Department of Virology, Haartman Institute, PL 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Helsinki, Finland1
Central Military Hospital2, Dextra Medical Centre3 and Departments of Anatomy, Oral Medicine and Rheumatology, University of Helsinki4, Helsinki, Finland
Author for correspondence: Klaus Hedman. Fax +358 9 1912 6491. e-mail klaus.hedman{at}helsinki.fi
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleotide sequence of the entire B19 genome has been determined only from blood, in an asymptomatic donor (Blundell et al., 1987 ) and in a child with aplastic crisis (Shade et al., 1986
). Shorter fragments have also been sequenced from other sources (Erdman et al., 1996
; Umene & Nunoue, 1993
, 1995
), but no disease-specific sequence variations have been observed (Umene & Nunoue, 1993
; Morinet et al., 1986
; Mori et al., 1987
; Haseyama et al., 1998
; Hemauer et al., 1996
; Nguyen et al., 1999
). In general, the unique part of VP1 is the most variable region in the B19 genome.
B19 infection is prevalent; more than half of the adults in industrialized countries have anti-B19 IgG antibodies (Cossart et al., 1975 ; Anderson et al., 1986
). The virus is the major aetiological agent of aplastic crisis, complicating haemolytic anaemias of various forms (Anderson et al., 1982
; Serjeant et al., 1981
; Pattison et al., 1981
), and is responsible for erythema infectiosum (fifth disease), a common epidemic disease in children and young adults (Anderson et al., 1984
). During pregnancy, B19 can be transmitted from mother to foetus, whereby the infection can lead to hydrops and foetal death (Brown et al., 1984
; Miller et al., 1998
). Although the infection in adults is often asymptomatic, especially among females it is frequently complicated by post-infectious arthropathy (Reid et al., 1985
; White et al., 1985
), typically affecting small joints. In most cases, parvovirus arthropathy is transient, but it can become chronic and fulfil the diagnostic criteria of rheumatoid or juvenile arthritis (Naides, 1993
).
The host cells for B19 virus replication in bone marrow are erythrocyte precursors CFU-E and BFU-E (Ozawa et al., 1986 ; Srivastava & Lu, 1988
). The cellular receptor has been identified as the glycolipid globoside (Brown et al., 1993
), while certain other glycosphingolipids may have a similar function (Cooling et al., 1995
). The tissue distribution of these surface receptors correlates with B19-associated disease, even though virus replication is restricted to the erythroid lineage (Brown et al., 1993
; Cooling et al., 1995
). Persistence of B19 DNA in blood and/or in bone marrow due to ongoing virus replication is well established in immunodeficiency (Kurtzman et al., 1988
). However, B19 persistence in bone marrow has also been described in some immunocompetent individuals with parvovirus-related symptoms, and occasionally without an apparent clinical association (Nikkari et al., 1995
; Lundqvist et al., 1999
).
In synovial tissue, B19 DNA has been shown to persist for years or decades after primary infection in a large proportion of subjects with or without chronic arthropathy (Saal et al., 1992 ; Kerr et al., 1995
; Söderlund et al., 1997
; Cassinotti et al., 1997
). However, the synovial persistence data have been based on PCR amplification of segregated, relatively small regions of viral DNA. For example, the previous study from our group utilized primers spanning a 1066 bp fragment at the junction area of the non-structural (NS1) and capsid protein (VP1 and VP2) genes, i.e. one-fifth of the B19 genome (Söderlund et al., 1997
). Neither the molecular and cell-biological mechanisms nor the possible clinical implications of synovial B19 DNA persistence are known.
In order to assess whether B19 DNA occurs in the synovial tissue as an intact molecule or in fragments, we performed PCRs at three different sites of the viral genome using DNA preparations diluted to the end-point as the template. To assess whether the synovial B19 is of a unique genotype or involves particular codon changes that could be causally related to tissue tropism or persistence, we amplified and sequenced 97% of the protein-coding region of the viral genome from four subjects with B19 carriership of varying duration and obtained partial sequence data from a fifth subject. The results were compared with each other and with previously reported B19 sequence data.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibody assays.
Anti-B19 IgG antibodies in serum were measured by a commercial enzyme immunoassay (EIA) (Dako) and anti-B19 IgM antibodies by the EIA of Biotrin. For further evidence of the time of B19 primary infection, the sera of six subjects examined in depth for synovial B19 DNA were studied for epitope-type specificity (ETS) of anti-VP2 IgG (Söderlund et al., 1995 ), as described recently (Kaikkonen et al., 1999
). An acute pattern in this assay indicates B19 primary infection within 36 months, whereas a non-acute pattern indicates B19 immunity of longer duration.
DNA purification.
DNA was purified by proteinase K digestion followed by phenol extraction and ethanol precipitation (Söderlund et al., 1997 ), but using glycogen as a carrier.
DNA controls.
DNA from a viraemic serum containing 1011 B19 genomes per ml was included as a positive control at a dilution of 10-5 in all PCR assays. Water was used as a negative control. Because of the high sensitivity of PCR, extreme precautions were taken to avoid false-positive results. Sample handling, in laminar-flow hoods, and preparation of the reaction mixtures occurred in isolated rooms. Aerosol-resistant pipette tips and disposable racks were used to avoid carry-over. The tissue samples were shown to be negative for PCR inhibitors by amplification of the human chromosomal gene for glyceraldehyde-3-phosphate dehydrogenase. For further controls, tissues from 12 B19-seronegative individuals were processed identically in the VP1 PCR assay, with no positive results.
Primer design and PCR optimization.
Primers were designed according to the sequence of the Au isolate (Shade et al., 1986 ) and our nucleotide numbering corresponds to this reference sequence. Optimal reaction conditions for each primer set were determined by using the control DNA as template.
We aimed to equalize the sensitivities of the NS1 and VP2 PCR methods to the level of the VP1 PCR, which has been shown to detect one target molecule in 2 µl template (Söderlund et al., 1997 ). The sensitivities of the assays were monitored with serially diluted control DNA. The detection sensitivities of all three PCRs were within one order of magnitude.
To compare the different B19 genomic regions within individual samples, DNA suspensions were diluted serially in 10-fold steps and each dilution was studied by the nested VP1 PCR. The last dilution (end-point) giving a positive signal by ethidium bromide staining was then used as a template in the NS1 and VP2 PCRs (Table 2).
|
NS1 PCR.
The outer primer pair was NSofwd and NSorev and the inner primer pair was NSifwd and NSirev (Table 1). Two µl of the end-point-diluted DNA suspension was added to 48 µl PCR mixture [0·2 µM both primers, 2·5 U AmpliTaq Gold (Perkin Elmer), 200 µM each nucleotide in Perkin-Elmer PCR buffer] and, after pre-heating at 94 °C for 10 min and 30 PCR cycles (94 °C for 30 s, 57 °C for 30 s, 68 °C for 1·5 min), 2 µl product was added to 48 µl reaction mixture with the inner primers (0·4 µM NSifwd, 0·8 µM NSirev, 200 µM each nucleotide and 2·5 U AmpliTaq Gold in PCR buffer). DNA was amplified for another 40 cycles (94 °C for 30 s, 57 °C for 30 s, 72 °C for 1·5 min).
|
VP2 PCR.
Two µl of the end-point-diluted DNA suspension was used as a template for the first-round PCR (pre-heated at 94 °C for 10 min, 30 cycles of 94 °C for 15 s, 57 °C for 30 s, 68 °C for 1·5 min), performed with 0·2 µM of each of the outer primer pair VP2ofwd and VP2orev. Two µl of the product was transferred to 48 µl reaction mixture with 0·4 µM inner primers VP2ifwd and VP2irev (Table 1). Conditions for the 40 cycles of the nested reaction after preheating were 94 °C for 30 s, 57 °C for 30 s and 72 °C for 20 s. Again, the products were detected with electrophoresis followed by Southern blotting. The probe was amplified with primers VP2pfwd and VP2prev (Table 1
).
Duplex PCR.
Using end-point-diluted DNA as the template, both the VP2 and NS protein-coding sequences were amplified simultaneously in one tube. The first-round PCR utilized the outer primer pairs of the VP2 and NS reactions. The product was purified with the High Pure PCR product purification kit (Boehringer Mannheim) and eluted with 100 µl sterile water, of which 4 µl was transferred to the second reaction tube. Primers for the nested reaction were NSifwd, NSirev, rt1 and VP2irev (Table 1). The primer concentrations were 0·5 µM except for NSirev, for which the optimal concentration was found experimentally to be 1 µM. The resulting amplicons were 439 and 639 nucleotides in size, respectively, and they were separated electrophoretically on a 1% TAEagarose gel and Southern blotted. Hybridization was done simultaneously with NS1 and VP2 probes.
Sequencing.
B19 DNA purified from the synovium of five subjects was amplified by non-nested PCR to give five partly overlapping amplicons of ~1000 bp, which together covered the whole protein-coding part of the genome. For these reactions, undiluted DNA samples were used to ensure that the resulting PCR product would represent the most common sequence of the viral DNA molecules present in the sample. Before sequencing, the PCR products were purified with the PCR product purification kit (Boehringer Mannheim). The same primers were used both for the PCR and the sequencing reactions. Sequencing was done (at the Institute of Biotechnology, University of Helsinki) from both ends of the amplicons. The primers were NSofwd and NSirev, NSsfwd and NSorev, p6 and p3, p9 and rtsrev and the last pair was rt1 and VP2orev (Table 1).
Phylogenetic analysis.
Phylogenetic analysis was performed on a 346 nt region corresponding to nt 22462789 of the viral genome. Sequence alignments were done by using CLUSTAL W version 1.75 (Thompson et al., 1994 ) and subsequent analyses were done by using the PHYLIP package (Felsenstein, 1989
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NS1 and VP2 PCR
The last dilutions of DNA that gave a positive VP1 signal (end-point dilutions) were used as the template in PCR assays for the VP2 and NS1 genes. The entire protein-coding area of the B19 genome could be found in all six samples (Table 2). With five of these samples, the last dilution that was positive in VP1 PCR also gave a positive result in VP2 and NS1 PCR assays. With the one sample positive in VP1 PCR in dilutions up to 1:10000, the NS1 and VP2 regions could be detected in dilutions up to 1:1000 (Table 2
).
Duplex PCR
In order to assess whether the tissues contained the entire B19 coding region and whether the DNA was intact or fragmented, both ends of the coding region were co-amplified in end-point-diluted templates. The NS1 and VP2 regions were detectable simultaneously in all four samples studied.
Sequencing
Sequencing was performed on five subjects as outlined in Methods. An almost-complete sequence was obtained for four of these samples, with just the two terminal primers and 438 nucleotides directly internal of them being unreadable. At approximately nt 3800 of the viral genome, i.e. near the extreme limit of sequence readability from either end of one particular amplicon, another small gap of 26 nt was not readable for three of four samples. In all, >97% of the B19 coding region of 4354 nt of the Au strain (corresponding to a coding sequence of 4362 nucleotides) was sequenced from each of the four isolates (Kati 1Kati 4).
Both in our patients with recent infection and in our subjects with past immunity, the sequence identity relative to the Au reference was >99%. Altogether, our synovial sequences differed from either reference (Au or Wi) by 27 conserved (i.e. occurring in every subject studied) nucleotide changes. Yet, all our conserved changes relative to either reference strain were found to agree with the other reference strain. Of the nine conserved changes relative to Au, five were within the NS gene and four within the VP1/2 gene (Fig. 1). While six of these mutations were silent, three changed an amino acid; nt 692 converted threonine to isoleucine, nt 3809 converted serine to threonine and nt 3182 converted serine to proline. Conversely, our synovial sequences differed from the Wi reference in 18 conserved changes. For all 18 nucleotides, our sequences were identical to the Au reference (Fig. 1
).
|
We next examined our sequence data for evidence of non-conserved mutations that might inactivate the B19 proteins functionally. Throughout our samples, non-silent mutations were rare, constituting <25% of all nucleotide differences (Table 3). Of the few that were found, none involved changes in amino acid side groups that would appear to cause gross disruption of the protein structure. In addition, no stop codons, frame-shifts, insertions or deletions were found.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The B19 genome has long palindromic repeats at both termini (Deiss et al., 1990 ; Shade et al., 1986
; Astell & Blundell, 1989
) consisting mainly of GC base pairs, which, together with their hairpin secondary structure, provide extremely difficult targets for PCR detection. We did not examine these non-coding termini. However, with an intact, full-length coding region present in synovium, viral mRNA and protein could potentially be produced; the B19 genomes could then possibly have a pathogenetic role. Recent findings suggesting such a role (Takahashi et al., 1998
) await confirmation.
A single point mutation in a viral coding region can alter host-cell tropism or might lead to organ-specific persistence. The host-range determinants of the non-human parvoviruses minute virus of mice (MVM) and feline and canine parvoviruses (FPV; CPV) have been characterized in detail. Just two amino acids of the capsid region determine the lymphoid-cell tropism of the MVM variant MVMi and the fibroblast tropism of MVMp (Ball-Goodrich & Tattersall, 1992 ). Likewise, a change of only a few amino acids yields canine tropism for FPV (Chang et al., 1992
). However, with the exception of a recently discovered variant strain, V9 (Nguyen et al., 1998
), the human B19 viruses from blood or bone marrow characterized until now have differed by conspicuously little in their sequences. Neither have disease-specific variants been reported. Similarly, the B19 coding sequences that we detected in synovium resembled closely those of two reference strains from blood, Au (Shade et al., 1986
) and Wi (Blundell et al., 1987
). This implies that the tropism for and genome persistence in synovial tissue are not related to specific mutations or strain variants. Such a conclusion is supported further by phylogenetic analysis of the VP1 unique region, in which our sequences showed no clustering apart from the known blood-derived sequences. Considering the immunological importance of the VP1 unique region, our data suggest further that neither synoviotropism nor persistence of the B19 virus is due to sequence-based evasion of host immunity. Interestingly, this same capsid protein region maintains IgG subclass 4 reactivity, a marker of chronic or recurrent antigenic stimulus (Franssila et al., 1996
).
Whereas in permissive cells, the VP transcripts outnumber the NS1 transcripts, the latter predominate in some non-permissive cell types (Liu et al., 1992 ; Leruez et al., 1994
). As NS1 regulates B19 gene expression via the p6 promoter (Doerig et al., 1990
), mutations in NS1 affecting its transactivation capacity could have pleiotropic effects on the pathogenicity of B19 infection, for example via modulation of its cytotoxicity. However, no conserved mutations pointing to this mechanism in synovial persistence were observed. With B19 detected in blood (presumably reflecting ongoing replication in the marrow), sequences of the NS1 region have been reported to be somewhat more variable during long-term persistence than in acute infection (Hemauer et al., 1996
). In our synovial samples, the recently and remotely infected individuals also resembled each other in extent of variability, although none of the sequences were identical. With these findings, it is tempting to speculate that the mechanisms of B19 persistence in bone marrow and synovium are different, so that only the former depends on continuous DNA replication. A larger sample is required for verification of this hypothesis.
Taken together, our data show that neither short-term synoviotropism nor long-term synovial persistence of parvovirus B19 require DNA sequence modifications. The single-stranded DNA would appear to enter the joint tissue at its full size and to be retained there for years or even decades (Saal et al., 1992 ; Kerr et al., 1995
; Söderlund et al., 1997
; Cassinotti et al., 1997
) without fragmentation. For such an innocuous role, macrophages or other actively hydrolytic cells would seem an unlikely candidate.
Synovial lining cells can be roughly divided into three populations according to morphology and surface antigen expression (Barland et al., 1962 ; Burmester et al., 1983
). Type A lining cells express antigens of the monocyte lineage and conform to the criteria of mature macrophages that are relatively long lived and originate from the bone marrow (Edwards & Willoughby, 1982
), where B19 replicates. Type B synoviocytes are fibroblastoid in morphology and express fibroblast-associated antigens (Burmester et al., 1983
). However, they differ from fibroblasts of other organs in a number of respects, e.g. in certain diseases and in vitro conditions they are rich in lysosomes (Fraser et al., 1979
), pointing to active extracellular uptake. The third, minor population includes cells with dendritic appearance and other characteristics of antigen presentation and/or storage (Wilkinson et al., 1990
). Our data would best fit retention of B19 virus genomes in synoviocytes of the third category, which in turn would underline the importance of synovium in immunological processes, possibly as a site for maintenance of memory. Indeed, identification of the cell type(s) hosting B19 virus persistence in the joint tissue and the transcriptional and replicative functionality of the viral genomes therein will be exciting subjects for future study.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, M. J., Lewis, E., Kidd, I. M., Hall, S. M. & Cohen, B. J. (1984). An outbreak of erythema infectiosum associated with human parvovirus infection.Journal of Hygiene 93, 85-93.[Medline]
Anderson, L. J., Tsou, C., Parker, R. A., Chorba, T. L., Wulff, H., Tattersall, P. & Mortimer, P. P. (1986). Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay.Journal of Clinical Microbiology 24, 522-526.[Medline]
Astell, C. R. & Blundell, M. C. (1989). Sequence of the right hand terminal palindrome of the human B19 parvovirus genome has the potential to form a stem plus arms structure.Nucleic Acids Research 17, 5857.[Medline]
Ball-Goodrich, L. J. & Tattersall, P. (1992). Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice.Journal of Virology 66, 3415-3423.[Abstract]
Barland, P., Novikoff, A. B. & Hamerman, D. (1962). Electron microscopy of the human synovial membrane.Journal of Cell Biology 14, 207-220.
Blundell, M. C., Beard, C. & Astell, C. R. (1987). In vitro identification of a B19 parvovirus promoter.Virology 157, 534-538.[Medline]
Brown, T., Anand, A., Ritchie, L. D., Clewley, J. P. & Reid, T. M. S. (1984). Intrauterine parvovirus infection associated with hydrops fetalis. Lancet ii, 10331034.
Brown, K. E., Anderson, S. M. & Young, N. S. (1993). Erythrocyte P antigen: cellular receptor for B19 parvovirus.Science 262, 114-117.[Medline]
Burmester, G. R., Dimitriu-Bona, A., Waters, S. J. & Winchester, R. J. (1983). Identification of three major synovial lining cell populations by monoclonal antibodies directed to Ia antigens and antigens associated with monocytes/macrophages and fibroblasts.Scandinavian Journal of Immunology 17, 69-82.[Medline]
Cassinotti, P., Burtonboy, G., Fopp, M. & Siegl, G. (1997). Evidence for persistence of human parvovirus B19 DNA in bone marrow.Journal of Medical Virology 53, 229-232.[Medline]
Chang, S.-F., Sgro, J.-Y. & Parrish, C. R. (1992). Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties.Journal of Virology 66, 6858-6867.[Abstract]
Cooling, L. L. W., Koerner, T. A. W. & Naides, S. J. (1995). Multiple glycosphingolipids determine the tissue tropism of parvovirus B19.Journal of Infectious Diseases 172, 1198-1205.[Medline]
Cossart, Y. E., Field, A. M., Cant, B. & Widdows, D. (1975). Parvovirus-like particles in human sera. Lancet i, 7273.
Cotmore, S. F., McKie, V. C., Anderson, L. J., Astell, C. R. & Tattersall, P. (1986). Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments.Journal of Virology 60, 548-557.[Medline]
Deiss, V., Tratschin, J.-D., Weitz, M. & Siegl, G. (1990). Cloning of the human parvovirus B19 genome and structural analysis of its palindromic termini.Virology 175, 247-254.[Medline]
Doerig, C., Hirt, B., Antonietti, J.-P. & Beard, P. (1990). Nonstructural protein of parvoviruses B19 and minute virus of mice controls transcription.Journal of Virology 64, 387-396.[Medline]
Edwards, J. W. C. & Willoughby, D. A. (1982). Demonstration of bone marrow derived cells in synovial lining by means of giant intracellular granules as genetic markers.Annals of the Rheumatic Diseases 41, 177-182.[Abstract]
Erdman, D. D., Durigon, E. L., Wang, Q.-Y. & Anderson, L. J. (1996). Genetic diversity of human parvovirus B19: sequence analysis of the VP1/VP2 gene from multiple isolates.Journal of General Virology 77, 2767-2774.[Abstract]
Felsenstein, J. (1989). PHYLIP Phylogeny inference package. (version 3.2).Cladistics 5, 164-166.
Franssila, R., Söderlund, M., Brown, C. S., Spaan, W. J. M., Seppälä, I. & Hedman, K. (1996). IgG subclass response to human parvovirus B19 infection.Clinical and Diagnostic Virology 6, 41-49.
Fraser, J. R., Clarris, B. J. & Baxter, E. (1979). Patterns of induced variation in the morphology, hyaluronic acid secretion, and lysosomal enzyme activity of cultured human synovial cells.Annals of the Rheumatic Diseases 38, 287-294.[Abstract]
Haseyama, K., Kudoh, T., Yoto, Y., Suzuki, N. & Chiba, S. (1998). Analysis of genetic diversity in the VP1 unique region gene of human parvovirus B19 using the mismatch detection method and direct nucleotide sequencing.Journal of Medical Virology 56, 205-209.[Medline]
Hemauer, A., von Poblotzki, A., Gigler, A., Cassinotti, P., Siegl, G., Wolf, H. & Modrow, S. (1996). Sequence variability among different parvovirus B19 isolates.Journal of General Virology 77, 1781-1785.[Abstract]
Kaikkonen, L., Lankinen, H., Harjunpää, I., Hokynar, K., Söderlund-Venermo, M., Oker-Blom, C., Hedman, L. & Hedman, K. (1999). Acute-phase-specific heptapeptide epitope for diagnosis of parvovirus B19 infection.Journal of Clinical Microbiology 37, 3952-3956.
Kerr, J. R., Cartron, J. P., Curran, M. D., Moore, J. E., Elliott, J. R. & Mollan, R. A. (1995). A study of the role of parvovirus B19 in rheumatoid arthritis.British Journal of Rheumatology 34, 809-813.[Medline]
Kurtzman, G. J., Cohen, B., Meyers, P., Amunullah, A. & Young, N. S. (1988). Persistent B19 parvovirus infection as a cause of severe chronic anaemia in children with acute lymphocytic leukaemia. Lancet ii, 11591162.
Leruez, M., Pallier, C., Vassias, I., Elouet, J. F., Romeo, P. & Morinet, F. (1994). Differential transcription, without replication, of non-structural and structural genes of human parvovirus B19 in the UT7/EPO cell line as demonstrated by in situ hybridization.Journal of General Virology 75, 1475-1478.[Abstract]
Liu, J. M., Green, S. W., Shimada, T. & Young, N. S. (1992). A block in full-length transcript maturation in cells nonpermissive for B19 parvovirus.Journal of Virology 66, 4686-4692.[Abstract]
Lundqvist, A., Tolfvenstam, T., Brytting, M., Stolt, C. M., Hedman, K. & Broliden, K. (1999). Prevalence of parvovirus B19 DNA in bone marrow of patients with haematological disorders.Scandinavian Journal of Infectious Diseases 31, 119-122.[Medline]
Miller, E., Fairley, C. K., Cohen, B. J. & Seng, C. (1998). Immediate and long term outcome of human parvovirus B19 infection in pregnancy.British Journal of Obstetrics & Gynaecology 105, 174-178.
Mori, J., Beattie, P., Melton, D. W., Cohen, B. J. & Clewley, J. P. (1987). Structure and mapping of the DNA of human parvovirus B19.Journal of General Virology 68, 2797-2806.[Abstract]
Morinet, F., Tratschin, J. D., Perol, Y. & Siegl, G. (1986). Comparison of 17 isolates of the human parvovirus B19 by restriction enzyme analysis.Archives of Virology 90, 165-172.[Medline]
Naides, S. J. (1993). Parvovirus B19 infection.Rheumatic Disease Clinics of North America 19, 457-475.[Medline]
Nguyen, Q. T., Sifer, C., Schneider, V., Bernaudin, F., Auguste, V. & Garbarg-Chenon, A. (1998). Detection of an erythrovirus sequence distinct from B19 in a child with acute anaemia.Lancet 352, 1524.[Medline]
Nguyen, Q. T., Sifer, C., Schneider, V., Allaume, X., Servant, A., Bernaudin, F., Auguste, V. & Garbarg-Chenon, A. (1999). Novel human erythrovirus associated with transient aplastic anemia.Journal of Clinical Microbiology 37, 2483-2487.
Nikkari, S., Roivainen, A., Hannonen, P., Möttönen, T., Luukkainen, R., Yli-Jama, T. & Toivanen, P. (1995). Persistence of parvovirus B19 in synovial fluid and bone marrow.Annals of the Rheumatic Diseases 54, 597-600.[Abstract]
Ozawa, K., Kurtzman, G. & Young, N. (1986). Replication of the B19 parvovirus in human bone marrow cell cultures.Science 233, 883-886.[Medline]
Ozawa, K., Ayub, J., Hao, Y.-S., Kurtzman, G., Shimada, T. & Young, N. (1987). Novel transcription map for the B19 (human) pathogenic parvovirus.Journal of Virology 61, 2395-2406.[Medline]
Pattison, J. R., Jones, S. E., Hodgson, J., Davis, L. R., White, J. M., Stroud, C. E. & Murtaza, L. (1981). Parvovirus infections and hypoplastic crisis in sickle-cell anaemia. Lancet i, 664665.
Reid, D. M., Reid, T. M. S., Brown, T., Rennie, R. A. N. & Eastmond, C. J. (1985). Human parvovirus-associated arthritis: a clinical and laboratory description. Lancet i, 422425.
Saal, J. G., Steidle, M., Einsele, H., Muller, C. A., Fritz, P. & Zacher, J. (1992). Persistence of B19 parvovirus in synovial membranes of patients with rheumatoid arthritis.Rheumatology International 12, 147-151.[Medline]
Serjeant, G. R., Topley, J. M., Mason, K., Serjeant, B. E., Pattison, J. R., Jones, S. E. & Mohamed, R. (1981). Outbreak of aplastic crises in sickle-cell anaemia associated with parvovirus-like agent. Lancet ii, 595597.
Shade, R. O., Blundell, M. C., Cotmore, S. F., Tattersall, P. & Astell, C. R. (1986). Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis.Journal of Virology 58, 921-936.[Medline]
Söderlund, M., Brown, C. S., Spaan, W. J. M., Hedman, L. & Hedman, K. (1995). Epitope type-specific IgG responses to capsid proteins VP1 and VP2 of human parvovirus B19.Journal of Infectious Diseases 172, 1431-1436.[Medline]
Söderlund, M., von Essen, R., Haapasaari, J., Kiistala, U., Kiviluoto, O. & Hedman, K. (1997). Persistence of parvovirus B19 DNA in synovial membranes of young patients with and without chronic arthropathy.Lancet 349, 1063-1065.[Medline]
Srivastava, A. & Lu, L. (1988). Replication of B19 parvovirus in highly enriched hematopoietic progenitor cells from normal human bone marrow.Journal of Virology 62, 3059-3063.[Medline]
Takahashi, Y., Murai, C., Shibata, S., Munakata, Y., Ishii, T., Ishii, K., Saitoh, T., Sawai, T., Sugamura, K. & Sasaki, T. (1998). Human parvovirus B19 as a causative agent for rheumatoid arthritis.Proceedings of the National Academy of Sciences, USA 95, 8227-8232.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research 22, 4673-4680.[Abstract]
Umene, K. & Nunoue, T. (1993). Partial nucleotide sequencing and characterization of human parvovirus B19 genome DNAs from damaged human fetuses and from patients with leukemia.Journal of Medical Virology 39, 333-339.[Medline]
Umene, K. & Nunoue, T. (1995). A new genome type of human parvovirus B19 present in sera of patients with encephalopathy.Journal of General Virology 76, 2645-2651.[Abstract]
White, D. G., Woolf, A. D., Mortimer, P. P., Cohen, B. J., Blake, D. R. & Bacon, P. A. (1985). Human parvovirus arthropathy. Lancet i, 419421.
Wilkinson, L. S., Worrall, J. G., Sinclair, H. D. & Edwards, J. C. (1990). Immunohistological reassessment of accessory cell populations in normal and diseased human synovium.British Journal of Rheumatology 29, 259-263.[Medline]
Received 25 November 1999;
accepted 23 December 1999.