Chronic autoimmune thrombopenia/neutropenia in a boy with persistent parvovirus B19 infection.

OBJECTIVE: We report an 11-year-old boy presenting with splenomegaly, chronic thrombocytopenia and concordant neutropenia. RESULTS: In contrast to autoantibodies against platelets, there were no detectable neutrophil-specific autoantibodies present in this patient. Extensive serologic investigations revealed increased IgM- and IgG-antibody titers against parvovirus B19. A nested polymerase chain reaction (PCR) showed parvovirus B19-specific sequences in the patient's bone-marrow cells but not in the serum. Specific antibodies against the structural proteins VP1 and VP2 in addition to those against non-structural protein NS1 of parvovirus B19 were detected by Western blot analysis. Thrombocytopenia and neutropenia responded to immunosuppressive therapy and subsequent splenectomy, the latter being necessary due to severe side-effects of steroid medication. CONCLUSION: Autoimmune thrombocytopenia/neutropenia may have been triggered and/or sustained by a chronic parvovirus B19 infection. Patients with this very rare disorder should be screened for this virus.

Seroprevalence of parvovirus B19 NS1-specific IgG in B19-infected and uninfected individuals and in infected pregnant women.

Parvovirus B19 is the causative agent of erythema infectiosum in children, but the virus is associated with an increasing range of different diseases. These include acute and chronic arthritis, hydrops fetalis in pregnant women, aplastic anemia, and thrombocytopenia. The host's immune response is directed against the viral structural proteins VP1 and VP2. This study investigated the presence of IgG against the viral nonstructural protein NS1 using Western blot. Serum panels from healthy individuals, B19-infected pregnant women, and various disease groups were tested. The disease groups included patients with symptoms that may be linked to parvovirus B19 infection. The results showed that IgG against the NS1 protein was present in 22% of healthy individuals with past B19 infection. In cases of persistent or prolonged B19 infections, the prevalence of NS1-specific antibodies was as high as 80%. It is concluded that NS1-specific IgG may be used as an indicator of chronic or more severe courses of parvovirus B19 infections.

Acute parvovirus B19 infection in connection with a flare of systemic lupus erythematodes in a female patient.

BACKGROUND: Since its discovery parvovirus B19-infections could be linked to a growing variety of diseases. Besides the harmless exanthema erythema infectiosum perferentially observed with B19-infections in childhood a panel of rather serious and also chronic courses that may be associated with anemia, thrombocytopenia, arthritis and others have been described. OBJECTIVE: In a 26-year-old female patient an acute parvovirus B19-infection was followed by a serious episode of systemic lupus erythematosus (SLE). Here we demonstrate the clinical and serological parameters which were observed in the patient during that episode in addition to the nucleotide sequence of the virus isolate. RESULTS AND CONCLUSION: In this patient parvovirus B19 was not the initial causative agent for SLE. However the B19 infection was followed by a severe flare of SLE and therefore may be considered as an enhancer of the autoimmune disease. The amount of nucleotide variability observed in the viral genome was in the range known from other B19 isolates. An elevated degree of mutations in antigenic domains was not detectable. Therefore, we would like to emphasize the possible role of parvovirus B19 in the aetiology or the enhancement of autoimmune diseases like SLE and the necessity of an according differential diagnosis.

[Imitation of an acute exacerbation of established systemic lupus erythematosus caused by parvovirus B19 infection]. / Imitation eines akuten Schubes eines bekannten systemischen Lupus erythematodes durch eine Parvovirus-B19-Infektion.

HISTORY AND ADMISSION FINDINGS: A 26-year-old woman, for 3 years known to have systemic lupus erythematodes (SLE), was admitted because of fever, muscle and joint pains as well as nausea and vomiting. Maintenance therapy of 50 mg azathioprine, three times daily, had been stopped by the patient several weeks earlier. Physical examination was unremarkable except for pale skin and mucosae as well as pain on pressure over muscle groups of the upper arms and thighs and paravertebrally. DIAGNOSIS, TREATMENT AND COURSE: Laboratory tests showed a pancytopenia with a decrease of the haemoglobin level of 3.0 g/dl. The differential diagnosis included an acute exacerbation of SLE, toxic damage to the bone-marrow by azathioprine or a viral infection involving the bone-marrow. Serological tests and the further course confirmed an acute paravirus B19 infection as the cause. CONCLUSION: Paravirus B19 infection should be included in the differential diagnosis when an acute exacerbation of SLE is suspected.

Infection of apheresis cells by parvovirus B19.

Parvovirus B19 is the only member of the Parvoviridae family known to cause disease in humans. Owing to the high level of cell tropism the virus can only replicate in proliferating and differentiating erythroid precursor cells, which are present in human bone marrow and foetal liver. As human bone marrow is very difficult to obtain, an alternative in vitro system for the propagation of B19 virus has been developed, based on the application of mobilized haemapoietic progenitor (apheresis) cells. These cells are routinely harvested from cancer patients after treatment with recombinant human granulocyte/macrophage colony-stimulating factor. Replication of parvovirus B19 in vitro is possible in these cells after stimulation with erythropoietin. Therefore, this system is an easily, accessible alternative to the use of human bone marrow in parvovirus B19 infection assays.

Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins.

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

Characterization of cis-acting and NS1 protein-responsive elements in the p6 promoter of parvovirus B19.

Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans-activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photinus pyralis). After transfection into HeLa, CEM, BJAB, and K562 cells, the p6 promoter was found to be highly active. When introduced into the erythroid cell line K562, p6-controlled transcription exceeded that of the simian virus 40 promoter-enhancer used as a control by more than 25-fold. Sequence elements relevant for promoter activity mapped to the regions from nucleotides (nt) 100 to 190 and 233 to 298. Also, the segment from nt 343 to 400 downstream of the TATA box was important for transcriptional activity in HeLa and K562 cells. By transfecting the promoter-luciferase constructs into a HeLa cell line stably carrying the viral NS1 gene under the control of an inducible promoter, transcriptional activity mediated by the p6 promoter rose significantly after induction of NS1 expression. The region from nt 100 to 160 proved to be essential for NS1-mediated transcriptional activation. Furthermore, NS1-mediated transactivation was dependent on the presence of two GC-rich elements arranged in tandem upstream of the TATA box. These data indicate that NS1-mediated p6 transactivation is dependent on a multicomponent complex combining NS1 with ATF, NF-kappaB/c-Rel, and GC-box binding cellular factors.

Sequence variability among different parvovirus B19 isolates.

Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins-NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.

Antibodies to the nonstructural protein of parvovirus B19 in persistently infected patients: implications for pathogenesis.

Three patients with persistent parvovirus B19 infection, as documented by the prolonged presence of IgM directed to the viral capsid proteins and detection of viral DNA in serum by dot-blot hybridization or polymerase chain reaction (PCR), were investigated for the presence of antibodies to the nonstructural protein NS-1 of parvovirus B19. This was done by using an ELISA based on recombinant NS-1 protein. Whereas control sera displayed no reactivity, sera from persistently infected patients showed a strong specific antibody response to NS-1. Patients were followed for 3-18 months, during which IgM titers declined but IgG directed to the nonstructural protein remained detectable. The appearance of NS-1-specific antibodies might indicate an altered course of viral infection leading to the establishment of persistently active infection and subsequent destruction of cells of nonerythroid lineage.