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Evaluation of five commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19.

The following commercial tests for detection of immunoglobulin M antibodies to human parvovirus B19 were evaluated: Ideia Parvovirus B19-IgM, MRL Diagnostics Human Parvovirus B19 IgM ELISA, Parvoscan-B19, and Biotrin Parvo B19 IgM EIA and IF. A total of 203 serum specimens from patients who probably have current B19 infections or have other viral infections and sera with rheumatoid factor were investigated. Between 75 and 79 of 102 serum samples from patients thought to have current B19 infections yielded positive results with the different tests. Ideia had the highest specificity (94.8%), while Parvoscan showed a specificity of only 70.1%. Our evaluation results show that Ideia, MRL, and Biotrin EIA and IF can be recommended for diagnostic purposes.

Diagnostic Errors↗

Detection of H-1 parvovirus and Kilham rat virus by PCR.

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.

Animals↗

Simultaneous detection of measles virus, rubella virus, and parvovirus B19 by using multiplex PCR.

We describe here a multiplex reverse transcription-PCR (RTMNPCR) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus B19. Serial dilution experiments with vaccine strains that compared cell culture isolation of measles in B95 cells and rubella in RK13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (TCID(50)) for measles virus and 0.04 TCID(50) for rubella virus. This RTMNPCR can detect as few as 10 molecules for measles virus and rubella virus and one molecule for parvovirus B19 in dilution experiments with plasmids containing inserts of the primary reaction amplification products. Five pharyngeal exudates from measles patients and 2 of 15 cerebrospinal fluid samples from measles-related encephalitis were found to be positive for measles virus by this RTMNPCR. A total of 3 of 27 pharyngeal exudates from vaccinated children and 2 pharyngeal exudates, plus one urine sample from a case of congenital rubella syndrome, were found to be positive for rubella virus by RTMNPCR, whereas 16 of 19 sera from patients with erythema infectiosum were determined to be positive for parvovirus B19 by RTMNPCR. In view of these results, we can assess that this method is a useful tool in the diagnosis of these three viruses and could be used as an effective surveillance tool in measles eradication programs.

Child↗

Human parvovirus B19 can infect cynomolgus monkey marrow cells in tissue culture.

The human pathogenic parvovirus B19 cannot be grown in standard tissue culture but propagates in human bone marrow, where it is cytotoxic to erythroid progenitor cells. We now show that parvovirus B19 can replicate in cynomolgus bone marrow. Cynomolgus monkeys may be a suitable animal model for pathogenesis studies of parvovirus B19.

Animals↗

Lymphoproliferative responses after infection with human parvovirus B19.

Immunity after infection with the parvovirus B19 is assumed to be conferred by a humoral immune response with development of neutralizing antibody. In contrast, little is known about the nature of T-cell-mediated responses to parvovirus B19 infection in humans. We used recombinant proteins VP1, VP2, and NS1, as well as a recombinant VP1-specific amino-terminal sequence, to test the proliferative responses of peripheral blood mononuclear cells after infection of otherwise healthy individuals with parvovirus B19. These proteins were used as antigens for the stimulation of freshly isolated cells. The results show that a B19 virus-specific cellular immunity develops that is directed against the capsid proteins VP1 and VP2. We also demonstrate that viral determinants are presented to CD4+ T cells by HLA class II molecules.

Antigens, Viral↗

Human parvovirus B19 nonstructural (NS1) protein induces apoptosis in erythroid lineage cells.

Infection of erythroid-lineage cells by human parvovirus B19 is characterized by a gradual cytocidal effect. Accumulating evidence now implicates the nonstructural (NS1) protein of the virus in cytotoxicity, but the mechanism underlying the NS1-induced cell death is not known. Using a stringent regulatory system, we demonstrate that NS1 cytotoxicity is closely related to apoptosis, as evidenced by cell morphology, genomic DNA fragmentation, and cell cycle analysis with the human erythroleukemia cell line K562 and the erythropoietin-dependent megakaryocytic cell line UT-7/Epo. Apoptosis was significantly inhibited by an interleukin-1beta (IL-1beta)-converting enzyme (ICE)/CED-3 family protease inhibitor, Ac-DEVD-CHO (CPP32; caspase 3), whereas a similar inhibitor of ICE (caspase 1), Ac-YVAD-CHO, had no effect. Furthermore, stable expression of the human Bcl-2 proto-oncogene resulted in near-total protection from cell death in response to NS1 induction. Mutations engineered into the nucleoside triphosphate-binding domain of NS1 significantly rescued cells from NS1-induced apoptosis without having any effect on NS1-induced activation of the IL-6 gene expression which is mediated by NF-kappaB. Furthermore, using pentoxifylline, an inhibitor of NF-kappaB activation, we demonstrate that the NF-kappaB-mediated IL-6 activation by NS1 is uncoupled from the apoptotic pathway. This functional dissection indicates a complexity underlying the biochemical function of human parvovirus NS1 in transcriptional activation and induction of apoptosis. Our findings indicate that NS1 of parvovirus B19 induces cell death by apoptosis in at least erythroid-lineage cells by a pathway that involves caspase 3, whose activation may be a key event during NS1-induced cell death.

Apoptosis↗

Identification of a novel cellular TPR-containing protein, SGT, that interacts with the nonstructural protein NS1 of parvovirus H-1.

The nonstructural protein NS1 of autonomous parvoviruses is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses. NS1 acts as nickase, helicase, and ATPase and upregulates P38-driven transcription of the capsid genes. We report here the identification of a novel cellular protein that interacts with NS1 from parvovirus H-1 and which we termed SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. The cDNA encoding full-length SGT was isolated through a two-hybrid screen with, as bait, the truncated NS1dlC69 polypeptide, which lacks the C-terminal transactivation domain of NS1. Full-length NS1 and SGT interacted in the two-hybrid system and in an in vitro interaction assay. Northern blot analysis revealed one major transcript of about 2 kb that was present in all rat tissues investigated. Rat sgt cDNA coded for 314 amino acids, and the protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 34 kDa. SGT could be detected in both the nucleus and the cytoplasm of rat cells, as determined by indirect immunofluorescence analysis and Western blotting of fractionated cellular extracts with an affinity-purified antiserum raised against recombinant SGT (AC1.1). In H-1 virus-infected rat and human cells, compared to mock-infected controls, differences in the migration of SGT polypeptides were revealed after Western blot analysis of total cellular extracts. Moreover, the transient expression of NS proteins was sufficient to induce SGT modification. These results show that cellular SGT, which we have identified as an NS1-interacting protein, is modified by parvovirus infection as well as NS expression.

Amino Acid Sequence↗

The VP1 unique region of parvovirus B19 and its constituent phospholipase A2-like activity.

Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca(2+) dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region.

Amino Acid Sequence↗

Parvovirus nonstructural proteins induce an epigenetic modification through histone acetylation in host genes and revert tumor malignancy to benignancy.

Several malignant tumor cells become apoptotic and revert to the benign phenotype upon parvovirus infection. Recently, we demonstrated that the rat parvovirus RPV/UT also induces apoptosis in the rat thymic lymphoma cell line C58(NT)D. However, a minority of cells that escaped apoptosis showed properties different from the parental cells, such as resistance to apoptosis, enhanced cell adherence, and suppressed tumorigenicity. The present study was performed to determine the molecular mechanism of parvovirus-induced phenotypic modification, including oncosuppression. We demonstrated that the nonstructural (NS) proteins of RPV/UT induced apoptosis in C58(NT)D cells and suppressed tumor growth in vivo. Interestingly, NS proteins induced the expression of ciliary neurotrophic factor receptor alpha, which is up-regulated in revertant cell clones, and enhanced histone acetylation of its gene. These results indicate that parvoviral NS regulate host gene expression through histone acetylation, suggesting a possible mechanism of oncosuppression.

Acetylation↗

Host-selected amino acid changes at the sialic acid binding pocket of the parvovirus capsid modulate cell binding affinity and determine virulence.

The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 A away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.

Amino Acid Substitution↗

Aplastic anaemia associated with parvovirus B19 infection.

Parvovirus B19 involvement was investigated in 30 children with severe aplastic anaemia. Active or recent parvovirus B19 infection, as shown by B19 DNA viraemia, positive B19 specific IgM antibodies, or both, was diagnosed in six patients. There were no other plausible causes. We suggest that parvovirus B19 infection might be associated with severe aplastic anaemia.

Adolescent↗

Influence of previous exposure to human parvovirus B19 infection in explaining susceptibility to rheumatoid arthritis: an analysis of disease discordant twin pairs.

OBJECTIVES: To assess the association between exposure to parvovirus B19 and susceptibility to rheumatoid arthritis (RA). METHODS: One hundred and fifty five twin pairs (76 monozygotic (MZ) and 79 dizygotic (DZ)), discordant for RA, were tested for the presence of IgG antiparvovirus antibodies using ELISA. The data obtained were analysed using conditional logistic regression, from which odds ratios and 95% confidence intervals were calculated. RESULTS: Overall, there was no association between exposure to parvovirus and RA (OR = 1.2, 95% CI: 0.7-1.7). However, in two subgroups there was a suggestion of an association. These were: (1) pairs where the affected twin was rheumatoid factor (RF) seronegative (OR = 2.0, 95% CI: 0.9-12.4) and (2) in opposite-sexed twin pairs where the affected twin was female (OR = 3.0, 95% CI: 0.9-11.6). CONCLUSION: Previous exposure to parvovirus infection did not explain disease susceptibility in both MZ and DZ discordant pairs with rheumatoid arthritis. This infection, however, might be relevant in some subgroups.

Antibodies, Viral↗

Association between human parvovirus B19 infection and arthritis.

OBJECTIVE: To gain information concerning the association between parvovirus B19 infection and arthritis. METHODS: Blood or synovial fluid, or both, from a total of 77 adult patients with various arthropathies (rheumatoid arthritis 13; mechanical arthropathies 11; crystal induced arthritis 13; idiopathic mono/oligoarthritis 25; suspicion of viral arthritis 15) were tested for the presence of the viral genome and anti-B19 antibodies. B19 DNA in blood and synovial fluid was investigated by nested polymerase chain reaction, and anti-B19 IgM and IgG antibodies were detected in blood by enzyme immunoassay. RESULTS: A recent parvovirus infection was documented by the presence of anti-B19 IgM antibodies in the blood of 13 patients. B19 DNA, together with anti-B19 IgM and IgG antibodies, were detected in the blood of seven patients who had an acute transient arthritis, putatively of viral origin. Viral DNA was detected in a synovial fluid sample and in the blood of one patient with monoarthritis who had an anti-B19 IgG response only. CONCLUSIONS: The prevalence of anti-B19 IgG antibody in these patients with various forms of arthritis (63%) was within the same range as that in the general population (blood donors). However, for the patients with clinical suspicion of viral arthritis, the increased seroprevalence of anti-B19 IgM and the presence of the B19 genome point to an association between human parvovirus infections and acute forms of arthritis.

Adult↗

Parvovirus B19 infection associated with unilateral cervical lymphadenopathy, apoptotic sinus histiocytosis, and prolonged fatigue.

This report describes the case of a 16 year old girl with a history of high fever, prolonged fatigue, and cervical lymphadenopathy of the right side. In addition, the patient showed neutropenia, thrombopenia, and pronounced reticulopenia. Cervical ultrasound showed unilateral hypoechoic lymph nodes up to 23 mm in diameter suspicious for malignant lymphoma. Histology of a cervical lymph node specimen revealed massive nodular histiocytic proliferation and prominent apoptosis without necrosis. Parvovirus B19 was detected by polymerase chain reaction and immunohistochemistry in the lymph node. In summary, this case is an unusual presentation of parvovirus B19 infection. The virus was identified as the potential causative agent of unilateral cervical lymphoma and apoptotic sinus histocytosis, thus broadening the clinicopathological spectrum of parvovirus B19 induced diseases.

Adolescent↗

Association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia.

AIMS: To investigate the association of acute parvovirus B19 infection with new onset of acute lymphoblastic and myeloblastic leukaemia. METHODS: Cerebrospinal fluid (CSF) samples from patients with acute myelogenous leukaemia (AML) at diagnosis (n = 2) and acute lymphoblastic leukaemia (ALL) at diagnosis (n = 14) were analysed for parvovirus B19 DNA by means of nested polymerase chain reaction. In addition, samples from patients with benign intracranial hypertension (BIH) (n = 10) and hydrocephalus (n = 13) were tested as controls. RESULTS: Four leukaemia cases were positive-common ALL (n = 2), null cell ALL (n =1), and M7 AML (n = 1)-whereas all controls were negative (Yates corrected chi(2) value, 3.97; p = 0.046; odds ratio, 16.92; confidence interval, 1.03 to 77.18). All four patients were significantly anaemic, but none was encephalitic or had evidence of central nervous system leukaemia. In three of these patients, serum tumour necrosis alpha, interferon gamma, interleukin 6, granulocyte-macrophage colony stimulating factor (range, 34.93-3800.06 pg/ml), and macrophage chemoattractant protein 1 were detectable. All of these four patients carried at least one of the HLA-DRB1 alleles, which have been associated with symptomatic parvovirus B19 infection. CONCLUSION: Erythroid suppression and immune cell proliferation are both associated with B19 infection and may also be important in the pathogenesis of acute leukaemia.

Acute Disease↗

Evidence for the role of demyelination, HLA-DR alleles, and cytokines in the pathogenesis of parvovirus B19 meningoencephalitis and its sequelae.

OBJECTIVE: To review the clinical and pathological features of parvovirus B19 meningoencephalitis and its sequelae in 12 previously published cases, and to perform additional tests to determine the pathogenesis of the disease. METHODS: Cases were reviewed and available serum and cerebrospinal fluid (CSF) tested for antiganglioside antibodies and a range of cytokines. In situ hybridisation for parvovirus B19 DNA was performed on postmortem brain tissue in two cases. HLA-DRB1 typing was undertaken on genomic DNA extracted from peripheral blood leucocytes. RESULTS: Cerebellar involvement was suggested either clinically or pathologically in four cases. In the two cases with postmortem histology, there was marked atrophy of the molecular and granular layers of the cerebellum with focal loss of Purkinje cells. Brain scanning by MRI or CT was done in six cases during the acute phase. Three were abnormal with evidence of demyelination. Three had markedly enlarged ventricles, in two of which there was high signal intensity from the white matter on both T1 and T2 weighted images. The three cases with abnormal brain scans had long term neurological sequelae (mental retardation, personality change, altered affect). In situ hybridisation on available postmortem brain tissue was negative in the two cases tested. All cases in which HLA-DR alleles were determined carried at least one of the following alleles: HLA-DRB1*01, *04, *07, *09, *15, *16. Available serum and CSF was tested for antiganglioside antibodies (all negative) and for a panel of cytokines, which had a similar profile in both serum (n = 5) and CSF (n = 1) during the acute phase. Cytokines that were consistently detectable were IL-6 (mean 726.20 pg/ml), TNFalpha (50.64 pg/ml), IFNgamma (39.64 pg/ml), GM-CSF (216.12 pg/ml), and MCP-1 (154.43 pg/ml); IL-1beta, IL-5, and IL-13 were undetectable. CONCLUSIONS: HLA-DR associations, an increased cytokine response, and benefit from immunomodulatory treatment (in one case) support a role for the immune response in the pathogenesis of parvovirus B19 meningoencephalitis.

Adolescent↗

Evidence of Muscovy duck parvovirus in Muscovy ducklings in California.

Muscovy duck parvovirus (MDPV) has been demonstrated in tissue samples from one- to four-week-old commercially reared Muscovy ducks that were weak, unable to walk and had a high mortality rate. On postmortem examination, the thigh and leg muscles, and the myocardium were found to be pale, and there was a fibrinous exudate on the capsule of the liver, and ascites. The parvovirus was isolated in embryonated Muscovy duck eggs and visualised by negative stain electron microscopy, detected by polymerase chain reaction (PCR) directly from the tissues, and antibodies to it were detected by immunoelectron microscopy, ELISA and immunofluorescence. In addition, the PCR products obtained that represented 1625 bp (74 per cent) of the capsid vP1 gene, including a hypervariable region between Derzsy's disease virus or goose parvovirus and MDPV, were sequenced and shown to be 100 per cent homologous with the MDPV 89384 reference strain, but only 82.3 per cent homologous with Derzsy's disease virus.

Animals↗

Parvovirus B19 in an immunocompetent adult patient with acute liver failure: an underdiagnosed cause of acute non-A-E viral hepatitis.

There are occasional pediatric reports of parvovirus B19-associated transient acute hepatitis and hepatic failure. A case of a 34-year-old immunocompetent woman who developed severe and prolonged but self-limited acute hepatitis and myelosuppression following acute parvovirus B19 infection is reported. Parvovirus B19 may be the causative agent in some adult cases of acute non-A-E viral hepatitis and acute liver failure.

Acute Disease↗