the other antigenic types of the FPV subgroup by the absence of reactivity with several monoclonal antibodies (MAbs). To characterize the antigenicity of CPV-2c, a panel of MAbs against CPV-2c was generated and epitopes recognized by these MAbs were examined by selection of escape mutants. Four MAbs were established and classified into three groups on the basis of their reactivities: MAbs which recognize CPV-2a, CPV-2b, and CPV-2c (MAbs 2G5 and 20G4); an MAb which reacts with only CPV-2b and CPV-2c(b) (MAb 21C3); and an MAb which recognizes all types of the FPV subgroup viruses (MAb 19D7). The reactivity of MAb 20G4 with CPV-2c was higher than its reactivities with CPV-2a and CPV-2b. These types of specificities of MAbs have not been reported previously. A mapping study by analysis of neutralization-resistant mutants showed that epitopes recognized by MAbs 21C3 and 19D7 belonged to antigenic site A. Substitution of the residues in site B and the other antigenic site influenced the epitope recognized by MAb 2G5. It was suggested that the epitope recognized by MAb 20G4 was related to antigenic site B. These MAbs are expected to be useful for the detection and classification of FPV subgroup isolates.

Animals↗

Chemiluminescence dot blot hybridization assay for detection of B19 parvovirus DNA in human sera.

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.

DNA Probes↗

Prokaryotic expression of a VP1 polypeptide antigen for diagnosis by a human parvovirus B19 antibody enzyme immunoassay.

To produce parvovirus B19 antigen for diagnostic purposes, partially overlapping segments covering the genes encoding the viral structural proteins VP1 and VP2 were cloned into expression vectors. The constructs were induced in Escherichia coli, resulting in the expression of beta-galactosidase fusion proteins. In immunoblotting experiments with sera from patients with erythema infectiosum, immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide of 235 amino acids at the N terminus of VP1. The DNA fragment encoding this polypeptide was amplified by the polymerase chain reaction and cloned into an expression vector. The viral capsid antigen expressed in E. coli was purified by preparative agarose gel electrophoresis and used in IgG and IgM solid-phase enzyme immunoassays. Comparison with reference gamma- and mu-capture radioimmunoassays using whole virus antigen showed that these antibody tests are suitable for the serodiagnosis of human infections caused by parvovirus B19.

Antibodies, Viral↗

Characterization of a nested polymerase chain reaction assay for detection of parvovirus B19.

The characterization and application of a nested polymerase chain reaction (PCR) assay for the detection of human parvovirus B19 DNA is described. The assay was evaluated with 149 diagnostic serum samples (collected up to 150 days after the onset of symptoms) previously tested by dot blot hybridization for B19 DNA and by class-specific capture radioimmunoassays for the detection of B19 immunoglobulin M (IgM) and IgG. B19 DNA was detectable by the PCR in 70% of the sera. There was a statistically significant association between the detection of B19 DNA by PCR and high B19 IgM values (P < 0.005), low B19 IgG values (P < 0.05), and a short interval between onset of symptoms and serum collection (P < 0.005). Serial serum samples, throat swabs, and peripheral blood mononuclear cells collected from 10 individuals during an outbreak of parvovirus B19 were also tested by the nested PCR. B19 DNA was detectable in the throat swabs at the time of the clinical illness and in the peripheral blood mononuclear cell fraction up to the end point of the study 6 months after infection. The location of the B19 DNA could not be determined in cytocentrifuge preparations of peripheral blood mononuclear cells with nonisotopic in situ hybridization and immunolabelling.

Antibodies, Viral↗

Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia.

Twenty-seven feline parvovirus (FPV) isolates were recovered from cats clinically diagnosed with feline panleukopenia (FPL) for assessing antigenic and genomic properties of FPL viruses (FPLV) recently prevalent among cats in Japan. All isolates, with the exception of one novel isolate, FPV-314, possessed homologous properties, and their subgroups in FPVs were identified as FPLV. The FPV-314 isolate, which was from a 1.5-year-old cat which manifested clinical signs of FPL and died on the 13th day after the first medical examination, was finally identified as canine parvovirus (CPV) because it lacked a specific antigenic epitope commonly detected in FPLV and mink enteritis virus and because the nucleotide sequence of the capsid protein gene was almost identical to those of CPV-2a and -2b antigenic type strains recently prevalent among dogs in Japan. The present result together with our previous findings (M. Mochizuki, R. Harasawa, and H. Nakatani. Vet. Microbiol. 38:1-10, 1993) indicates the possibility that CPV and FPLV undergo mutual interspecies transmission between dogs and cats, and it is postulated that they may cause disease in some adventitious hosts.

Animals↗

Acute-phase-specific heptapeptide epitope for diagnosis of parvovirus B19 infection.

The major capsid protein VP2 of human parvovirus B19, when studied in a denatured form exhibiting linear epitopes, is recognized exclusively by immunoglobulin G (IgG) antibodies of patients with acute or recent B19 infection. By contrast, conformational epitopes of VP2 are recognized both by IgG of the acute phase and by IgG of past immunity. In order to localize the VP2 linear epitope(s) specific for acute-phase IgG, the entire B19 capsid protein sequence was mapped by peptide scanning using well-characterized acute-phase and control sera. A unique heptapeptide epitope showing strong and selective reactivity with the acute-phase IgG was detected and characterized. By using this linear epitope (VP2 amino acids 344 to 350) and virus-like particles exhibiting conformational VP2 epitopes, an innovative approach, second-generation epitope-typing enzyme immunoassay, was set up for improved diagnosis of primary infections by human parvovirus B19.

Acute Disease↗

Increased risk of parvovirus B19 infection in young adult cancer patients receiving multiple courses of chemotherapy.

An increased human parvovirus B19 infection rate has been observed in immunocompromised hosts. In this study, we sought to determine the prevalence of parvovirus B19 infection in adult cancer patients receiving multiple courses of systemic chemotherapy. From March 1999 through April 2000, 59 men and 68 women, with a median age of 49 (18 to 79) years, were enrolled in this study. They had received an average of 7.1 (4 to 32) courses of systemic chemotherapy. The median duration from the date of starting chemotherapy to the date of blood sampling was 11 (4 to 88) months. Serum B19 immunoglobulin G (IgG) and IgM levels were examined by an enzyme-linked immunosorbent assay, and B19 DNA was examined by a nested PCR. A group of 400 healthy blood donors served as the control group. The overall prevalences of anti-B19 IgG in adult cancer patients and healthy blood donors were 61.4 and 25.0%, respectively (P < 0.01). Anti-B19 IgM and B19 DNA were not detectable in these anti-B19 IgG-seropositive individuals. A further age-stratified comparison revealed that only patients younger than 40 years had a significantly higher anti-B19 IgG seropositivity rate than the controls (19 of 39 versus 53 of 310; P < 0.001). The increased prevalence of B19 infection in these 39 adult patients younger than 40 years might be clinically significant, since unexplained anemia, a common sequela of B19 infection, was detected in 3 of 20 seronegative patients (15.0%) and in 12 of 19 seropositive patients (63.2%) (P < 0.005). The results of this study suggest that adult patients younger than 40 years and receiving multiple courses of systemic chemotherapy may have a significantly increased risk of B19 infection. Prospective studies to define the time course and clinical consequence of B19 infection in this group of patients are needed.

Adolescent↗

Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial 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 known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

Blood Donors↗

Evaluation of a real-time PCR assay using the LightCycler system for detection of parvovirus B19 DNA.

We evaluated the artus RealArt Parvovirus B19 LC PCR reagent (artus biotech USA, San Francisco, Calif.) for real-time PCR detection of parvovirus B19 DNA by retesting 71 specimens previously submitted to our laboratory. The artus assay, which produces a quantitative result and provides an internal PCR control, appeared to be slightly more sensitive than our conventional qualitative PCR assay.

DNA, Viral↗

Conformational changes in the VP1-unique region of native human parvovirus B19 lead to exposure of internal sequences that play a role in virus neutralization and infectivity.

The unique region of the capsid protein VP1 (VP1u) of human parvovirus B19 (B19) elicits a dominant immune response and has a phospholipase A(2) (PLA(2)) activity, which is necessary for the infection. In contrast to the rest of the parvoviruses, the VP1u of B19 is thought to occupy an external position in the virion, making this region a promising candidate for vaccine development. By using a monoclonal antibody against the most-N-terminal portion of VP1u, we revealed that this region rich in neutralizing epitopes is not accessible in native capsids. However, exposure of capsids to increasing temperatures or low pH led to its progressive accessibility without particle disassembly. Although unable to bind free virus or to block virus attachment to the cell, the anti-VP1u antibody was neutralizing, suggesting that the exposure of the epitope and the subsequent virus neutralization occur only after receptor attachment. The measurement of the VP1u-associated PLA(2) activity of B19 capsids revealed that this region is also internal but becomes exposed in heat- and in low-pH-treated particles. In sharp contrast to native virions, the VP1u of baculovirus-derived B19 capsids was readily accessible in the absence of any treatment. These results indicate that stretches of VP1u of native B19 capsids harboring neutralizing epitopes and essential functional motifs are not external to the capsid. However, a conformational change renders these regions accessible and triggers the PLA(2) potential of the virus. The results also emphasize major differences in the VP1u conformation between natural and recombinant particles.

Antibodies, Monoclonal↗

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

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 normal 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.

Cell Differentiation↗

Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1-VP2 junction regions.

Presentation of linear epitopes of the B19 parvovirus capsid proteins as peptides might be a useful vaccine strategy. We produced overlapping fusion proteins to span the viral capsid sequence, inoculated rabbits, and determined whether the resulting antisera contained antibodies that neutralized the ability of the virus to infect human erythroid progenitor cells. Antibodies that bound to virus in an enzyme-linked immunosorbent assay were present in antisera raised against 10 of 11 peptides; strongest activity was found for antisera against the carboxyl-terminal half of the major capsid protein. However, strong neutralizing activity was elicited in animals immunized with peptides from the amino-terminal portion of the unique region of the minor capsid protein and peptides containing the sequence of the junction region between the minor and major capsid proteins. The development of neutralizing activity in animals was elicited most rapidly with the fusion peptide from the first quarter of the unique region. A 20-amino-acid region of the unique region of the minor capsid protein was shown to contain a neutralizing epitope. Multiple antigenic peptides, based on the sequence of the unique region and produced by covalent linkage through a polylysine backbone, elicited strong neutralizing antibody responses. Synthetic peptides and fusion proteins containing small regions of the unique portion of the minor capsid protein might be useful as immunogens in a human vaccine against B19 parvovirus.

Antibodies, Viral↗

The transcriptional regulator YY1 binds to the 5'-terminal region of B19 parvovirus and regulates P6 promoter activity.

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 chromatography and identified as YY1, a multifunctional transcription factor. Of multiple possible YY1 binding sites in the upstream region of the P6 promoter, three showed specific strong binding. Transcription enhancement by YY1 was demonstrated in vitro by transient transfection assays. In studies using truncated and mutated versions of this region, YY1 activity was diminished by the alteration of any two of these three sites and abolished by the alteration of all three sites. Our results suggest that YY1 is a positive regulator of B19 parvovirus transcription.

Base Sequence↗

Transcriptional transactivation of parvovirus B19 promoters in nonpermissive human cells by adenovirus type 2.

The pathogenic human parvovirus B19 contains a promoter at map unit 6 (B19p6) of the viral genome, expression from which is largely restricted to human cells in the erythroid lineage, whereas a putative promoter at map unit 44 (B19p44) is inactive during a natural viral infection. Although nonerythroid human cells, such as HeLa and KB, allow expression from the B19p6 promoter but not from the B19p44 promoter following DNA-mediated transfection, little expression from the B19p6 promoter occurs following recombinant virus infection (S. Ponnazhagan, X.-S. Wang, M.J. Woody, F. Luo, L.Y. Kang, M.L. Nallari, N.C. Munshi, S.Z. Zhou, and A. Srivastava, submitted for publication). However, significant expression from the B19p6 promoter as well as the B19p44 promoter could be detected in a human 293 cells line that expresses the adenovirus early gene products, suggesting that coinfection with adenovirus might mediate transcriptional transactivation of the B19 promoters in nonpermissive cells. Expression of the firefly luciferase reporter gene from the B19 promoters was evaluated either following plasmid transfection or following infection with the recombinant adeno-associated virus type 2 vectors. Both B19p6 and B19p44 promoters could be transactivated by coinfection with adenovirus in nonpermissive human cells, although the extent of transactivation of the B19p44 promoter was significantly lower than that of the B19p6 promoter. Expression of the adenovirus E1A proteins was necessary and sufficient for the observed transactivation of the B19 promoters. These studies further illustrate that the underlying molecular mechanisms of transactivation of parvovirus promoters in general by the adenovirus early proteins have similarities with those of the well-documented transactivation of the adeno-associated virus type 2 promoters.

Adenovirus E1A Proteins↗

Symmetric-strand packaging of recombinant parvovirus LuIII genomes that retain only the terminal regions.

LuIII is an autonomous parvovirus which encapsidates either strand of its genome with similar efficiency in NB324K cells. Two parvoviruses closely related to LuIII, minute virus of mice (MVM) and H-1 virus, encapsidate primarily the minus strand of their genome when grown in the same cell type. It has been postulated that an AT-rich region unique to LuIII is responsible for symmetric encapsidation of plus- and minus-strand genomes by LuIII. To address this hypothesis, recombinant LuIII-luciferase genomes containing or lacking the AT-rich sequence (AT) were packaged into LuIII virions. Hybridization of strand-specific probes to DNA from these virions revealed that either strand of the genome was packaged regardless of the presence of AT. In addition, encapsidation of both strands of the AT+ LuIII-luciferase genome into MVM and H-1 virions was observed, suggesting that MVM and H-1 viral proteins are not responsible for the minus-strand packaging bias of these two viruses. Alignment of the published LuIII and MVMp sequences shows that AT exists as an insertion into an element that, in MVM, binds cellular proteins. We suggest that in LuIII, AT disrupts binding of these cellular proteins, allowing encapsidation of either strand.

Base Sequence↗