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PubMedDate not supplied

Production of digoxigenin-labelled parvovirus DNA probe by PCR.

A 560-bp digoxigenin(Dig)-labelled DNA-probe was produced by PCR using a 699-bp parvovirus DNA fragment as template with introduction of Dig-dTUP into the PCR reaction mixture. It was found to be very important to pay close attention to the amount of template employed, the number of cycles used, predenaturation of target DNA and optimization of the percentage of dTTP substituted by Dig-DUTP in the reaction mixture. The same 560-bp DNA fragment produced by PCR without the incorporation of Dig-DUTP in the reaction mixture, was subsequently labelled with Dig-dUTP by the random primed labelling method. Both of the Dig-labelled parvovirus DNA probes described above showed the same DNA detection level (about 1 pg), but production of the probe with Dig-DUTP incorporated in the PCR reaction mixture was much simpler.

Base Sequence↗
PubMed2005

[Parvovirus B19 thrombocytopenic purpura complicated with a cerebral hemorrhage].

UNLABELLED: Severe hemorrhage complications are rare in idiopathic thrombocytopenic purpura. This pathology is often considered as benign. CASE REPORT: We report the case of a four-year-old boy presenting a parvovirus B19 idiopathic thrombocytopenic purpura. Despite early and repeated use of intravenous immunoglobulin, the evolution was characterized by the secondary apparition of a cerebral hemorrhage. It was lethal seven days after the initial diagnosis. CONCLUSION: Parvovirus B19 should be investigated as an etiologic agent of idiopathic thrombocytopenic purpura, using PCR. The unpredictive aspect of severe hemorrhage complications, especially cerebral hemorrhages, explains the potential severity of this disease.

Cerebral Hemorrhage↗
PubMed2004

Assembly of fluorescent chimeric virus-like particles of canine parvovirus in insect cells.

Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1, VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two proteins are produced by differential splicing of a primary transcript of the right ORF of the viral genome. The VP2 protein can be further proteolytically cleaved to form VP3. Previous studies have shown that VP1 and VP3 are unnecessary for capsid formation and consequently, that VP2 alone is sufficient for assembly. We have hypothesized that insertion of the enhanced green fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out without altering assembly. To investigate the possibility to develop fluorescent virus-like particles (fVLPs) from such chimeric VP2 proteins, the corresponding fusion construct was abundantly expressed in insect cells. Confocal imaging indicated that the EGFP-VP2 fusion product was assembled to fluorescent capsid-like complexes. In addition, electron micrographs of purified EGFP-VP2 complexes showed that they displayed a very similar size and appearance when compared to VP2 VLPs. Further, immunolabelling of purified EGFP-VP2 VLPs showed the presence of EGFP within the structure. Fluorescence correlation spectroscopy (FCS) studies confirmed that fVLPs were very similar in size when compared to authentic CPV. Finally, feeding of mammalian cells susceptible to CPV infection with these fVLPs indicated that entry and intracellular trafficking could be observed. In summary, we have developed fluorescent virus-like nanoparticles carrying a heterologous entity that can be utilized as a visualization tool to elucidate events related to a canine parvovirus infection.

Amino Acid Sequence↗
PubMed2026

Tracing the evolution and diversity of human parvovirus B19 across human history.

Human parvovirus B19 (B19V) is an ubiquitously spread, exclusively human pathogen, mainly posing risks to children, as well as pregnant and immunocompromised individuals. Despite evidence of B19V infection of human populations as far back as 7,000 years, the evolutionary history of B19V remains poorly understood. In this study, we present B19V genomic data from the remains of 53 globally distributed individuals spanning more than 8,000 years, including 7 children. Our findings suggest that the most recent common ancestor of all present B19V lineages existed around 12,000 years ago, at the end of the last Ice Age. Additionally, we identified an extinct Eurasian clade that participated in the recombination event that led to the emergence of B19V genotype 2 (GT-2). We date this event to ∼3,200-1,800 BP, potentially in the greater Mediterranean area. Our study shows aspects of how ancient parvovirus variants arose, disseminated, and impacted human health through time.

ancient DNA↗
PubMed2006

Development of an improved method of detection of infectious parvovirus B19.

BACKGROUND: Parvovirus B19, the only known pathogenic human parvovirus is the aetiologic agent of erythema infectiosum, transient aplastic crisis, pure red cell aplasia, and hydrops fetalis. Transmission is either by respiratory secretions or, as it can be present at high titre in plasma, by blood and blood products. B19 is only cultured with difficulty in vitro, and there is no readily available assay for detecting B19 infectivity or neutralizing antibodies. OBJECTIVES: In this study, we evaluated different methods to detect viral infection for the purpose of developing automated methods for large-scale testing of viral infectivity, development of neutralizing antibody and viral inactivation assays. STUDY DESIGN: Different cell lines were evaluated for their ability to support B19 infection and assays tested for sensitivity and ease of performing. A high-throughput assay was validated by determining infectious virus in blood pools and for determining neutralizing antibody in sera. RESULTS: B19 protein production was detected by immunofluorescence (IF) staining and increased viral DNA production by dot blot hybridization and quantitative PCR. The detection of RNA transcripts by RT-PCR assay and quantitative RT-PCR (qRT-PCR) was used as an indirect marker for infection. Of the cell lines tested, the subclone UT7/Epo-S1 showed the greatest sensitivity to B19 infection, with detection of viral transcripts by qRT-PCR the preferred assay. The assays were validated by experiments to determine the infectious titre of sera from acutely infected humans, to evaluate the presence of infectious virus in human donor plasma pools and to measure neutralizing antibodies.

Antibodies, Viral↗
PubMed2006

Association of hypospadias and cardiac defect in a Parvovirus B19-infected stillborn: a causality relation?

Parvovirus B19 intrauterine infection is a known cause of hydrops fetalis and fetal death. It is also associated with congenital malformations, although the teratogenic potential seems to be low. Postmortem examination of a male stillborn of 29 gestational weeks revealed mild subcutaneous edema, malformed micropenis, perineoscrotal hypospadias and atrial septal defect, along with fetal erythroblastosis and villitis. Polymerase chain reaction detected Parvovirus B19 DNA genome in tissues from the fetus and the placenta, confirming the hypothesis of an intrauterine infection.

Abnormalities, Multiple↗
PubMedDate not supplied

Parvovirus B19 in pregnancy.

Pregnant women who report possible exposure to a viral illness can present a clinical predicament for obstetric providers. Although some viruses are benign in pregnancy, others can have serious consequences. Parvovirus B19, the causative agent of fifth disease, is one of the more serious of the common viruses. Understanding the pathophysiology and appropriate testing for parvovirus will help providers better counsel their patients.

Adult↗
PubMed2007

Human parvovirus B19, varicella zoster virus, and human herpesvirus-6 in mesenchymal stem cells of patients with osteoarthritis: analysis with quantitative real-time polymerase chain reaction.

OBJECTIVE: To investigate whether there is a possible viral transmission using mesenchymal stem cells (MSCs) in autologous or allogeneic transplantation in the context of osteoarthritis (OA) patients. The presence of parvovirus B19 (B19), varicella zoster virus (VZV), and human herpesvirus-6 (HHV-6) was studied in MSCs from bone marrow of patients with OA and healthy controls. METHODS: MSCs were prepared from bone marrow aspirates obtained from 18 patients undergoing joint replacement as a result of OA and from 10 healthy controls. DNA was extracted from primary MSCs' culture established from these cells and quantitative real-time polymerase chain reaction was performed to analyse the prevalence and viral load of B19, VZV and HHV-6. RESULTS: The prevalence of total viral DNA among patients with OA was 16.7% (3/18), with a mean viral load of 29.7 copies/microg of DNA. One out of 18 was positive for B19 (viral load, 61.2 copies/microg of DNA), two for VZV (mean viral load, 14.4 copies/microg of DNA), and none for HHV-6. The prevalence of total viral DNA in the control group was 20% (2/10), with a mean viral load of 13.4 copies/microg of DNA. Both positive results were of B19 parvoviruses. There were no statistically significant differences among patients and controls. CONCLUSIONS: This first approach to the viral prevalence in MSCs of bone marrow in OA patients and healthy controls seems to show a very low risk of viral transmission or reactivation in a possible MSCs' transplantation.

Adult↗
PubMed2006

Effects of probe binding mutations in an assay designed to detect parvovirus B19: implications for the quantitation of different virus genotypes.

Quantitative real-time PCR is being widely used in the identification of plasma donations that contain high levels of parvovirus B19, to ensure their exclusion from start pools used in the manufacture of plasma derived medicinal products. In this study, the primers and probe of one such published assay, are examined for their ability to quantify different genotypes of parvovirus B19. Under standard assay conditions, there is a failure to detect and quantify one genotype 3 subtype. Alterations in assay conditions can restore quantitation of this subtype.

Genotype↗
PubMed2005

Lupus-like presentation of parvovirus B19 infection.

OBJECTIVES: To describe 2 cases of parvovirus B19 (B19) infection mimicking systemic lupus erythematosus (SLE) and to identify all cases of SLE imitated by and/or associated with B19 in the medical literature. METHODS: A computer-assisted (PubMed) search of the medical literature from 1975 to 2003 was performed using the following key words: parvovirus, B19, SLE, lupus, antibodies, auto-immunity. RESULTS: Thirty-eight patients were identified: 35 women, 3 men; mean age = 28.8 years. Clinical manifestations were as follows: fever (24 patients); articular involvement (36 patients); cutaneous lesions (28 patients); lymphadenopathy (9 patients); hepato- and/or splenomegaly (6 patients); serositis (6 patients); renal involvement (4 patients); cerebral impairment (10 patients). Cytopenia was observed in 23 cases. Antinuclear antibodies were detected in 34 patients, anti-double-stranded DNA antibodies in 20 patients, anti-Sm antibodies in 4 patients, antinuclear ribonucleoprotein antibodies in 5 patients, anti-Ro-SSA antibodies in 4 patients, anti-La-SSB antibodies in 4 patients, and anticardiolipin and/or anti-beta2-glycoprotein I antibodies in 8 patients. Hypocomplementemia was found in 15 of 26 patients. In 19 cases, the B19 infection had a self-limiting course. In 6 cases, B19 infection occurred in a context of previously established SLE, simulating SLE exacerbation. In 6 observations, symptoms persisted several months after the viral infection. In 7 cases, the exact relationship between SLE and B19 could not be determined. CONCLUSIONS: B19 infection may present a clinical and serological tableau making it difficult to distinguish between a viral infection and the first episode of SLE. Although B19 may modulate the clinical and biological features of rheumatic disease, studies in large series do not support a causative role for B19 in the pathogenesis of SLE.

Adult↗
PubMed2004

T-helper cell-mediated interferon-gamma, interleukin-10 and proliferation responses to a candidate recombinant vaccine for human parvovirus B19.

Recombinantly expressed virus-like particles of human parvovirus B19 containing the two structural proteins VP1 and VP2 (VP1/2 capsids) or VP2 alone (VP2 capsids) elicit vigorous antibody responses in animal models, whereas only VP1/2 capsids elicit neutralizing antibodies. VP1 is, therefore, essential for protective B-cell immunity. In this study, we determined the ability of VP1/2 capsids containing VP1 and VP2 in the ratio recommended for vaccine use, and of sole VP2 capsids to stimulate T-helper (Th) cells to proliferate and to secrete interferon gamma (IF-gamma) and interleukin-10 (IL-10) in humans long after natural infection. Similar proliferation, IF-gamma and IL-10 responses were found with the VP1/2 and VP2 capsids. We conclude that, whereas VP1 contains important B-cell epitopes, VP2, the major structural protein of human parvovirus B19, appears to provide the major target for B19-specific Th-cells years or decades after natural infection.

Adult↗
PubMed2002

Detection of human parvovirus B19 infection in first-trimester fetal loss.

OBJECTIVE: To investigate the frequency of parvovirus B19 infection in first-trimester fetal loss, as measured by B19 DNA polymerase chain reaction in placental tissue in a prospective descriptive study from a nonendemic area. METHODS: Placental tissues from first-trimester fetal losses were examined for presence of B19 DNA by polymerase chain reaction in a prospective study during 30 months. For comparison, placental tissues from second-trimester fetal losses, as well as from full-term normal pregnancies, were also studied. RESULTS: B19 DNA was detected by polymerase chain reaction in one of 36 (3%) placental tissues from first-trimester fetal losses. In second-trimester fetal losses, eight of 64 (12%) samples were B19 DNA positive. None of the 53 placental tissues from full-term normal pregnancies were B19 DNA positive. In first-trimester fetal losses, maternal serum from the B19 DNA-positive sample was B19 immunoglobulin (Ig)G positive but B19 IgM negative. In second-trimester fetal losses, six of six tested B19 DNA-positive samples were both B19 IgG and IgM positive. CONCLUSION: The frequency of first-trimester fetal loss associated with parvovirus B19 infection was low, 3%, during a nonepidemic period in Sweden, as measured by B19 DNA-specific polymerase chain reaction in placental tissue.

Abortion, Spontaneous↗
PubMed1995

Peptides derived from the unique region of B19 parvovirus minor capsid protein elicit neutralizing antibodies in rabbits.

B19 parvovirus is pathogenic in humans. The virus propagates in the bone marrow, where it is cytotoxic to erythroid progenitor cells. Antibodies appear in blood after infection and neutralize virus in vitro; infection appears to confer lasting immunity. The predominant immune response on immunoblot is to the minor capsid protein (VP1), which differs from the major capsid protein (VP2) by an additional 227 amino acids. We previously demonstrated that antisera directed to a fusion protein containing this unique region or to more limited fusion peptides of 50-100 amino acids each neutralized virus. In the current work, we tested synthetic peptides of about 20 amino acids derived from the VP1 unique region for their ability to elicit a neutralizing antibody response in rabbits. Individual peptides were covalently linked to a lysine core to produce a multivalent antigen. Animals produced antibodies to all 13 synthetic peptides, as determined by ELISA. At 12 weeks, animals injected with one of three peptides--two from the far amino terminus and the third from the center of the unique region--had produced antibodies that completely neutralized virus; by 16 weeks, antisera elicited with another four peptides also were effective. In summary, we identified regions containing neutralizing epitopes within the first 80 amino acids and amino acids 148-205 of the unique region. Our data suggest that synthetic peptides might be useful vaccine reagents for protection against parvovirus infection in humans.

Amino Acid Sequence↗
PubMed1999

Parvovirus infection mimicking systemic lupus erythematosus in a pediatric population.

OBJECTIVES: To assess the striking similarities of presentation in a pediatric population between human parvovirus B19 (HPV-B19) infection and systemic lupus erythematous (SLE). METHODS: Medical records of seven patients (ages 6 to 15) with HPV-B19 infection were reviewed retrospectively. RESULTS: Six of seven cases presented with a history of malar rash, and all seven had prolonged arthralgias and fatigue. Six of seven had a positive antinuclear antibody (ANA) titer ranging from 1:40 to greater than 1:640, with two patients having antibodies to Scl-70 and others to Sm, RNP, SS-A (Ro), or SS-B (La). The erythrocyte sedimentation rate (ESR) varied from 2 to 68 mm/h. Two patients presented with elevated rheumatoid factor (RF) titers of 24 and 271 IU/mL, respectively. All had elevated IgM antibody levels to parvovirus at the onset, and markedly elevated IgG levels when evaluated throughout their disease course. Over the course of 2 to 3 months, three improved, but the other four continued to have symptomatology for 14, 40, 78, and 120 weeks, respectively. Treatment was symptomatic, and no one developed classic SLE. CONCLUSIONS: HPV-B19 infection in a pediatric patient group may present with SLE-like symptomatology and positive serology suggestive of SLE. The course of the disease is usually self-limited, though it may be prolonged in some for up to 120 weeks.

Adolescent↗
PubMed1999

Serologic study of human parvovirus B19 infection in pregnancy in Japan.

OBJECTIVES: To clarify the relationship between hydrops fetalis and parvovirus outbreaks in the community, seroprevalence of B19 antibody among women of childbearing age, and adverse effects of intrauterine B19 infection. METHODS: Sera were collected from 168 cases of hydrops fetalis which were diagnosed between 1987 and 1997 in Miyagi prefecture, Japan, from 232 healthy pregnant women in 1987 and 277 healthy pregnant women in 1997 in Miyagi, and from 48 women infected with B19 during pregnancy. The sera were examined for B19 IgG and IgM antibodies by enzyme-linked immunosorbent assay and for B19 DNA by polymerase chain reaction. The number of cases of erythema infectiosum in Miyagi had been monitored each month. RESULTS: Thirteen of the 168 cases of hydrops fetalis were found to be caused by intrauterine B19 infection and 12 of the 13 cases clustered in two periods of outbreaks of erythema infectiosum in the community. The positive rates of B19 IgG antibody between 1987 and 1997 were significantly different: 33% in 1987 and 46% in 1997. Nine of the 48 women infected during pregnancy showed adverse effects of the fetus: eight hydrops fetalis and one early abortion with positive B19 DNA. The fetal death rate (>12 weeks of gestation) among them was 15% (7/48), far higher than the calculated 1% among the general population. The nine mothers with adverse fetal outcomes had contact with the infectious source at the 16 weeks of gestation or earlier. CONCLUSIONS: These data clearly showed a relationship between hydrops fetalis and parvovirus outbreaks in the community, and it may be important to follow the seroprevalence for an extrapolated period time to predict occurrence of hydrops fetalis caused by B19. Also the data indicated that the gestational week infection occurred is the most important determinant of an adverse effect to the fetus as described previously.

Adolescent↗
PubMed2003

Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new lightcycler nested-PCR assay for detection of PPV.

Porcine parvovirus (PPV) is a major causative agent in a syndrome of reproductive failure in swine. In validation (viral clearance) studies, PPV is a model of non-enveloped viruses and is widely used instead of human parvovirus B19, which causes a variety of illnesses including erythema infectiosum (fifth disease) in children and hydrops fetalis in pregnant women. To improve the sensitivity of current PCR-based assays for detection of PPV and to standardize the quantification of PPV, we have developed a lightcycler (LC) nested-PCR (nPCR) assay and constructed a PPV DNA standard evaluated in the LC nPCR assay. The PPV DNA standard, a plasmid termed pPPV, encodes a 3.3 kb PPV NADL-2 genome fragment. One genome copy equivalent (gce) of PPV equals 6.7 attograms of pPPV. The LC nPCR assay is a simple and specific method developed for detection of PPV strains but not any other viruses including members of Parvoviridae. The first 25-cycle PCR with outer primers chose by comparative analysis of 12 primers in 21 different combinations and a second 45-cycle PCR with inner primers amplify 286 and 251 bp fragments of PPV genome, respectively, for 40 min with a sensitivity of approximately 100 gce per assay (ml). By using the LC nPCR assay for analysis of PPV samples with known infectivity, we found that one 50% tissue culture infectious dose (TCID(50)) equals 1.93 +/- 0.24 log(10) gce.

Animals↗
PubMed1998

Mapping the antigenic structure of porcine parvovirus at the level of peptides.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Amino Acid Sequence↗
PubMed2003

[Multiple pseudo-cellulitis plaques and Koplick's sign: a particular form of parvovirus B19 primo-infection in adults].

INTRODUCTION: clinical presentation which seems to be more specific of this infection. EXEGESIS: A 35-year-old woman, 12 weeks pregnant, presented with a primary infection of parvovirus B19. The clinical presentation was characterized by pseudo-cellulitis plaques of the buttocks and the vulva, buccal enanthema with ulcerations and Koplick spot. CONCLUSION: This is the second observation which describes such cutaneous and mucosal manifestations associated with parvovirus B19 infection. This kind of clinical presentation should be systematically reported to become well known by the physicians as erythema infectiosum of fifth disease or "gloves and socks" syndrome.

Adult↗